The purpose of this study was to compare the effects of supplementation with Gaspari Nutrition's SOmaxP Maximum Performance™ (SOmaxP) versus a comparator product (CP) containing an equal amount of creatine (4 g), carbohydrate , and protein (7 g whey protein hydrolysate) on muscular strength, muscular endurance, and body composition during nine weeks of intense resistance training.a priori at p ≤ 0.05.Using a prospective, randomized, double-blind design, 20 healthy men  were matched for age, body weight, resistance training history, bench press strength, bench press endurance, and percent body fat and then randomly assigned via the ABBA procedure to ingest 1/2 scoop (dissolved in 15 oz water) of SOmaxP or CP prior to, and another 1/2 scoop (dissolved in 15 oz water) during resistance exercise. Body composition (DEXA), muscular performance , and clinical blood chemistries were measured at baseline and after nine weeks of supplementation and training. Subjects were required to maintain their normal dietary habits and follow a specific, progressive overload resistance training program  during the study. An intent-to-treat approach was used and data were analyzed via ANCOVA using baseline values as the covariate. Statistical significance was set 1-RM bench press ; lean mass ; RTF ; and fat mass . No statistically significant differences in vital signs  or clinical blood chemistries were noted.When adjusted for initial differences, significant between group post-test means were noted in: These data indicate that compared to CP, SOmaxP administration augments and increases gains in lean mass, bench press strength, and muscular performance during nine weeks of intense resistance training. Studies designed to confirm these results and clarify the molecular mechanisms by which SOmaxP exerts the observed salutary effects have begun. Both SOmaxP and the CP were well-tolerated, and no supplement safety issues were identified. The use of pre- or peri-workout supplements among recreational and elite athletes have become increasingly popular due to studies suggesting improvements in aerobic and anaerobic performance and recommendations from expert panels in sports nutrition . Among tBased on the findings of such research and expert recommendations, supplement manufacturers have developed sports drinks combining the same three core ingredients and have added proprietary ingredients to be used in the peri-workout time period to increase muscle strength, lean mass, and/or endurance. Aside from the convenience of having multiple ingredients in one product, there is potential for the components to exert additive or synergistic effects. Because different dietary supplement products contain differing quantities of the core and proprietary components, it is often difficult to perform valid head-to-head studies. However, because most products purporting to build strength and/or endurance contain the same three core ingredients, and the preponderance of evidence suggests that these three ingredients are the most important contributors to observed ergogenic gains, then it is reasonable to assume that if similar quantities of the core ingredients were compared, a valid comparison could be made. If differences were found between two products, then a likely explanation for the difference would be some effect of the proprietary ingredients, since the core ingredients are matched by dose. Proprietary ingredients could contribute to a difference either by exerting independent effects or by enhancing the effects of the core ingredients in a differential way or both.Size On Maximum Performance™ (SOmaxP) is a product manufactured by Gaspari Nutrition containing creatine, carbohydrate, whey protein and other proprietary ingredients, and was used during the peri-workout period only on the days when resistance training occurs. The comparator product was standardized to contain similar amounts of creatine, carbohydrate and whey protein. The study compared the effects of SOmaxP to a comparator product (CP), which was standardized to contain equal amounts of creatine (4 g creatine monohydrate), carbohydrate  and protein (7 g whey protein hydrolysate), and given with identical timing. We hypothesized that subjects in the SOmaxP groups would outperform the subjects in the CP during post-testing after adjusting for baseline differences.Twenty subjects, ten in each group, were randomized to receive either SOmaxP or CP during this 9-week study. Key elements of the inclusion criteria included: male or female subject in good health; aged between 18-45; a body fat of 10%-25% inclusive; who had undergone regular resistance training for at least two years; who had signed an informed consent; who were willing and able to comply with the training and supplement protocol; possessed normal vital signs; and had a fluent understanding of English. Physical activity levels and health history were determined using standardized questionnaires adapted from Kent State University, Purdue University, and Eastern Michigan University at baseline and weeks 3, 6 and 9. The protocol was in compliance with the Helsinki Declaration, and was approved by the IntegReview Ethical Review Board . Although the inclusion criteria allowed for female subjects, no females enrolled in the study. The actual age range of subjects who participated in the