Patients who do not complete one cycle of therapy on Phase I trials for reasons other than dose limiting toxicity (DLT) are considered inevaluable for toxicity and must be replaced.Individual records from patients enrolled to NCI-sponsored Phase I trials activated between 2000 and 2010 were used. Early discontinuation was defined as the failure to begin cycle 2 for reasons other than a DLT during cycle 1. A multinomial logistic regression with a 3-level nominal outcome  was used with continuation to cycle 2 serving as the reference category. The final model was used to create two risk scores. An independent external cohort was used to validate these models.P = .0315 and P = .0007), creatinine clearance , alkaline phosphatase , AST , hemoglobin , albumin , and platelets  were predictors of early discontinuation. The c-index of the final model was 0.63.Data from 3079 patients on 127 Phase I trials were analyzed. ECOG performance status (1, ≥ 2, two-sided Knowledge of risk factors for early treatment discontinuation in conjunction with clinical judgment can help guide Phase I patient selection. Phase I eligibility criteria are intended to select fit patients with good performance status, near normal organ function, and minimal co-morbidities in order to accurately characterize the toxicity of the investigational drug –5. In adUnfortunately, early trial discontinuation remains a fairly common event in Phase I studies despite strict entry criteria. In one large series, 16% of patients enrolled to Phase I trials discontinued protocol therapy within the first 21 days of beginning treatment . Althoug9/L, hemoglobin ≥ 8 g/dL, platelet count ≥ 75 × 109/L, aspartate aminotransferase (AST) or alanine aminotransferase (ALT) ≤ 5x upper limit of normal (ULN), and total bilirubin ≤ 2x ULN. This led to the exclusion of 5% of patients in the derivation dataset, reducing the number of patients from 3910 to 3717. Patients with incomplete data for one or more of the covariates included in the final multivariate model were also excluded. On this basis, 17% (N = 638) of the 3717 patients in the derivation set were excluded. Baseline characteristics of patients with complete and missing data were largely similar (data not shown). All patients had regular follow-up visits as specified by the protocol to which they were enrolled. Patients must have received at least one dose of study drug(s) to be included in the analysis.A multicenter cohort of all patients treated on NCI-sponsored  Phase I An independent cohort of 232 patients consecutively enrolled to 20 Phase I trials between 2009 and 2012 in the Developmental Therapeutics Clinic at Memorial Sloan Kettering Cancer Center was used as a validation set. CTEP-sponsored studies included in the derivation set were excluded from this cohort. Patients and trials in the validation set were required to meet the same eligibility criteria as the derivation set.The primary outcome for each patient was early discontinuation (yes/no), which was defined as: 1) the inability to begin cycle 2 of therapy, and 2) the absence of any dose limiting toxicity (DLT) during cycle 1. The DLT criteria were standardized to allow for a uniform outcome definition across all trials and were defined as a grade ≥ 3 non-hematologic or grade ≥ 4 hematologic toxicity attributed as at least possibly related to study treatment, excluding asymptomatic electrolyte abnormalities. Toxicity level data were used to determine whether each patient had an adverse event that qualified under this definition of DLT. These DLT criteria are commonly used in contemporary Phase I studies , 18.The following categories of baseline patient characteristics were investigated for their association with early trial discontinuation: 1) commonly utilized Phase I eligibility criteria, 2) established prognostic factors, 3) prior treatment exposure, and 4) disease burden.All variables except ECOG performance status (PS) were dichotomized to facilitate the final goal of creating a simple risk score. Cutoffs were chosen based on upper limit of normal and/or cutoffs defined by the NCI Common Terminology Criteria of Adverse Events (CTCAE), most commonly using the criteria for grade 1 abnormalities. For certain covariates, grade 1 abnormalities were very common and would therefore have been of limited discriminatory utility and in those cases criteria for grade 2 abnormalities were utilized. Similarly, cutoffs that would identify a small minority of patients and thus have limited clinical utility and statistical properties were avoided. ECOG PS was treated as a categorical variable with three levels: 0, 1, and ≥ 2. Due to concern about the heterogeneity of the patients who did not discontinue early (this group contains both patients who experienced a DLT during cycle 1 and patients who began cycle 2 without a DLT), multinomial logistic regression with a 3-level nominal outcome  was used to assess relationships between factors and outcome. Continuation to cycle 2 was the reference category.P-value  were sequentially removed until all discontinuation-specific P-values were < 0.10. The final logistic model assigns each patient a predicted risk of cycle 1 DLT, early discontinuation, and continuation to cycle 2. The binary outcome for model validation was early discontinuation (yes/no), with cycle 1 DLT and continuation to cycle 2 combined. Predictive accuracy of the multinomial logistic model was evaluated with the concordance-index (c-index), which estimates the proportion of pairs where the patient who discontinued early has a higher model-predicted risk of early discontinuation than the patient who did not discontinue early.Candidate factors for the multivariate model were selected based on clinical reasoning and univariate results, and the final model was selected using a backward selection procedure where variables with the largest discontinuation-specific The final multinomial model (based on Hosmer equations 8.1 to 8.5 ) was useThe risk scores were dichotomized at various cutoff points to define patients at low and high risk of early discontinuation. Overall correct classification rate was defined as the percent of patients who were classified correctly: either as low risk and did not discontinue early, or as high risk and did discontinue early. Two cutoffs  were selected based on their performance in the CTEP data, with an emphasis on high specificity, which translates to a low probability of classifying patients who did not discontinue early as high risk. These were then validated on an external dataset. C-index, sensitivity, specificity, and overall correct classification rate were used to assess full model performance and risk score performance in the external data.P-values were two-sided, and P-values less than 0.05 were considered significant.All statistical analysis was performed in SAS 9.2  and R 3.0.1  using the pROC and verification packages. All N = 312) discontinued early for clinical progression or death, 23% (N = 115) for an adverse event that was not a DLT, and 2% (N = 10) for other reasons. No off study reason was provided for 14% (N = 71) of the patients who discontinued early.Data on 3079 patients treated on 127 Phase I trials were analyzed. Baseline patient characteristics are presented in Table P = 0.0008 and P < .0001 for PS 1, ≥ 2 respectively), WBC (P = 0.0005), platelet count (P < .0001), AST (P < .0001), alkaline phosphatase (P < .0001), ALT (P = 0.0305), number of prior lines of therapy  and lower hemoglobin (P < .0001), creatinine clearance (P = 0.0289), number of metastatic sites , and albumin (P < .0001) were significantly associated with the risk of early discontinuation. Primary tumor site was not considered a candidate for the multivariate model because we did not wish to discriminate against specific tumor types. Similarly, prior lines of therapy and number of metastatic sites were not candidates for the final model because we did not wish to exclude broad groups of patients who constitute a significant proportion of the Phase I eligible population from participation in these studies. Other significant variables were carried forward to a multivariate model, where WBC, ALT, and absolute lymphocyte count (ALC) were not independent predictors of early discontinuation (P > 0.10) and were subsequently removed from the model, yielding the final model. The final multivariate model, accounting for ECOG PS , creatinine clearance , alkaline phosphatase , AST , hemoglobin , albumin , and platelet count  had a c-index of 0.63 in the derivation set. The c-index of this model with all the covariates included as continuous except ECOG PS was 0.64, and the c-index of our final model with the additional covariates number of prior lines of treatment and number of metastatic sites is 0.64; thus we feel that our modeling choices did not lead to decreased model performance. The odds ratios for both outcomes of the final multinomial model are shown in N = 232). The rate of early discontinuation in the validation set was 15%. The derivation and validation sets were well balanced with regards to the presence of factors independently associated with the risk of early discontinuation . A simplified risk score further condenses the multivariate model by assigning 1 point each for the four characteristics with the largest impact on the risk of early discontinuation: ECOG PS (≥ 2), albumin (< 3.5 g/dL), alkaline phosphatase (> 2.5xULN), and hemoglobin (< 10 g/dL).In order to create a user-friendly clinical decision aid, two risk scores were developed utilizing the final multivariate model and assessed for performance. The two proposed risks scores and their associated sensitivity, specificity, and overall correct classification rate (OCCR) in both the derivation and validation sets are shown in Table Figure Utilizing individual patient records from a very large, multi-institutional, contemporary cohort of North American patients enrolled to Phase I trials, we identified baseline clinical characteristics independently associated with the risk of early trial discontinuation. To our knowledge, this is the first such analysis performed to date. We identified several factors independently associated with early trial discontinuation including higher ECOG PS, alkaline phosphatase, AST, and platelets and decreasing albumin, hemoglobin, and creatinine clearance. These results offer important insights to physicians charged with selecting appropriate patients for Phase I trials.9/L) is frequently cited as a prognostic factor, it did not predict for early discontinuation after adjusting for other covariates. Creatinine clearance, an independent predictor of early discontinuation, is not a well established prognostic factor. Moreover, the likelihood of early discontinuation due to progression or death was similar in our overall population and for patients with ≥ 2 points on our simplified risk score . Patients with ≥ 2 of these risk factors prior to enrollment had an observed rate of early discontinuation of 31%, approximately twice the rate of the overall population (16.5%). By comparison, patients with none of these risk factors had only a 12% chance of discontinuing early, a relative risk reduction of 27% compared to the overall population. Both risk scores had similar performance in the derivation and validation sets, suggesting they are generalizable to new patient populations. Additionally, the risk scores performed similarly when assessed in the subset of patients who received molecular targeted agents only, indicating that these scores will continue to be relevant as drug development becomes more focused on these agents.To illustrate how using the simplified risk score would impact patient selection and the composition of Phase I trials, Figure Unfortunately, the number of patients seeking Phase I trials often exceeds study availability at high volume centers. As such, physicians often must select from multiple potentially eligible patients for a limited number of study spots. In doing so, physicians attempt to identify which patients are most likely to remain on study long enough to potentially benefit. Currently, physicians must rely solely on clinical experience to make these difficult judgments. In these circumstances, we believe that even highly expert Phase I investigators can benefit from the knowledge of how a limited number of objective patient characteristics may increase the risk of early treatment discontinuation.