study was 19-31 years.Key exclusion criteria included: a history of various metabolic conditions or diseases; the concomitant use of a variety of medications, including but not limited to those with androgenic and/or anabolic effects; the use of nutritional supplements known to improve strength and/or muscle mass  within six weeks prior to the start of the study; a weight gain or loss of more than 10 lbs. within the past 30 days; known allergy to any ingredients in SOmaxP Maximum Performance™ or CP; participation in other research studies within the last 30 days; the current use of tobacco products; and the presence of any orthopedic limitations or injuries.The study was a prospective, randomized, double-blind, parallel-group clinical trial. Subjects were matched into two groups according to body mass, age, and resistance training experience. Subjects were then randomly assigned (via the ABBA procedure ) to recePrior to pre-testing, subjects were instructed to refrain from heavy exercise for 48 hours and fast for at least 12-hours. The assessment of upper body muscular strength (1-RM) and repetitions to failure (RTF) testing was performed after a general warm-up of 3-5 minutes of light activity involving the muscle(s) to be tested . Next, the subject performed several minutes of static stretching exercises of the involved musculature. The subject then performed a specific warm-up set of 8 repetitions at approximately 50% of the perceived 1-RM followed by another set of 3 repetitions at 70% of the perceived 1-RM. Subsequent lifts were single repetitions of progressively heavier weights until failure. The initial increments in weight were evenly spaced and adjusted such that at least two single lift sets was performed between the three repetition warm-up set and the estimated 1-RM. At failure, a weight approximately midway between the last successful and failed lift was attempted. This process was repeated until the 1-RM was determined. The rest interval between sets was between 3-5 minutes  . ResultsBody composition was assessed at baseline, and weeks 3, 6 and 9. Standing height was determined using a wall-mounted stadiometer. Body weight was measured using a SECA™ Medical Scale. Lean mass and fat mass were assessed using dual energy x-ray absorptiometry . For each subject, the same technician performed all four DEXA measurements.SOmaxP contains creatine monohydrate (4 g), carbohydrate (39 g), and whey protein (7 g), and a number of proprietary ingredients. Subjects randomized to the SOmaxP group took 1 serving of SOmaxP + 30 ounces of water starting 10-15 minutes before the workout and finishing before the end of the workout, and used the product only on the days when resistance training occurs. The comparator product (CP) was standardized to contain equal amounts of creatine monohydrate (4 g), carbohydrate  and protein (7 g whey protein), and given with 30 ounces of water, with identical timing, and similarly used only on resistance training days. The CP was virtually indistinguishable in taste, color and consistency to SOmaxP. The supplements were prepared in powder form and packaged in coded generic containers for double-blind administration by an independent company . Compliance to the supplementation protocol was monitored by a research nurse/dietician who contacted the study subjects on a weekly basis by telephone. Subjects were required to bring in their supplement bottles on workout days at weeks 3, 6 and 9 for visual inspection by study personnel to assess compliance with the protocol.A questionnaire was completed at weeks 3, 6 and 9  to monitor individual changes in DOMS and assess potential adverse events and change in sleep habits, general attitude, irritability, appetite, thirst, muscle soreness, muscle cramping, stomach distress, and headache, as well as any other idiosyncratic responses to the supplementation/training protocol. If identified, events were recorded as adverse events. In addition, subjects were contacted on a weekly basis by phone contact to inquire if they had experienced any adverse events, and were told to call at any time during the study to report side effects.The research dietitian met with each subject to explain the proper procedures for recording dietary intake. Each subject's baseline diet (3-days: two weekdays & one weekend day) was analyzed using the NutraBase IV Clinical Edition,  to determine its energy and macronutrient content. Additional 3-day diet records were analyzed at weeks 3, 6 and 9 to verify that eating habits had remained consistent throughout the study.1, lower body1, upper body2, lower body2) with gradual increases in volume and intensity. The workout consisted of at least 12 exercises, including but not limited to: bench press, lat pulldown, shoulder press, seated row, shoulder shrug, dip, biceps curl, triceps push down, leg press, squat, deadlift, lunge, leg curl, leg extension, and calf raise. For each exercise, subjects performed 3-6 sets of 8-15 repetitions with as much weight as they could handle with good form . As subject strength and endurance improved, training resistances were progressively increased to maintain the required repetition range. Rest periods between exercises were 1-3 minutes, and between sets were 60-120 seconds. Training was conducted at the subject's local training facility, documented in training logs, and signed off by fitness instructors/gym personnel to verify compliance. Two different facilities were utilized and identical equipment was available at both facilities. In addition, at each session, the subject completed a physical activity question, which described their physical activity during the preceding month. A schematic of the training program is displayed below in Figure All subjects followed a specific 4-day per week workout designed by a Certified Strength and Conditioning Specialist (CSCS). The workout involved training the upper and lower body twice per week using a 4-day split  with differential and platelet count, and a chemistry panel, which included sodium, potassium, chloride, carbon dioxide, calcium, AP, AST, ALT, bilirubin, glucose, blood urea nitrogen, creatinine, albumin, globulin, and estimated glomerular filtration rate, The lipid panel  was drawn at baseline and at week 9. Quest Diagnostics  was utilized to transport and analyze all blood samples.Separate analyses of co-variance (ANCOVA), using baseline scores as the covariate were used to analyze between-group differences in body composition, muscular performance, and clinical markers of safety. Data was considered statistically significant when the probability of a type I error was less than or equal to 0.05 (P ≤ 0.05). If a significant group, treatment and/or interaction was observed, least significant differences (LSD) post-hoc analyses were performed to locate the pair-wise differences between means.The demographic characteristics of the two cohorts were similar, and these are presented in Table A summary of the performance and outcome measures at baseline ("Pre") and at week 9 session ("Post") are presented in Table The measures of muscular performance  increased in both the SOmaxP and CP cohorts, though by a higher percentage in the SOmaxP group. The 1 RM for the SOmaxP cohort increased from 233.5-283.5 lbs. [106.1-128.9 kg] from pre- to post-testing (21.4% increase), while the CP cohort increased from 256.5-292.5 lbs. [116.6-132.9 kg], (14.0% increase). The RTF for the SOmaxP cohort increased from 19.6 to 30.25 from pre- to post-testing (54.3% increase), while the CP cohort increased from 26.3 to 30.8 (17.1% increase).Several measures of body composition differed statistically between the two cohorts, with the SOmaxP cohorts demonstrating favorable improvements. The body fat percentage in the SOmaxP group decreased from 16.8% to 15.5% from pre- to post-testing (7.7% decrease), while the CP cohort increased slightly from 16.5% to 16.9% (2.4% increase). Lean body mass increased in the SOmaxP group from 62.7 kg to 64.2 kg (2.4% increase), while the CP cohort increased marginally from 62.6 kg to 62.8 kg (0.3% increase). Body weight did not change significantly in either group, with the SOmaxP group experiencing a drop of 1.5 kg from a baseline of 81.1 kg to 80.8 kg (0.2 kg decrease), while the CP cohort gained 1.5 kg from a baseline of 79.9 kg to 80.2 kg (0.2 kg increase). Finally, in the SOmaxP cohort, fat mass decreased from 13.5 kg to 12.2 kg (9.6% decrease), while the CP cohort increased from 13.3 kg to 13.8 kg (3.8% increase). The percentage change from baseline (Post minus Pre × 100) in strength measures (RTF(t) and 1-RM) are presented in Figure There were no clinically meaningful changes in vital signs or laboratory results from baseline to Week 9. One subject experienced an adverse event. The subject was a 20 year-old male, (SOmaxP group) who experienced seasonal flu symptoms during Week 8 of the study. Symptoms included nausea, vomiting, and decreased appetite, and the events were not assessed as related to study product. Symptoms were resolved at the Week 9 post-testing visit. There were no significant changes in dietary intake for the subjects in either cohort, based on dietary diary evaluation.This double-blind, comparator study showed that nine weeks of supplementation with SOmaxP resulted in statistically significant improvements in muscular performance (1-RM and RTF), decreases in body fat and fat mass, and increases in lean mass, versus a comparator product matched with similar amounts of creatine, carbohydrate and whey protein. Both the SOmaxP and CP were well-tolerated, and there were no changes in laboratory measures or vital signs during the study. There were no adverse events assessed as related to either product, and no significant changes in body weight occurred during the study period in either group.1, and yet strength gains were noted in both the SOmaxP and CP groups. Typical protocols recommend ingesting approximately 0.3 g/kg/day of creatine monohydrate for 5-7 days as a loading dose , followed by 3-5 g/day thereafter [The SOmaxP cohort experienced an increase in strength and a concomitant increase in lean muscle mass and loss in body fat, without a significant change in body weight. These changes are consistent with a desired anabolic effect. Improvements in strength were also noted with the CP, though significantly less than with SOmaxP. The dose of creatine in this study (4 g/workout or 16 g/week) for both the SOmaxP and CP cohorts is lower than what is recommended by some of the more commonly described creatine protocolsereafter . A few sereafter , or muscereafter ,11. A loThe CP cohort gained strength, but only had a slight increase