Legionella in drinking water distribution systems, the growth and survival of Legionella under various conditions was studied. When incubated in tap water at 4 °C, 25 °C, and 32 °C, L. pneumophila survival trends varied amongst the temperatures, with the stable populations maintained for months at 25 °C and 32 °C demonstrating that survival is possible at these temperatures for extended periods in oligotrophic conditions. After inoculating coupons of PVC, copper, brass, and cast iron, L. pneumophila colonized biofilms formed on each within days to a similar extent, with the exception of cast iron, which contained 1-log less Legionella after 90 days. L. pneumophila spiked in a model drinking water distribution system colonized the system within days. Chlorination of the system had a greater effect on biofilm-associated Legionella concentrations, with populations returning to pre-chlorination levels within six weeks. Biofilms sampled from drinking water meters collected from two areas within central Arizona were analyzed via PCR for the presence of Legionella. Occurrence in only one area indicates that environmental differences in water distribution systems may have an impact on the survival of Legionella. These results document the impact of different environmental conditions on the survival of Legionella in water.To examine the impact of environmental factors on  Legionella genus contains over 50 species of gammaproteobacteria [Legionella protection from harmful substances such as disinfectants as well as more ready access to eukaryotic host organisms thought to be required for their replication in the environment [Hartmanella [Legionella are also capable of infecting a wide variety of eukaryotes, with humans being a potential incidental host. Although able to survive in a wide range of temperatures, Legionella typically require relatively warm environments—often found in anthropogenic water systems—to reach population levels capable of posing public health risks to humans [The bacteria , many ofironment . The typironment , notablytmanella . DespiteLegionella pneumophila [Legionella being responsible for more drinking water related disease outbreaks in the United States than any other microbe [The first reported outbreak of legionellosis occurred at an American Legion convention in Philadelphia, PA during the summer of 1976. Several months later, the causative agent of the pneumonia cases linked to the outbreak was found to be a previously unidentified environmental bacterium, umophila . DrasticLegionella require temperatures above 20 °C to grow, contamination is often associated with heated artificial water systems such as cooling towers, spas, and hot tap water lines [Legionella replication are more likely to occur in these environments, non-heated engineered water systems are also common sources of outbreaks [Legionella at the in-premise level, a relatively small amount of research has been focused on Legionella within main distribution systems [Legionella in drinking water distribution systems via the following experiments: (1) a bench-scale experiment to measure the capability for Legionella to survive in tap water at varying temperatures; (2) a bench-scale experiment to determine the association and survival of Legionella within biofilms on various water pipe materials; (3) a pilot-scale experiment examining the interactions between Legionella in flowing water and biofilms of a model drinking water distribution system (MDS); and (4) a field study of the occurrence of Legionella in drinking water distribution system biofilms from residential water meters.As er lines . As a reutbreaks . Due to  systems , leadingLegionella was observed at any temperature /mL for the remainder of the study period. At 25 °C, Legionella concentration decreased exponentially for 30 days and stabilized at approximately 105 CFU/mL. At 32 °C, Legionella concentration decreased at an exponential rate for 18 days and relatively stabilized at approximately 5 × 103 CFU/mL. After the initial decrease, the variation in concentration was more pronounced at 32 °C than for the other temperatures. After 11 months, Legionella concentrations at 4 °C, 25 °C, and 32 °C were <1 CFU/mL, <1 CFU/mL, and >103 CFU/mL, respectively, suggesting that long-term survival of Legionella was supported by the higher temperature.During the first week of incubation, no significant decrease in the concentration of perature . At 4 °CLegionella biofilm association (105 to 2.7 × 105 CFU/cm2) was observed three days after inoculation  followed a similar trend to brass, increasing to 1.6 × 105 CFU/cm2 by day 21 before decreasing. Final concentrations on copper, brass, and PVC were similar at day 90, each near 104 CFU/cm2, approximately a 1-log reduction from the initial spiked concentrations. Legionella levels in water surrounding each coupon  were detected in samples collected from both sampling ports  decreased to 1.1 × 102 CFU mL−1. After six weeks, concentrations in both biofilms and the MDS flowing water increased to pre-chlorination levels. In addition, culturable Legionella were detected for over a year in the MDS, with approximately 1.5 × 103 CFU mL−1 in flowing water and 2 × 103 CFU cm−2 in biofilm samples 13 months after the initial inoculation.Within hours of inoculation, similar ng ports  of the Mmeasured . ConcentLegionella in 9 m, with five samples also testing positive for L. pneumophila  during the initial three months of incubation may have been due to lowered metabolically linked cell turnover or similar factors. Cell cycling may have also contributed to the differences in concentrations observed at the two temperatures, as the highly pleomorphic Legionella spp. are known to alter cellular morphology in response to environmental stresses to aid survival under extreme conditions [Legionella [Legionella. This is particularly relevant in regards to filamentous cells, as a single multinucleate filament capable of giving rise to dozens of individual bacilli under specific environmental conditions may only produce a single colony when cultured. Regardless of cell cycling dynamics, replication could have contributed to the increased survival observed at both higher temperatures compared to 4 °C. This appears to have been demonstrated by multiple concentration increase in Legionella population in the 32 °C incubation, including a one log increase between days 67 and 82. No such increase in population was recorded for the cultures incubated at the other temperatures. It should be noted that the long-term survival and population stabilization from this experiment occurred in the absence of mature biofilm formation, as the frequent agitation of the tubes prior to sampling should have disrupted and minimized biofilm formation.Data from the initial three months of the experiment indicate a more stable population of perature , raisingnditions . High tegionella , which mLegionella within biofilms on each coupon examined demonstrates the capability of this organism to establish a population on a wide variety of pipe materials. Two materials chosen due to their assumed antimicrobial properties [Legionella in biofilms than on the cast iron coupon. While this may have been due to a combination of replication, different rates of colonization/release, or impacts on culturability within biofilms formed on cast iron, it appears that Legionella may have increased affinity for these presumed antibacterial materials than previously thought. Overall, Legionella concentrations were highest on the PVC coupon, although the final recorded concentrations were similar for PVC, brass, and copper. These results suggest that, while pipe material may be a factor in the long-term survival of Legionella populations, the dynamics of Legionella persistence in distribution systems may rely more significantly on other factors, such as pipe age, water quality, and microbial community, which were unaccounted for in this batch study. The net concentration changes were similar between water and biofilms for each coupon, with the exception of cast iron, which demonstrated an additional 1-log reduction compared to the other coupons. Overall, steady reductions in Legionella concentration were measured for the water surrounding each material, while concentrations in biofilms experienced more drastic fluctuations. This discrepancy between concentrations over time in biofilms and water suggests that dynamics such as biofilm release/re-colonization, replication, or cell cycling (including transitions to viable but non-culturable states) may have been occurring, potentially most noticeably within biofilms on the cast iron coupon. As expected, Legionella population dynamics substantially varied amongst biofilms and water, characterized by periods of concentration increase, stability, and sharp decrease in biofilms, as opposed to steady decreases observed in water samples.The association of operties ,17, brasLegionella over the first 10 days after inoculation can be explained by dispersal throughout the MDS and colonization of biofilms. The spikes in concentration occurring at days 58 and 131 were potentially due to release of organisms from biofilms resulting from incidental agitation while removing pipe segments for biofilm sampling. This suggests that analogous disruption events within a drinking water distribution system could cause increases in Legionella concentration in flowing water, resulting in an increased risk of exposure to Legionella. Because the vast majority of Legionella in the MDS were always biofilm associated , the results also suggest that monitoring of only water samples may not provide an accurate estimation of the true level of contamination within a distribution system. Despite the two sampling ports used being located on opposite ends of the MDS with differing flow conditions, similar concentrations (1.44 × 103 CFU/mL and 1.48 × 103 CFU/mL) of Legionella were seen in the flowing water samples collected two hours after inoculation, suggesting a rapid and even dispersal throughout the MDS and within dead ends. This was further supported by the fact that mean Legionella concentrations in samples collected from these two flow conditions remained relatively similar throughout the study. In addition, similar concentrations were observed in water samples collected before and after flushing the ports. It should be noted that the largest variation in water concentrations was measured in the samples collected after chlorination on day 131, possibly related to both differential dispersal of chlorine and sedimentation depending on flow conditions of pipe sections. After fluctuations, Legionella concentrations in both flowing water and biofilms of the MDS reached stable levels for extended periods of time, with an initial increase, then decrease, followed by stabilization. However, in biofilms the periods of increase and decrease occurred 14 days earlier than in the flowing water samples. Chlorination resulted in significant reduction of Legionella within biofilms  and flowing water . The difference in the effectiveness of chlorination against Legionella present in water and biofilm seems contrary to the notion that biofilms typically provide additional protection from disinfection [Legionella in the flowing water in the system by inducing the release of Legionella containing biofilm fragments into the flowing water. This demonstrates a potential scenario in which the concentration of Legionella or other biofilm-dwelling pathogens could increase in the flowing water of a distribution system following increased disinfection. The eventual increase and stabilization observed for concentrations in both biofilms and flowing water following chlorination coincides with previous evidence highlighting the difficulty of treating distribution systems for Legionella [The initial fluctuation in the concentration of nfection . The reaLegionella demonstrates a practical approach for future studies on the occurrence and prevalence of this pathogen in drinking water distribution system biofilms  liquid medium. Cell concentration in stock cultures was estimated using optical density measurement at 600 nm. The spread plate technique was used to quantify Legionella after incubation at 37 °C for 72 h, with up to seven additional days as needed. All laboratory experiments utilized the L. pneumophila serogroup 1 strain Knoxville-1 (American Type Culture Collection 33153).All evention . CulturiLegionella spp. and L. pneumophila specific primers were used in this study . is study . For bot7L. pneumophila cells, duplicate tubes were incubated at 4 °C, 25 °C, and 32 °C. The temperatures chosen for this experiment were intended to represent the range of temperatures commonly recorded in water distribution systems across the United States. The study parameters simulate two temperature conditions possible in an actual distribution system where growth of Legionella may occur (25 °C and 32 °C) and a condition under which replication is impossible (4 °C) [A series of 50-mL polystyrene conical tubes were filled with 50 mL of dechlorinated tap water from the city of Tempe, AZ, USA. Following inoculation with 3 × 10e (4 °C) . Over th8L. pneumophila cells were spiked in each container. One-milliliter water samples and 2 cm2 biofilm samples were collected from different sections on the coupons in each container periodically over 94 days. Prior to sampling, the water in each container was gently agitated to mix in the suspended Legionella while maintaining biofilm integrity. Biofilm samples were collected with sterile cotton swabs and resuspended in sterilized deionized water. CFU/mL and CFU/cm2 were determined for each sample via spread plate technique on BCYE agar medium.A series of 5-L polyethylene containers were filled with 1 L of tap water from the city of Tempe, AZ, USA. Single coupons of PVC, cast iron, copper, and brass were placed in the containers and incubated at 25 °C for three weeks to allow biofilm development. The materials studied were chosen to represent a range of pipes and water fixtures commonly found in drinking water distribution systems in the United States. Following the initial biofilm incubation, 3 × 10Legionella to colonize and survive within distribution system water and biofilms was established using a laboratory scale model water distribution system. The MDS has a loop with PVC piping, consisting of a 5.5 m long and 5.1 cm diameter main pipe, with a 1.4 m long dead end, and a total volume of 50 liters. Distribution mains and dead-end lines consisted of six and two removable pipe sections (65 cm each in length), respectively. The main pipe is connected to a reservoir. A self-priming, thermally protected, magnetic-drive pump  continuously re-circulated water between the main loop and the reservoir. Pressure, flow rate (0.304 m/s), and temperature (25 °C) were kept constant through external controls. The MDS had been used continuously for 11 years and had well established biofilm communities before the start of this experiment.The ability of introduced L. pneumophila, water and biofilm samples were tested to confirm the absence of culturable Legionella in the MDS. After inoculation with approximately 107L. pneumophila cells, water and biofilm samples from the MDS were periodically collected over 131 days and cultured on BCYE to determine Legionella concentrations. Water samples were collected from two sampling ports: (1) one located in a dead end segment of the system approximately 3 m from the reservoir and (2) one located in an open segment of the system approximately 1.2 m from the reservoir. Two 5-mL water samples were collected from each port for each sampling event, one before and one after flushing 1 L of water, with Legionella concentrations averaged between the two. This form of sampling was performed to assess any variation between Legionella in the samples due to stagnant water or biofilms in the ports. When necessary, water samples were concentrated via the membrane filtration technique using 0.45-micron pore size filters resuspended in 10 mL of sterilized deionized water. Biofilm samples were collected from PVC coupons using sterile cotton swabs and resuspended in sterilized deionized water prior to spread plating. After 131 days, the MDS was treated with 5 mg/L of chlorine introduced into the reservoir. Two hours later, water samples were collected along with biofilm samples from a coupon, a dead-end section, and an open end section of pipe.City of Tempe, AZ, USA tap water was circulated in the MDS, following dechlorination. PVC coupons 2.5 cm in diameter and 5 cm in length were suspended in the reservoir for several weeks to allow biofilm formation. Prior to inoculation with a laboratory strain of 2 of biofilm were collected using sterilized nylon wire brushes and resuspended in 20 mL of sterile water. DNA extraction was performed on 750 µL of this suspension, followed by PCR amplification using specific primers for Legionella spp. and L. pneumophila. PCR reactions producing bands visible on a DNA gel were sequenced to identify the presence of Legionella or L. pneumophila. Biofilm samples positive for Legionella were cultured on BCYE via the spread plate technique.Residential drinking water meters were collected from two general areas located in central Arizona: 35 from area A and 32 from area B. Sampling occurred over a period of approximately one month during June/July 2011. Collection was possible due to water meter recycling programs in which water utilities replace mechanically worn meters with new ones. All water meters collected were made of brass, with some containing rubber washers on their inlet rims. Upon removal by utility personnel, meters were submerged in tap water and transferred to the environmental microbiology laboratory at Arizona State University. Biofilm samples were collected from the inlet of each meter within two hours of removal. Approximately 40 cmt-tests were performed to determine the statistical significance of select data.Excel  was used for all data analysis and graph generation. Student’s Legionella contamination of in-premise plumbing, the dispersal and contamination of Legionella in these systems is clearly an important factor to consider. The very water distribution systems that introduce these pathogens to building plumbing systems are a logical target to help prevent legionellosis incidence. By examining Legionella survival in tap water, association in pipe material biofilms, interactions in a model distribution system, and prevalence in drinking water distribution system biofilms, the results of this study provide valuable information relevant to the design of monitoring and control procedures for Legionella in public drinking water.With legionellosis cases continually on the rise in the United States and across the world, the development of appropriate public health protocols to curtail the incidence of this theoretically preventable disease is more important now than ever. Despite the fact that a substantial proportion of efforts focus on monitoring and treatment procedures aimed at preventing


Objective: Gut–brain axis (GBA) is very important in creation and modulation of gastrointestinal problems. Aloe vera gel has gastroprotective properties. The purpose of this study was to evaluate the effect of aqueous extract of Aloe vera leaves on the gastric acid secretion and brain and intestinal water content following acetic acid gastric ulcer induction.Materials and Methods: Gastric ulcer was induced by injection of 20% acetic acid into the subserosal layer in male rats. Rats were randomly assigned into three groups: intact group, gastric ulcer group and Aloe vera group (treatment with Aloe vera following gastric ulcer induction). The acid levels and brain and intestinal water content of each sample were measured eight days after the gastric ulcer induction.Results: Gastric acid levels were significantly decreased in Aloe vera group when compared with gastric ulcer group (p<0.05). However, there were no differences in acid output between gastric ulcer and Aloe vera groups with intact group. After Aloe vera administration, the amount of brain water content had no difference with intact and gastric ulcer groups (p<0.05). The duodenal water content in Aloe vera group was significantly reduced compared with intact group (p<0.05) but gastric ulcer group had no significant difference with intact and Aloe vera group.Conclusions: The administration of Aloe vera has an inhibitory effect on the gastric acid output.  