in lean mass, body fat % and body weight. A possible explanation for this is that the CP group, taking a similar 16 g/week of creatine monohydrate experienced physiologic changes sufficient to increase strength, but not sufficient to measurably increase lean mass. This finding is consistent with work by Rawson et al. (2010), who found that subjects who received low dose creatine (2.3 g/day or 16.1 g/week) for six weeks, experienced a significant increase in plasma creatine, and statistically significant enhanced fatigue resistance without weight gain compared to a matched placebo group .There are several possible explanations for the statistically significant difference between the SOmaxP group and CP, and these may be explained in part by several of the proprietary ingredients. SOmaxP contains a large quantity of branched chain amino acids. Branched chain amino acids (BCAAs), particularly leucine, have been shown to have anabolic effects, presumably through reducing protein breakdown . BCAAs hIn addition, SOmaxP contains isomaltulose  as part of its carbohydrate moiety. This carbohydrate is present in honey and has been associated with delayed digestion and absorption, which may account for the difference in body fat changes between the SOmaxP group and the CP group. Oizumi and colleagues 2007) developed a palatinose-based balanced formula (PBF) for use in human subjects with impaired glucose tolerance . During 07 develo2 max experienced significantly lower oxygen consumption with palatinose compared to sucrose, resulting in a lower plasma insulin response at 30 minutes compared to sucrose. Subjects consumed either water or 1 of 2 carbohydrate solutions  providing 1.1 g/min of carbohydrate. The authors concluded that the lower carbohydrate delivery and a small difference in plasma insulin may have resulted in a higher endogenous carbohydrate use and higher fat oxidation during the isomaltulose trial than during the sucrose trial.In addition, Achten et al. (2007) compared the oxidation rates from orally ingested sucrose and palatinose  during moderately intense exercise . The autper se. Furthermore, LOLA improved performance in Number Connection Test-A as well as mental state gradation in patients with more advanced hepatic encephalopathy. Stauch et al (1998) found an improvement in cerebral ammonia levels compared to placebo using an oral dose of 6 gm per day [Another possible ingredient the SOmaxP that may contribute to the results of this study is L-ornithine-L-aspartate (LOLA), a substance shown to be effective in lowering blood ammonia concentration, particularly in patients with hepatic encephalopathy . LOLA wa per day .In another published trial, LOLA decreased protein breakdown and stimulated protein synthesis in muscle in patients with hepatic encephalopathy . The theWe acknowledge some limitations to the study. No females enrolled in the study, although some were approached for possible inclusion. The study group was small and homogenous, with a relatively tight age range, on the younger side of the eligibility criteria. No attempts were made to identify the physiologic mechanism for any differences between the two groups. The study attempted to control for the use of other supplements during the study, but did not perform any testing to verify non-use of other supplements.The use of SOmaxP four times per week for nine weeks resulted in statistically significant improvements in strength, muscle endurance, lean muscle mass, and percentage body fat versus a comparator with identical quantities of creatine, whey protein and carbohydrate. Given that the quantities of the core components were identical, and these components are presumed to contribute most to ergogenic effects, the differences between the SOmaxP and CP groups may be due to additive or synergistic effects of the proprietary ingredients in SOmaxP. Additional research is needed to further elucidate these effects. A double-blinded, comparator controlled study of six weeks duration which includes muscle biopsy measurements is currently underway to examine and possibly help identify genetic and pharmacological mechanisms by which SOmaxP may exert these effects.S. Schmitz is not affiliated with any institution. J. Hofheins and R. Lemieux are associated with The Center for Applied Health Sciences, Division of Sports Nutrition and Exercise Science. Mr. Lemieux works as the strength coach for Kent State University.Stephen Schmitz declares he has a potential competing interest as he is non-employee, part-time, paid consultant for Gaspari Nutrition, working specifically in the areas of dietary supplement adverse event monitoring and reporting for the company. Jennifer Hofheins and Robert Lemeiux declare that they are employed by the Center for Applied Health Sciences, which conducted the study. However, neither individual was compensated above and beyond their customary amount as a result of this study. Gaspari Nutrition is paying the JISSN article processing charges; however, no Gaspari Nutrition employee was involved in the writing of this article.SS was the primary author of the manuscript. JH worked at the study site, was involved in subject recruitment, data collection and editing of the manuscript. RL developed the workout routine for the protocol. All three authors have read and approved the manuscript.