Gut–brain axis (GBA) is very important in creation and modulation of gastrointestinal problems. It was described that GBA is a bidirectional neurohumoral communication system that integrates brain and gastrointestinal (GI) functions. The brain is the most influential organ within the axis. Peptic ulcer disease as gastrointestinal problem is created by imbalance pepsin and gastric acid secretion , C, and E, which are antioxidants. It provides calcium, copper, magnesium, potassium, and zinc which are essential for the proper functioning of various enzyme systems in different metabolic pathways and a few of them are antioxidants  were purchased from Animal Center of Mashhad University of Medical Sciences, Iran. The rats were housed in temperature and humidity controlled animal quarters with a 12-hour light/dark cycle with free access to food and water. All procedures were approved by the Institutional Animal Care Committee of North Khorasan University of Medical Sciences and were in accordance with the guidelines of the National Institutes of Health on the care and use of animal’s surgery.Experimental groupsThe rats were randomly divided into three groups (n= 7 in each group):1- Intact group: animals that were not given any drugs (they received distilled water freely).2- Gastric ulcer group: animals without treatment.Aloe vera group: gastric ulcer animals that were treated with oral administration of Aloevera   separated from locally available plant, identified by botanist of Ferdowsi university of Mashhad, Iran. Whole leaves of Aloe vera were cut into thin pieces, put onto a glass plate and then ground into a fine powder for further use. The dried powder (120 g) was mixed with distilled water in a balloon and was shaken for two days. The preparation was then filtered off using a Gauze mesh and the solvent was dried by evaporation under reduced pressure at 40 °C. The final product yielded an 8% w/w dried extract. Whole fresh leaves of Induction of ulcerThe animals were fasted but allowed only water 12 hours before experiment. On the day of experiment, the animals were anesthetized with ether. The animals were located on the surgical table . The abdominal wall at midline epigastric line was incised and the stomach was extended and fixed. After exposing the stomach, 20% acetic acid (0.02 ml) was injected into the subserosal layer of the glandular portion of the anterior wall with a microsyringe (0.05 ml). At the time of injection, a thumb was placed tightly on the inserted needle to avoid solution leakage. Accuracy of the injection was confirmed by observation of wheal-like swelling at the injection site. After closing the abdomen, the animals were routinely maintained .Evaluation of gastric acid secretion+/15 min  had been stable for 30 min, it was considered as basal acid secretion. Throughout the experiment, the gastric secretions were collected in consecutive 15-min samples. The acid content of each gastric washout sample was measured with a manual titrator to an endpoint pH of 7 with 0.01 N NaOH and expressed as μmol HDetermination of brain water contentTo measure the brain edema, the brain water content was determined as the animal was anesthetized and the brain was extracted 8 days after ulcer induction. The weight of wet tissue was measured first and then incubated in 60 °C in an incubator  for 72 hours to evaporate the tissue water and become dry. The brain was weighed again after drying and water content was calculated by % using formula  were obtained, and their contents were removed. Wet weights of the intestinal walls were determined, then the samples were dried in a vacuum oven for 48 h at 60°C and weighted immediately to determine the dry weights. Water content of duodenum was calculated by % using formula  group (6.10±1.288 μmol/15 min) when compared with gastric ulcer group (9.66±0.411 μmol/15 min) (p<0.05). However, there were no differences in gastric acid levels between gastric ulcer and Aloe vera groups with intact group (8.25±0.869 μmol/15 min).Changes in gastric acid secretion levels (acid output) for different groups are shown in Effect on brain water contentAloe vera administration, the amount of brain water content was 77.06±0.582% but had no difference with intact and gastric ulcer groups (p<0.05).Effect on duodenal water contentAloe vera group (81.87±1.516%) was significantly reduced compared with intact group (78.50±0.643%) (p<0.05). However, gastric ulcer group (79.35±0.622%) had no significant difference with intact and Aloe vera groups.As shown in Aloe vera extract inhibits gastric acid secretion. This may suggest that, at this dose, the plant may possess gastroprotective activity against gastric acid as an aggressive factor . The mechanism of cytoprotection is presently unknown. Several hypotheses have been suggested, such as increased mucus synthesis , bicarbonate secretion , increased mucosal blood flow , and increased phospholipids mucosal coating  may be shared with other anti-ulcer drugs . It is possible that the antiulcer effect of Aloe vera should be mediated through the decrease in aggressive factors and increase in protective factors which provide the advantages for patient with gastric ulcer. In addition, the Aloe vera has been demonstrated to have wound healing properties  can enter the systemic circulation through the portal vein and the mesenteric lymph, and result in sepsis and multiple organ dysfunction syndrome (MODS). The intestinal tract, therefore, is regarded as “initiator” of MODS and its potential pathogenicity has been given more attention (Davidson et al, 2004Aloe vera has an inhibitory effect on the gastric acid output. The inhibitory effect of Aloe vera was mostly due to the component of the formula and the plant gastroprotective activity. In general, authors confirmed that administration of


Advanced oxidation protein products (AOPP) are newly identified efficient oxidative stress biomarkers. In a longitudinal birth cohort the effects were investigated of genetic polymorphisms in five oxidative pathway genes on AOPP levels.GSR), glutamylcysteine synthetase (GCLC), glutathione S-transferase (GST) P1, haem oxygenase 1 (HMOX1) and superoxide dismutase 2 (SOD2) in 298 children. There were 284 children assessed for anaemia and clinical malaria infection at the age of 24 months.This study is part of a three-arm randomized, double-blind, placebo-controlled trial. Three hundred and twelve children were included in the present study with AOPP levels measured at 2.5, 5.5, 10.5, 15 and 24 months of age. Twelve polymorphisms were genotyped in five oxidative stress pathway genes: glutathione reductase (GSR RS3594 polymorphism (p = 0.037). Mixed linear regression analyses found that the single nucleotide polymorphisms GCLC RS10948751 and HMOX1 RS17885925 were significantly associated with AOPP levels .Two principal components (PCA1 and PCA2) were derived from the AOPP levels measured at the five time points. PCA1 was significantly associated with anaemia (p = 0.0002), and PCA2 with clinical malaria infection (p = 0.047). In the K-Means Cluster Analysis based on levels of AOPP, children were clustered into two groups: Group A (lower AOPP levels) and Group B (higher AOPP levels). The cluster membership was significantly associated with anaemia (p =0.003) as well as with the GCLC, GSR and HMOX1 genes were associated with oxidative stress status of these children.Plasma AOPP levels were predictive for anaemia and oxidative stress markers for clinical malaria infection in two year old children. Several polymorphisms in  Plasmodium falciparum has developed an elaborate reduction-oxidation (redox) system to maintain adequate antioxidant defence throughout its complex life cycle [Oxidative stress is a common pathogenic mechanism underlying the development of many diseases and conditions including malaria infection, in which both the host and the parasite are under its effects. Adapting to the oxidative stress exerted by the host immune response against malaria infection, fe cycle . In humafe cycle ,3, contrfe cycle . AlthougGST) genes have been reported to be associated with susceptibility to many chronic inflammatory conditions and other diseases including malaria [GSR [GCLC [HMOX1 [SOD2 [Oxidative pathway genes have been extensively investigated for associations with different phenotypes for their anti-oxidative effects on a range of conditions and diseases ,6. Genet malaria ,8, anaem malaria , asthma  malaria , allergy malaria , chronic malaria , diabete malaria , cardiov malaria  and canc malaria . Glutathria [GSR , GCLC [1SR [GCLC ,15, HMOXC [HMOX1  and SOD2X1 [SOD2  with oxiX1 [SOD2 ,18,19.Evidence of oxidative stress, or an antioxidant status, is usually assessed using biomarkers that reflect the actual oxidative stress in a tissue or the whole body. Plasma proteins, predominantly albumin and fibrinogen, undergo molecular modifications by oxidation which can be measured as advanced oxidation protein products (AOPP), which were first identified by Witko-Sarsat et al. as efficient oxidative stress biomarkers in uraemic patients ,21. FurtGCLC and HMOX1 were associated with AOPP concentrations measured at five time points in the first two years of life. The AOPP biomarker was associated with anaemia and clinical malaria infection ascertained at two years of age. There is evidence that oxidative stress pathway genes contribute to the balance between a pro- and anti-oxidant status that is related to the development of anaemia and clinical malaria infection in young children.In the AgeMal  collaborative study , the genetic effects were investigated in young children of 12 functionally important polymorphisms in five oxidative pathway genes regarding their functional relevance for oxidative stress status measured as AOPP levels, as well as for the two phenotypes of anaemia and clinical malaria infection. Genetic variants in P. falciparum during the first year of life for the development of naturally acquired immunity [P. falciparum is perennial with marked seasonality and moderate intensity, and the entomological inoculation rate of 38 infective bites/person/year [P. falciparum infection was fulfilled with different protocols for administration of monthly chemoprophylaxis with sulphadoxine-pyrimethamine plus artesunate to the children during the first year of life in a three-arm intervention trial. A total of 312 infants were included in the study, with AOPP measured at 2.5, 5.5, 10.5, 15 and 24 months of age, and > 88% of children analysed at each of the five time points. There were 284 children who had their anaemia and clinical malaria infection status determined at the age of 24 months, and DNA samples were available from 298 children. The study was approved by the National Mozambican Ethics Committee, the Hospital Clínic of Barcelona Ethics Review Committee, the Bioethical Committee of the Torino University School of Medicine, and the Princess Margaret Hospital for Children Ethics Committee (1473/EP) in Perth.This study is part of the AgeMal project consisting of a three-arm randomized, double-blind, placebo-controlled trial aimed at determining the importance of time of exposure to immunity . The stuson/year . The stuson/year ,26,27. BGSR, GCLC, GST P1, HMOX1 and SOD2 based on their potential functional importance and estimated minor allele frequency (>10%) in Africans. Two single nucleotide polymorphisms (SNPs) were genotyped in GSR, 3 in GCLC, 3 in GST P1, 2 SNPs and 1 AG deletion in HMOX1, and 1 SNP in SOD2. The genomic locations of these polymorphisms in the five genes are shown in Additional file Genomic DNA was extracted from peripheral blood mononuclear cells by an automated DNA extraction instrument . Genetic polymorphisms were selected from the five oxidative stress pathway genes: Spectrophotometric determination of AOPP plasma levels was performed by modification of the Witko-Sarsat’s method in a micro-plate reader ,29. BrieP. falciparum asexual stage parasites of any density, determined by microscopy following standard quality control procedures [A clinical malaria episode was defined as axillary temperature ≥ 37.5°C, or history of fever within the prior 24 h, plus the presence in peripheral blood of For the 12 polymorphisms in the five oxidative stress pathway genes, Hardy-Weinberg equilibrium was examined using the online tool . The levt test, when appropriate. The associations of PCA scores of AOPP levels were also investigated with cluster membership, anaemia and clinical malaria infection at the age of 24 months using independent sample t tests. Chi-square tests were employed to investigate the associations of genotypes, anaemia, and malaria infection with cluster membership. A mixed linear regression model was further employed to investigate the associations of genotypes with levels of AOPP adjusting for gender and presence/absence of anaemia. Other potential confounding factors were investigated for their association with levels of AOPP. Analyses were conducted using SPSS (PASW Statistics 18). All p-values are two-sided and were considered significant when <0.05.The associations between genotypes and PCA scores of AOPP levels were investigated using analysis of variance and independent sample HMOX1 was not in Hardy-Weinberg Equilibrium so it was excluded from further analyses. There was also complete linkage disequilibrium for the RS10948751 and RS7742367 SNPs in the GCLC gene, therefore, only RS10948751 was included in further analyses.Eleven single nucleotide and 1 insertion/deletion polymorphisms were genotyped in five functionally important oxidative stress pathway genes in 298 young children. Table Plasma AOPP levels were measured in 302 children at 2.5 months, 300 at 5.5 months, 296 at 10.5 months, 283 at 15 months and 274 at 24 months of age, showing decreasing levels by age  significantly and positively contributed to the first component score (PCA1) that accounted for 30% of the variation of plasma AOPP levels at the five time points. The levels of AOPP at 24 months of age significantly and positively contributed to the second component score (PCA2) that accounted for 20% of the variation of the plasma AOPP levels at the five time points. In addition, the levels of AOPP at 2.5 and 5.5 months of age negatively contributed to the PCA2. Then, the PCA1 was related to plasma AOPP levels before 15 months of age, and the PCA2 was positively correlated with plasma AOPP levels at 24 months of age and negatively correlated with plasma AOPP levels in infants less than 6 months of age. Additional file Based on the levels of AOPP at the five time points, the children were clustered into two groups: Group A (lower AOPP levels) and Group B (higher AOPP levels). Group A included 161 children and Group B included 151 children. It was expected that the cluster membership would be associated with the levels of AOPP at the five time points as well as to the two PCA components, as the membership was defined from the levels of AOPP. Figure Among the 284 children who were followed up to the age of 24 months, 23 (8.1%) had anaemia and 32 (11.3%) had clinical malaria infection at this last time point. Generally, children with anaemia at 24 months had higher levels of plasma AOPP at all five time points, with significant differences at ages 2.5 months (p = 0.014) and 15 months (p = 0.001) . The odds ratio of B membership for anaemia was 4.23 . No association was found between cluster membership and clinical malaria infection at age 24 months.GCLC RS10948751 was significantly associated with PCA2 . Figure Using a mixed linear regression model, the associations of genotypes were investigated with the AOPP plasma levels measured at the five time points. The model showed that the variables age (the five time points) and anaemia (presence/absence) were significantly associated with the plasma AOPP levels, with the presence of anaemia being associated with higher AOPP levels and age associated with decreased AOPP. After adjusting for age and presence/absence of anaemia the SNPs GCLC, GSR and HMOX1 genes are associated with the host oxidative stress status, which was evidenced by their associations with AOPP levels. This study provides valuable data to explain the complex oxidative stress phenomena in children regarding anaemia and clinical malaria infection.This is the first study to investigate the complex relationships between oxidative stress pathway genes, the oxidative stress biomarker AOPP, anaemia and clinical malaria infection in a longitudinal cohort of young children. The findings in this study are: (1) Plasma AOPP is a valuable oxidative stress biomarker of anaemia and clinical malaria infection in children two years old living in a malaria endemic area; (2) Preceding levels of AOPP predict to some extent the development of anaemia in children two years old; and (3) Variants in AOPP are biomarkers for oxidative stress, and were first detected in the plasma of chronic uraemic patients . In addiGCLC SNP RS10948751 was associated with PCA2, and the further mixed linear regression model analysis confirmed the association of this SNP with plasma AOPP levels. There are no reports investigating associations of variations of the GCLC gene with susceptibility to malaria or with the biomarker AOPP. However, a few studies have reported that the SNP -129 C/T and the TNR (GAG trinucleotide repeat) polymorphism in the GCLC gene are associated with several clinical phenotypes such as type 1 diabetes [GSR gene (RS3594) was associated with cluster membership based on AOPP levels measured longitudinally during the first two years of life. Polymorphisms in the GSR gene have been associated with postmenopausal bone mineral density values [P-falciparum-parasitized RBCs with these GSR phenotypes were reported to show increased in-vitro phagocytosis rates due to membrane oxidations [in vivo and explain the association of the GSR polymorphism with anaemia. A few studies have investigated the associations of the HMOX1 gene polymorphisms with malaria susceptibility in humans [HMOX1 RS17885925 polymorphism was associated with AOPP levels. However, considering the linkage disequilibrium between polymorphisms in this gene, the effect of HMOX1 RS17885925 on the AOPP levels may be attributed to other intragenic variants [The main goal of this study was to investigate the associations of 12 functionally important polymorphisms in five oxidative stress pathway genes with plasma AOPP levels. One polymorphism which violated the Hardy-Weinberg equilibrium and one redundant polymorphism were excluded, thus only 10 were included in the analyses. To circumvent multiple tests, the relationships were examined of these 10 polymorphisms with the two PCA scores derived from the AOPP levels at the five time points. The diabetes , chronicdiabetes , depressdiabetes  and lungdiabetes . This stdiabetes  the polydiabetes , enhancediabetes  and defey values  and COPDy values . This stidations . Similarn humans ,19,51. An humans ,19. The variants ,19.The main limitation of this study was that the longitudinal cohort had a small sample size (less than 300 children at age two years). As the subjects were recruited from a general African community, the prevalence of anaemia and clinical malaria infection was less than 12%, with only 23 anaemic children and 32 children with clinical malaria infection at age two years. This study, therefore, did not have sufficient statistical power to investigate the associations of the different genotypes with the anaemia and clinical malaria infection phenotypes. However, AOPP plasma levels were longitudinally measured, and this has provided a comprehensive assessment of the children’s oxidative status and allowed a systematic examination of the complex interrelationships between AOPP plasma levels, polymorphisms in several oxidative stress pathway genes and anaemia and clinical malaria infection in the longitudinal cohort.GCLC, GSR and HMOX1 genes that are associated with oxidative stress status in the blood plasma of two-year old children from a malaria endemic region. In addition, plasma AOPP is an oxidative stress marker for clinical malaria in these children and that AOPP levels are predictive for anaemia.In conclusion, the present study has identified several polymorphisms in The authors do not have any commercial or other association that might pose a conflict of interest. The funding sources did not have any involvement in study design, collection, analysis and interpretation of data, writing of the report, or in the decision to submit the paper for publication. The researchers are independent from the funders.GZ, ES, PNL, CD, PLA, JG contributed to conception and design of the study; TM, RA, OAS, ES, KSK, SW, CMH, AN, MNM, AB, CG contributed to acquisition of data; SW, KSK, CMH contributed to selection of genes and polymorphisms; GZ, MSM, LQ analyzed the data; all authors contributed to interpretation of data; GZ drafted the article; all authors revised the article critically for important intellectual content; all authors read and gave final approval of the version to be published.Table S2. Component matrix of levels of AOPP at five times with two principal component scores.Polymorphisms in the five oxidative pathway genes. Click here for file


Phelsuma guimbeaui (lowland forest day gecko), is now restricted to 30 small isolated subpopulations following severe forest fragmentation and isolation due to human colonisation. We used 20 microsatellites in ten subpopulations and two mitochondrial DNA (mtDNA) markers in 13 subpopulations to: (i) assess genetic diversity, population structure and genetic differentiation of subpopulations; (ii) estimate effective population sizes and migration rates of subpopulations; and (iii) examine the phylogenetic relationships of haplotypes found in different subpopulations. Microsatellite data revealed significant population structure with high levels of genetic diversity and isolation by distance, substantial genetic differentiation and no migration between most subpopulations. MtDNA, however, showed no evidence of population structure, indicating that there was once a genetically panmictic population. Effective population sizes of ten subpopulations, based on microsatellite markers, were small, ranging from 44 to 167. Simulations suggested that the chance of survival and allelic diversity of some subpopulations will decrease dramatically over the next 50 years if no migration occurs. Our DNA-based evidence reveals an urgent need for a management plan for the conservation of P. guimbeaui. We identified 18 threatened and 12 viable subpopulations and discuss a range of management options that include translocation of threatened subpopulations to retain maximum allelic diversity, and habitat restoration and assisted migration to decrease genetic erosion and inbreeding for the viable subpopulations.Genetic structure can be a consequence of recent population fragmentation and isolation, or a remnant of historical localised adaptation. This poses a challenge for conservationists since misinterpreting patterns of genetic structure may lead to inappropriate management. Of 17 species of reptile originally found in Mauritius, only five survive on the main island. One of these,  It is important to determine whether the observed genetic structure is a consequence of recent population fragmentation and isolation, or a remnant of historical localised adaptation. Misdiagnosing the former when the latter is true risks disrupting patterns of local adaptation and outbreeding depression if incompatible populations are mixed. Conversely, interpreting structured patterns to be signatures of local adaptation when they are a consequence of isolation and drift risks inappropriate management to maintain existing genetic patterns, when maximising gene