Automatic identification of various perfusion compartments from dynamic susceptibility contrast magnetic resonance brain images can assist in clinical diagnosis and treatment of cerebrovascular diseases. The principle of segmentation methods was based on the clustering of bolus transit-time profiles to discern areas of different tissues. However, the cerebrovascular diseases may result in a delayed and dispersed local perfusion and therefore alter the hemodynamic signal profiles. Assessing the accuracy of the segmentation technique under delayed/dispersed circumstance is critical to accurately evaluate the severity of the vascular disease. In this study, we improved the segmentation method of expectation-maximization algorithm by using the results of hierarchical clustering on whitened perfusion data as initial parameters for a mixture of multivariate Gaussians model. In addition, Monte Carlo simulations were conducted to evaluate the performance of proposed method under different levels of delay, dispersion, and noise of signal profiles in tissue segmentation. The proposed method was used to classify brain tissue types using perfusion data from five normal participants, a patient with unilateral stenosis of the internal carotid artery, and a patient with moyamoya disease. Our results showed that the normal, delayed or dispersed hemodynamics can be well differentiated for patients, and therefore the local arterial input function for impaired tissues can be recognized to minimize the error when estimating the cerebral blood flow. Furthermore, the tissue in the risk of infarct and the tissue with or without the complementary blood supply from the communicating arteries can be identified. The advent of modern imaging modalities has enabled studies to unravel temporal hemodynamic patterns in different regions of the brain to assist in the assessment of cerebrovascular diseases. Dynamic-susceptibility-contrast MR (DSC-MR) imaging records signal changes associated with different blood supply patterns following the intravenous injection of a bolus of contrast agent. Based on the bolus profile in the arterial compartment and on indicator dilution theory Classification of brain hemodynamics into different components is essential to facilitate the analysis and assessment of brain perfusion. In our previous studies, fast independent component analysis (FastICA) The segmentation of brain tissues using perfusion images relies on the clustering of bolus transit-time profiles to discern areas of different tissue types. However, cerebrovascular diseases would change the local perfusion of the impaired tissues with different levels of delay and dispersion, and therefore complicate the overall distribution of the signal profiles. The delay and dispersion phenomena were resulted from the defect of the blood vessel structures, such as the internal carotid artery (ICA) stenosis, moyamoya disease, and arteriovenous malformation. Assessing the accuracy of the segmentation technique under delay/dispersed circumstance is critical to accurately evaluate the severity of the vascular disease and locate the impaired tissues. However, previous literatures have focused on the effects of delay and dispersion in the deconvolution algorithm for estimating the rCBF Brain regions were extracted from perfusion images using Otsu’s method Similar to PCA, the data whitening process first calculates eigenvalues and corresponding eigenvectors for the covariance matrix of the zero-mean normalized data. The zero-mean data were then transformed via a whitening matrix, which was constructed of eigenvectors and eigenvalues, so that the covariance matrix became an identity matrix x. Initially, HC was carried out in the data set During HC, the compressed data are denoted by a matrix p and q, respectively, p and q, respectively, which are defined byith featured vector in cluster p and jth objects in cluster q. The two clusters with the smallest between-group distance are grouped together to form a new group. The algorithm proceeds until all of the feature vectors fall within a single group, thus forming a hierarchical clustering tree. Third, the level or scale of clustering is determined by cutting the hierarchical cluster tree. The HC results for the condensed data, n,θj−1)p was modeled from the averaged concentration-time curve of artery in order to compute the rCBV, rCBF, and MTT of each segmented tissue types The rCBF can be computed based on the relationship with concentration-time curve for each pixel:Simulated dynamic images based on a region of interest (ROI) in one of the raw data sets were adopted. In those images each simulated data set accommodated the possibilities of having four to nine