flow between populations might reduce genetic loss and the risk of extinction. Identifying the origins of genetic structure is particularly important when deciding whether to use translocation to reinforce existing populations and/or establish new populations, and to determine how many founding individuals are required to retain the existing genetic diversity. It is also important to interpret genetic patterns alongside ecological factors such as habitat loss when deciding on the most appropriate management option.Phelsuma), and these support a wide range of ecological functions such as pollination, predator-prey dynamics and seed dispersal Phelsuma guimbeaui (lowland forest day gecko) is the species most vulnerable to extinction. It persists in only 30 small and isolated subpopulations, many of which may be lost within the next decade due to increasing urbanisation, habitat loss, and the impact of invasive species such as Phelsuma grandis (giant Madagascar day gecko), introduced to Mauritius in the 1990s .In Mauritius, successive occupations by the Dutch (1638–1710), French (1721–1810) and British (1810–1968) have destroyed a large part of the ecosystem through habitat destruction and the introduction of invasive alien species P. guimbeaui and consider the most appropriate strategy for its long-term conservation. We used a suite of microsatellite markers for P. guimbeauiP. guimbeaui.Here, we characterise genetic diversity and structure for The capture and tissue sampling were approved by the University of Bristol's Ethical Review Committee (University Investigation Number UB/11/031) and the National Parks and Conservation Service, Ministry of the Agro-Industry, Mauritius.Pinus taeda (loblolly pine) and Psidium cattleianum (strawberry guava), or human habitation. Between 2007 and 2009, we searched the western part of Mauritius and found a total of 30 subpopulations .For the purpose of this study, we defined a subpopulation as inhabiting a piece of wooded habitat separated by barriers such as a major road, large area of agriculture, non-habitable planted/non-planted exotic trees such as ulations  covering2  was removed with a sterile scalpel blade, placed in a labelled 1.5 ml microfuge tube containing 94% alcohol and stored at −20°C. We only caught one gecko in subpopulation L11 and three each in L12 and L13 ; these w2 .DNA was extracted using an ammonium acetate salt precipitation protocol P. guimbeauica 10 ng DNA, 1 µl Qiagen Master Mix  We selected 41 polymorphic microsatellite loci with high amplification success, developed specifically for Forty samples were randomly selected, re-PCRed and genotyping error rates per allele A), allelic richness (AR) and private allelic richness (PA) were calculated using a rarefaction approach in the software HP-RARE O) and expected heterozygosities (HE) for each subpopulation were estimated in CERVUS v3.0 and the mean inbreeding coefficient (FIS) was calculated using GENETIX v4.05 (http://kimura.univ-montp2.fr/genetix/), based on 10,000 permutations. Differences in AR and HE were examined using a parametric ANOVA conducted in R 2.15.3 (R Development Core Team 2013).To detect genetic diversity within each subpopulation, the number of different alleles, mean number of different alleles  against the log-transformed geographical distances among the ten subpopulations.Weir and Cockerham's FR and HE) were related to patterns of habitat loss and isolation, we compiled data on forest cover between 1773 and 1935 from P. guimbeaui is a habitat specialist and is mostly reliant on native habitats that have high tree diversity and tall, large trees with cavities . The maximum dispersal distance of 28 adult P. guimbeaui monitored for a year was <15 m . There are no data on dispersal in sub-adult and juvenile P. guimbeaui, but the maximum recorded dispersal distance for P. ornata (ornate day gecko), a similar-sized Mauritian species, was 87 m P. guimbeaui also persists in the presence of four exotic tree species, i.e. Eucalyptus tereticornis , Haematoxylum campechianum (campeche), Mangifera indica (mango) and Terminalia arjuna , these trees are usually scattered and probably act as ecological traps; there are no data on the density of P. guimbeaui in these exotic plantations, and it has never been recorded on buildings or in agricultural land.To determine whether levels of genetic diversity  g = 0.46) NThree different techniques were used to estimate contemporary rates of migration between the subpopulations. First, we used a Bayesian approach implemented in BAYESASS v1.3 e by ten We first used simulations in the R package AlleleRetain cyt b and 16S rRNA, two mitochondrial genes frequently used in phylogenetic research Partial regions of st were calculated in GenAlEx 6.5; analyses were performed separately per subpopulation  and for the entire population.The start and end of sequences were trimmed. A consensus sequence was obtained by aligning the forward and reverse sequence for each individual in CodonCode Aligner  and complementary alignment was conducted in MEGA v5.05 cyt b and 16S rRNA, respectively. For maximum likelihood, a phylogenetic tree with 1,000 bootstraps was used to explore the robustness of tree topology. Bayesian analysis was conducted with four chains of 10,000,000 replications, with sample frequency of 2,000 and discarding the first 25% of replicates as burn-in. Two independent runs were conducted to produce a consensus tree that we used to explore relationships between haplotypes. We considered the runs as having converged when their split frequency was <0.01 and the potential scale reduction factor was close to 1 P. ornata (Genbank AY221451.1). We also built a statistical parsimony network using TCS v1.7 Phylogenetic trees of different haplotypes were inferred by applying the maximum-likelihood method in MEGA 5.05 S) Neutrality tests  and three subspecies of Phelsuma sundbergi (Seychelles giant day gecko), all of which are common in the Seychelles To compare the genetic diversity of Pgu 043, which amplified only 57.6% of the time and so was excluded from subsequent analyses , except for analyses . Amplifir allele ; these lSignificant LD was observed in three pairs of loci. However, we retained these loci because the LD was not consistently high across all subpopulations. The number of loci in HW disequilibrium  and signE varied from 0.844 to 0.891, HO from 0.792 to 0.876, NA from 10.6 to 15.5 and AR from 8.4 to 10.7. PA was observed in all the subpopulations and mean PA varied from 0.400 to 0.960  and AR  between the following subpopulations: (i) subpopulation L10 had significantly lower HE and AR compared to all other subpopulations except L1 and L7; and (ii) L1 and L7 had significantly lower HE and AR compared to subpopulations L2, L3, L4, L5 and L6. There was also a significant positive FIS varying between 0.006 and 0.108 in all subpopulations .Based on the maps of habitat loss , the dif2 = 0.182, P = 0.010) , with Fst estimates between 0.016 and 0.072 9 = 0.042, P<0.001) of variation occurring between subpopulations, 10% among individuals  and 86%  within individuals.The high membership coefficients to genetic clusters (0.705 to 0.899) found using STRUCTURE supported strong genetic differentiation of subpopulations. The highest delta K and log probability values were observed at K = 9, suggesting that all subpopulations except L8 and L9 were genetically distinct . On aver= 0.010) . Pairwise for the different subpopulations were small, ranging from 44 to 167 in Ne ESTIMATOR and 19 to 96 in MIGRATE (Using the mutation rate estimate for reptilian microsatellite loci  MIGRATE .cyt b with 464 base pairs (accession numbers HG779461-HG779540) and 16S rRNA with 313 base pairs (accession numbers HG518676-HG518755). We analysed concatenated partial cyt b and 16S rRNA sequences (777 base pairs) of 80 individuals from 13 subpopulations  has had an impact on genetic differentiation in many subpopulations by limiting migration. There are two plausible non-mutually exclusive explanations that may account for the level of genetic differentiation observed among the subpopulations. First, restricted dispersal can produce a pattern of IBD P. guimbeaui. The initial founder effect of these colonisations, together with the cumulative effects of genetic drift and low effective population size, may have promoted genetic differentiation.Our analyses suggest that either nine (STRUCTURE) or all ten (TESS and GENECLASS2) of the sampled subpopulations were genetically distinct. There was a relatively moderate or high level of admixture between subpopulations L2 and L6, and between L8 and L9, which may be due to the relative proximity of these paired sites or connectivity. While most sites were separated by large expanses of urbanisation or agriculture, subpopulations L2 and L6 were in a continuous forest within the Black River mountains but isolated by forest dominated by invasive plants, predominantly e ESTIMATOR were consistently higher than those obtained with MIGRATE, and the former LD-based method may provide a better estimate of Ne because it records recent Ne (up to two generations) and so is less likely to be affected by historical events e estimates to be small and so these subpopulations are at high risk of extinction P. guimbeaui should be interpreted with caution.Values for NP. guimbeaui prior to habitat loss. Shared haplotypes among the subpopulations suggest that they originally formed part of a panmictic population. For example, Hap_3 was shared by five subpopulations, among which L3 and L11 were more than 20 km apart. The absence of genetic structure within the mtDNA data is also supported by historical vegetation maps, which show that most of the western part of Mauritius had continuous forest cover, and P. guimbeaui may have occurred throughout these lowland dry forests prior to human colonisation. This also suggests that P. guimbeaui was widely dispersed when the forest was not fragmented.Phylogenetic analyses provided strong evidence that the spatial genetic structure and differentiation were absent in P. guimbeaui  and the observed strong differentiation of nuclear markers.Contrasting signals of genetic structure between nuclear and mtDNA data have been observed in other species P. guimbeaui was a panmictic population until relatively recently, which has become differentiated through anthropogenic habitat loss and isolation. There is, therefore, no reason to maintain the genetic identity of individual subpopulations and little risk of outbreeding depression should subpopulations be mixed. Conservation management is a priority, since there is a high risk of genetic erosion and extinctions in the next 50 years, even without further habitat loss and other anthropogenic changes. Possible interventions based on our genetic data are: (i) the construction of habitat corridors linking closely-neighbouring subpopulations; (ii) the restoration and expansion of the native habitats in and around existing subpopulations; and (iii) the translocation of individuals to enhance the genetic diversity of viable subpopulations and/or establish one or more new populations.Our genetic data suggest that Continuing habitat loss in Mauritius suggests that some subpopulations are likely to be lost in the next decade: we sampled nine such subpopulations. Given the immediacy of the threat, and that the construction of habitat corridors linking some of them and/or habitat restoration are not realistic options, the only practical solution is to translocate individuals from these nine subpopulations to reinforce viable subpopulations, if there is habitat to support additional geckos, or move them to suitable habitat patches to establish new population(s) However, translocation of a selected number of animals is only practical if the host subpopulation is viable in the medium to longer term. Since these nine subpopulations  are highly threatened by further habitat loss, it is unlikely that any will persist for more than a decade. So it may be more logical to translocate as many geckos as possible from each subpopulation in one operation, even if it hastens the ultimate loss of that subpopulation. Nine other subpopulations not sampled during this study  are equaP. guimbeaui to expand its range in the Black River mountains. The most practical solution would be to create habitat corridors linking those areas of native forest that still harbour relatively large subpopulations of P. guimbeaui. While this will increase migration between the 12 viable subpopulations and decrease intra-subpopulation inbreeding, this is a long-term strategy and so assisted migration within the Black River mountains should be considered as an interim option. However, prior to any such intervention, data are needed to confirm the viability and breeding success of translocated geckos following release into a new population. Post-release data are also needed to assess whether greater genetic diversity is retained by releasing geckos “rescued” from non-viable subpopulations into a viable subpopulation or using them to establish a new population.Only four of the subpopulations we examined  were still viable, and they were all within the Black River mountains, the largest area of remnant native forest in Mauritius. There were eight other subpopulations within the Black River mountains, which we did not sample but were probably also viable. Habitat restoration and expansion within this mountain range would support the long-term management of the species and enable P. guimbeaui is habitat destruction, our data highlight the importance of genetic studies in guiding conservation management. Maximising the retention of genetic diversity is important and we identified two conservation measures to maximise the retention of genetic diversity in P. guimbeaui. We recommend: (i) a short-term rescue action by translocating as many geckos as possible from the 18 subpopulations threatened by imminent habitat loss; and (ii) a long-term action to restore habitats in the Black River mountains to link the 12 potentially viable subpopulations by habitat corridors. Since habitat management is a long-term strategy, especially since P. guimbeaui is a habitat specialist relying on high native tree diversity and large tall trees with numerous cavities, assisted migration among the 12 viable subpopulations should reduce the risks of genetic degradation in the short term. However, data are needed on the survival and breeding success of translocated geckos used to reinforce viable subpopulations or to establish new populations prior to the implementing any program of assisted migration.Although the immediate threat facing Table S1Phelsuma guimbeaui: (i) initial number of translocated individuals (StartN) varied from 10–40; (ii) number of assisted migrants after translocation (MigrN) ranged from 0–30; and (iii) frequency at which assisted migrants were translocated (Frequency) confined from one to five years. R and simulations codes are given below the table.Simulation results from R package AlleleRetain. The table shows the probability of survival and genetic diversity after 50 years (1000 replications) in different scenarios in each subpopulation (Subpop) of (DOC)Click here for additional data file.Table S2Phelsuma guimbeaui.Mitochondrial DNA primers in (DOC)Click here for additional data file.Table S3Phelsuma guimbeaui.Screening for data quality to select the best loci for analyses of molecular variation in (DOC)Click here for additional data file.Table S4Phelsuma guimbeaui not in Hardy-Weinberg equilibrium (P<0.05) at the different loci.The number of subpopulations of (DOC)Click here for additional data file.Table S5Phelsuma guimbeaui.Null allele frequencies in ten subpopulations of (DOC)Click here for additional data file.Table S6Phelsuma guimbeaui.Number of alleles observed for the 20 loci used in the microsatellite analyses of (DOC)Click here for additional data file.Table S7Phelsuma guimbeaui obtained in GENECLASS2.The mean probability of no migration and migration among ten subpopulations of (DOC)Click here for additional data file.Table S8Phelsuma guimbeaui.Haplotype distribution and frequency in 80 individuals from 13 subpopulations of (DOC)Click here for additional data file.


Hanseniaspora vineae T02/19AF, which has been shown to positively contribute to aroma and wine quality.The use of novel yeast strains for winemaking improves quality and provides variety including subtle characteristic differences in fine wines. Here we report the first genome of a yeast strain native to Uruguay,  Saccharomyces cerevisiae produces most of the ethanol in wine, apiculate yeasts of the genus Hanseniaspora are the main species present on grapes and they play a significant role at the beginning of fermentation  >100 nucleotides were predicted using Augustus  trained H. vineae and S. cerevisiae, 87 of those genes (68%) were found through BLASTp in the genome of H. vineae T02/19AF. The accurate analysis of these genes will help further the understanding of the “flavor phenotype” of this and other yeast species. To the best of our knowledge, this is the first report of a Saccharomycodaceae yeast family genome.One hundred twenty eight genes associated with fermentation, such as those participating in glycolysis/gluconeogenesis, citrate cycle, pentose pathway, steroid biosynthesis, fatty acid degradation, and fatty acid biosynthesis pathways , from KEJFAV00000000. The version described in this paper is version JFAV02000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no.


Lolium arundinaceum), and to determine the physiological mechanisms of citric acid effects on heat stress tolerance. The grasses were subjected to four citric acid levels  and two temperature levels  treatments in growth chambers. Heat stress increased an electrolyte leakage (EL) and malonaldehyde (MDA) content, while reduced plant growth, chlorophyll (Chl) content, photochemical efficiency (Fv/Fm), root activity and antioxidant enzyme activities . External citric acid alleviated the detrimental effects of heat stress on tall fescue, which was evidenced by decreased EL and MDA content, and improved plant growth under stress conditions. Additionally, the reduction in Chl content, Fv/Fm, SOD, POD, CAT and root activity were ameliorated in citric acid treated plants under heat stressed conditions. High temperature induced the expression of heat shock protein (HSP) genes, which exhibited greater expression levels after citric acid treatment under heat stress. These results suggest that exogenous citric acid application may alleviate growth and physiological damage caused by high temperature. In addition, the exogenously applied citric acid might be responsible for maintaining membrane stability, root activity, and activation of antioxidant response and HSP genes which could contribute to the protective roles of citric acid in tall fescue responses to heat stress.Citric acid may be involved in plant response to high temperature. The objective of this study was to investigate whether exogenous citric acid could improve heat tolerance in a cool-season turfgrass species, tall fescue ( Under h 114 kDa . There i 114 kDa . LMW-HSPentgrass . In addiin wheat  and withbidopsis .Lilium (Festuca arundinacea), and demonstrated that the citric acid served majorly as an antioxidant respiratory metabolism intermediate involving the defense pathways in response to high temperature  is one of the widely used cool season forage and turfgrass species because of its adaptability, yield and persistence. Natural populations are scattered from Northern Europe including the Mediterranean region to North Africa, the Middle East, central Asia and Siberia. The optimum growth temperature of tall fescue ranges from 15 to 25°C. High temperatures of over 35°C in warm climate regions during summer are usually detrimental to turf quality and growth. Many approaches have been applied to improve whole-plant stress tolerance in cool-season turfgrass species, including exogenous application of cytokinins and ascorbic acid  filled with peat soil and sand with the ratio of 1:1 (v/v). After germination, 15 uniform seedlings were allowed to grow and watered every other day with tap water. All plants were placed in a controlled walk-in growth room for 50 days with the temperature of 25/20 ± 0.5°C (day/night), a 12-h photoperiod, and a PAR of 240 μmol m per pot . After 5-2 s-1, and 70 to 80% relative humidity.Plants were subjected to the following two temperature treatments for 15 days: control: plants were maintained at the optimal temperature ; heat stress: plants were exposed to supraoptimal temperatures of 10°C above the optimum range . All plants were watered daily to maintain the soil water content at field capacity. The two temperature treatments were repeated three times on three different sets of plants. All growth chambers had a 12-h photoperiod, PAR of 400 μmol mCitric acid treatments were applied to the plants 3 days before the imposition of high temperature treatment, and then applied weekly during the period of temperature treatments. For citric acid treatment, plants in each plastic pot were foliar sprayed with 20 ml of 0, 0.2, 2, and 20 mM sodium citrate solution  until the turf grass canopy was saturated and some dripping occurred. Treatments were arranged as a split-plot design with temperature treatments as main plots and citric acid treatments as subplots. Each temperature and citric acid treatment had four replicates. All pots were randomized within the growth chamber.After 15 days of heat stress, the shoots and roots were harvested for physiological analysis.Vertical shoot growth rate was estimated by measuring the difference in average turf canopy height before and after treatment using a ruler according to the method described by Cmax) was determined after the solution cooled to room temperature. Relative EL was calculated using the formula: EL (%) = (Ci/Cmax) × 100.To determine EL, 0.1 g fully expanded leaves were washed three times with deionized water and placed in tubes filled with 15 ml deionized water after cut to 0.5 cm long segments, and then shaken for 24 h. The initial conductivity (Ci) was determined using a conductivity meter . The tubes were autoclaved at 121°C for 20 min, and the conductivity of the incubation solution with killed tissues . The measurement points were randomly selected on the third fully developed leaves from top, and four points were selected on each leaf for all leaves in one pot .Fv/Fm (maximum quantum efficiency of PSII) was estimated by measuring the ratio of variable to maximum fluorescence of Chl (Fv/Fm) with a Chl fluorometer . Measurements were made on intact leaves with the fluorometer after plants were adapted in darkness for 30 min.Leaf 2SO4 was added to stop the reaction with the root. The root was dried with filter paper and then extracted with ethyl acetate. The red extract was transferred into the volumetric flask to reach 10 ml by adding ethyl acetate. The absorbance of the extract at 485 nm was recorded. Root activity was expressed as TTC reduction intensity. Root activity = amount of TTC reduction (μg)/fresh root weight (g) × time (h).Root activity was determined by the TTC method . Brieflyg for 15 min at 4°C. The supernatants were collected to determine soluble protein, enzyme activities, and MDA content. Protein content was quantified using the method described by For the determination of SOD, POD, CAT activity and MDA content, about 0.3 g of fully developed leaves was sampled at 15 days of treatment, frozen immediately with liquid nitrogen, and then stored at -80°C for subsequent analysis. Frozen leaves were homogenized in 50 mM ice-cold phosphate buffer (pH 7.0) with a pre-chilled mortar and pestle. The homogenate was centrifuged at 12,000 × The SOD activity was determined using the method described by 2O2 was measured by the decline in absorbance at 240 nm for 1 min. The 3-ml reaction mixture contained 50 mM phosphate buffer (pH 7.0), 15 mM H2O2, and 0.1 ml enzyme extract, which initiated the reaction. For POD, the oxidation of guaiacol was measured by the increase in absorbance at 470 nm for 1 min. The reaction mixture contained 50 μl of 20 mM guaiacol, 2.8 ml of 10 mM phosphate buffer (pH 7.0), and 0.1 ml enzyme extract. The reaction was started with 20 μl of 40 mM H2O2.The activities of CAT and POD were measured using the method of g for 10 min. The supernatant was read for absorbance at 532 and 600 nm. The absorbance for non-specific absorption at 600 nm was subtracted from the value at 532 nm. The content of MDA was calculated using the adjusted absorbance and the extinction coefficient of 155 mM-1cm-1 (The MDA content was determined by the TBA reaction using the method described by mM-1cm-1 .Leaf citric acid content was determined according to the high-performance liquid chromatography (HPLC) method described by Table 1) were designed based on the target gene sequences using Primer 5 software  and 2 μM of the forward and reverse primers. Three independent biological replicates of each sample and two technical replicates of each biological replicate were used for real-time PCR analysis. The thermal cycling conditions were as follows: 40 cycles of 95°C denaturation for 5 s, and 52–55°C annealing and extension for 20 s. After the PCR, a melting curve was generated by gradually increasing the temperature to 95°C to test the amplicon specificity. To determine relative fold differences for each sample, the CT value for each gene was normalized to the CT value for the reference gene and was calculated relative to a calibrator using the DDCT method as described by Total RNA was extracted from fresh tissues by using Trizol reagent  according to the user manuals. After extraction, the RNA pellet was dissolved in 100 μl of RNase-free water. RNase-free DNase I was added to the total RNA to remove DNA contamination. The total RNA concentration was then determined by absorbance at 260 nm and RNA quality was evaluated on a 0.8% agarose gel. The first strand cDNA fragments were synthesized from 2 μg of total RNA using oligo(dT)12-18 primer using cDNA synthesis kit  according to the user manual. Gene-specific primers  according to the general linear model procedure of SAS  to determine the effects of temperature, citric acid and their interactions. Treatment means were separated using the Duncan’s multiple range test at the Figure 1). This result indicated that citric acid might be involved in heat stress response. Additionally, treatments with higher concentrations of citric acid resulted in higher accumulation of endogenous citric acid in both control and heat stressed plants, and the accumulation of citric acid content by exogenous application of citric acid was in a concentration-dependent manner .In this study, to investigate the relationship between heat stress and citric acid content, the endogenous citric acid levels in leaves of tall fescue were quantified after treatments with high temperature  for 15 days. In tall fescue leaves, heat stress significantly increased endogenous citric acid content when compared to the control plants . High temperature remarkably reduced plant growth in tall fescue as indicated by the lower canopy height and plant dry weight . However, the citric acid treatment alleviated the decline in canopy height and plant dry weight under heat stress as compared to the non-treated plants, particularly at 2 mM concentration.Under optimum growth conditions, tall fescue growth was generally equivalent to the non-citric acid treated plants irrespective of foliar concentration of citric acids, as showed by the canopy height and plant dry weight . Heat stress treatment significantly increased EL and induced the accumulation of MDA in leaves of tall fescue , while citric acid treated plants displayed significantly lower EL and MDA content in comparison with non-treated plants .Leaf EL and MDA levels were generally employed to assess the extent of membrane damage caused by environmental stress. Treatment with citric acid alone had no effect on the leaf EL and MDA content under control growth conditions  was measured by analysis of Chl fluorescence. A significant decline in Chl content and Fv/Fm in leaves of tall fescue was observed under heat stress, while treatment with different concentration of citric acid alone had no effect under control condition . Under stress condition, however, citric acid treated plants displayed an increased Chl content and Fv/Fm, particularly for the concentration of 2 and 20 mM, when compared to the 0 mM citric acid in tall fescue .Leaf senescence was assessed from a decline in the Chl content based on a SPAD values. The Figure 5). Heat treatment plus citric acid at 2 and 20 mM increased root activity by 32 and 25%, respectively, when compared with heat treat treatment alone. No significant difference among the four citric acid application levels was observed under control conditions .Reduction of TTC has been used frequently employed to assess the root metabolic activities in under various growth conditions. Heat treatment with or without citric acid addition resulted in a decreased root activity when compared to the control plants, irrespective of citric acid concentrations . Heat treatment led to the deactivation of these antioxidant enzymes irrespective of citric acid concentration, when compared to the control plants . On the other hand, foliar application of exogenous citric acid improved the activities of SOD, POD, and CAT under heat stress, when compared to the non-citric acid treated plants, particularly with the concentration of 2 and 20 mM .On one hand, foliar application of exogenous citric acid alone had no effect on SOD, POD, and CAT activity under control condition . Heat stress induced higher expression levels of HSP HMW-HSP, LMW-HSP, HSP74.8, and HSC70 genes when compared to the control plants. Evidently, foliar application of 2 and 20 mM concentration of exogenous citric acid significantly induced HSP gene expressions, when compared to the 0 and 0.2 mM concentration of citric acid under heat stress condition .Exogenously applied citric acid alone had no significant effect on the expression levels of HSP gene families, including HMW-HSP, LMW-HSP, HSP74.8, and HSC70 under control condition . However, leaf EL and MDA in tall fescue increased under heat stress, but exogenous application of citric acid reduced lipid peroxidation, as indicated by the less accumulation of MDA and lower EL when compared to the untreated plants .These results suggested that application of citric acid would reduce loss of grasses under high temperature environments by mitigating oxidative stress.Heat stress often triggers drastic changes in the cell membrane stability, and ultimately influences the sensors present in the membrane . It is g2- into H2O2 in plants . These results suggested that the exogenous citric acid ameliorated the heat-induced injury to plants. This could be possibly attributed to the improvement of root viability and functions, which were associated with water and nutrient uptake and transport to shoots . However, exogenous application of citric acid resulted in the alleviation of Chl and Fv/Fm reduction in leaves of tall fescue under heat stress. These results suggested that external citric acid treatment might have been related to the inhibition and loss of Chl under heat stress, which damaged the reaction centers , wheat (Triticum sp.), barley (Hordeum sp.) and oat (Avena sp.) (Agrostis stolonifera) and fescues (Festuca sp.) (The HSPs are molecular chaperones which played an important role in the heat shock tolerance under high temperatures conditions . The preena sp.) . Some stuca sp.) . In thisuca sp.) .Figure 8). On one hand, heat stress had detrimental effects on the cell membrane, root activity, induced leaf senescence and ROS production. On the other hand, exogenous application of citric acid could promote antioxidant enzyme activities  and HSP family gene expressions, improve Chl biosynthesis and photosynthetic capacity. All these changes in turn alleviated cell membrane damages, ROS accumulation, leaf senescence and increased root activity all induced by high temperature.Based on the above observations, a model for citric acid-mediated heat stress response in tall fescue was proposed in this study (Heat stress and external application of citric acid induced the accumulation of endogenous citric acid levels in tall fescue leaf tissues. Heat stress was detrimental to plant growth, cell membranes, root activity, and antioxidant enzyme activities in tall fescue. However, external application of citric acid alleviated the detrimental effects of heat stress on tall fescue. The beneficial effect of external application of citric acid in tall fescue in response to heat stress may attribute to its protective effects on peroxidation-linked membrane deterioration, free radicals scavenging, maintenance of membrane stability, increase in root activity and activation of antioxidant response and HSP genes.LH and ZY conceived and designed the experiment. LH and ZZ conducted the experiment. ZX and LH analyzed data, LH and ZY wrote the manuscript. The authors read and approved the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.