hypothetical clusters . Pixels ith cluster and ith class. The classification rate for a set of simulated dynamic images, denoted by The parameters of MoMG for EM segmentation were initialized by 2 different means: 1) HC results from whitened data; 2) random sampling of columns from the matrix representing the PCA data, which was used in our previous EM-MoMG method Delay and dispersion are two popular phenomena in the hemodynamics of subjects with cerebrovascular diseases. Thus, Monte Carlo simulations were conducted to investigate the influences of these two factors on the proposed HC-EM-MoMG method. The concentration-time curves, tissueb denote time delays relative to AIF and dispersion, respectively. The values of tissueb were estimated to simulate the concentration-time curves of the delayed/dispersed artery (dArtery), delayed/dispersed gray matter (dGM), and delayed/dispersed white matter (dWM). Specifically, we calculated the values of rCBF and MTT for artery, GM, and WM, based on the raw data of hypothetical compartments. The normal rCBF and MTT values of artery, GM, and WM were also used for dArtery, dGM and dWM, respectively. The derived simulated concentration-time curves were then converted into signal-time curves using the following equation0S is the baseline signal and TE is echo time. In addition to the signal-time curves for dArtery, dGM, and dWM, signal-time curves for artery, GM, WM, vein combined with sinus (vein+sinus), sinus, as well as for CSF including the corpus plexus (CSF+cp) tissues were also used in these simulations. Hence, there were 9 hypothetical tissue types in the test of delay and dispersion effects. With delayt values of 0, 1, 2, 3, 4, and 5 s and dispersion b values of 0, 1, 2, 3, 4, and 5 s, overall 36 combinations of abnormal conditions were simulated (delayt’s) b’s) delayt and b for the impaired compartments were both 5 s.imulated . The numi, GE Medical Systems, Milwaukee, WI, USA) was used to acquire dynamic perfusion images. For the healthy subjects, trans-axial imaging was used with TE/TR = 60/1000 ms, flip angle = 90 degrees, FOV = 24 cm ×24 cm, matrix = 128×128, slice thickness/gap = 5/5 mm for 7 slices, one acquisition, and 70 images per slice location with a one second temporal resolution. Twenty ml of Gd-DTPA-BMA  followed by 20 ml of normal saline were delivered administratively using a power injector  at a flow rate of 3–4 ml/s in the antecubital vein. Some imaging protocol settings were changed for the ICA patient, i.e., TE/TR = 40/1000 ms, flip angle = 60 degrees, slice thickness/gap = 7/14 mm for 7 slices. All routines were implemented using MATLAB  codes and carried out on a 2.66 GHz Intel-Core2-based personal computer.This study received prior approval from the Institutional Review Board of Taipei Veterans General Hospital. Each participant provided written informed consent before participating in this study. Five healthy volunteers  aged from 18 to 47 years, one 78 year-old male with a unilateral ICA stenosis, and one 57 year-old female with moyamoya disease participated in this study. A multi-slice gradient-echo echo-planar-imaging pulse sequence on a 1.5 Tesla scanner  b, delayt) = , , , and were over 85% when  = , , , . The results also show that as noise increased from the noise-free condition to SNR = 40, the mean classification rates also decreased. As examples, the mean classification rates decreased from 87.0b, delayt) = . In addition, the mean classification rates obtained from the six normal tissues and the three impaired compartments  under different levels of delay and dispersion were 4% to 20% lower than the rates from the 9 normal tissues shown in The classification rates that were determined using different percentages of abnormal voxels are presented in Data from the five normal participants were processed and their perfusion images segmented into different compartments based on minimizing MDL values. Various tissue types, such as artery, GM, WM, vein+sinus, sinus, CSF+cp, CSF, vein with noise, sinus with noise, as well as artifacts, were segmented from normal data sets. The ratios of GM to WM for rCBV, rCBF, and MTT were 2.196±0.097, 2.259±0.119, and 0.968±0.023, respectively. These ratios were in agreement with other published reports th and 28th images, whereas the left hemisphere  had a signal drop and recovery between the 12th and 22nd images, i.e., the signal drop occurred 2 s earlier and recovery occurred 6 s earlier on the normal side than on the stenosis-affected side. In addition, a trans-axial T2-weighted MR image (−1⋅min−1) was lower than that of the normal GM (54.26−1⋅min−1), and the MTT of the dGM  and rCBF (9.95⋅100 g−1⋅min−1) but higher MTT (8.50⋅100 g−1) and rCBF (29.98±12.68 ml⋅100 g−1⋅min−1), which implies this area is worth in therapy to recover. These values can be re-computed after treatment to assess therapeutic effects.Another case was a 57 year-old female patient with moyamoya disease. She suffered from sudden vision loss on her left side for 3 days. Her right cerebral angiogram at the early arterial phase with lateral projection showed stenosis of the distal internal carotid artery, proximal segments of the anterior and middle cerebral arteries  vectors from the whitened data. Through that similarity, each component in the subtraction of each paired vectors contributed equally to the calculation of Euclidean distance. On the contrary, in PCA data, the first components were the dominant terms in any pair of column vectors, and they overwhelm the contributions from the remaining components, producing results in which the features from the rest of components of each column vector have been suppressed. Thus, clusters resolved from the dissimilarity matrix can be better discriminated based on the whitened data. In brief, the simulation results suggest that the HC-EM-MoMG method performed well, particularly when the SNR was higher than 40.Various tissue types, such as artery, GM, WM, vein+sinus, sinus, CSF+cp, CSF, vein with noise, sinus with noise, as well as artifacts, were segmented from normal data sets. Additionally, impaired tissue types, namely, dArtery, dGM, dWM, were segmented from an ICA subject’s data, and the infarct and risk of infarct were distinguished from others normal tissues in the moyamoya data. Since the slice locations from each of the normal subjects were not exactly the same, the segmented tissue types and the optimal number of clusters (between 7 and 8) varied slightly among subjects. Nevertheless, the artery, GM, WM, CSF+cp (or CSF) could be consistently segmented from the five normal data sets and the delivery of the contrast agent appeared in the following order: artery, GM, WM, and CSF+cp (or CSF).The proposed HC-EM-MoMG method successfully showed spatial and temporal hemodynamic patterns for each of the dissected tissue compartments. Three advantages of using the proposed method are as follows. First, the averaged signal-time curve for arterial tissue can be used as an AIF; in particular, the dAIF can be identified in the subject with an ICA stenosis. That allows calculation of the hemodynamic parameters of abnormal tissues, which can be used to compensate for delay and dispersion effects. In the ICA stenosis subject, for example, the rCBF values of the dGM and dWM obtained from the normal AIF were 46.21In future studies, the concept of the bargaining problem can be implemented for the optimisation of the parameters to assess the delay and dispersion effects. From the perspective of Game theory In summary, we improved the EM method by using the results of HC on whitened data to initialize the parameters of MoMG models, termed as HC-EM-MoMG method. The results of Monte Carlo simulations confirmed that the mean classification rates of the proposed method can achieve over 93.6% and small standard deviations, which was superior to that using the random sampling as initializations. Moreover, this is the first study to assess the delay and dispersion effects on the hemodynamic segmentation, which is critical to the cerebrovascular diseases. Our results of Monte Carlo simulations showed that most classification rates of the HC-EM-MoMG method were greater than 80% whenever the differences in delay or dispersion between normal and corresponding impaired compartments were longer than one second. Finally, the analysis of data from the patients with unilateral ICA stenosis and moyamoya disease illustrated the effectiveness of the method for identifying impaired compartments. The proposed method can quantify impaired hemodynamics and serve as an aid in diagnosis and therapeutic assessment, such as evaluation of the integrity of the circle of Willis and identification of the risk of infarct area.


The UCG wastewater may pose a potential threat to the groundwater since it contains high concentrations of inorganic  and organic  contaminants. The principal component analysis and hierarchical clustering analysis enabled to effectively explore the similarities and dissimilarities between the samples generated in lignite and hard coal oxygen gasification process in terms of the amounts and concentrations of particular components. The total amount of wastewater produced in lignite gasification process was higher than the amount generated in hard coal gasification experiments. The lignite gasification wastewater was also characterized by the highest contents of acenaphthene, phenanthrene, anthracene, fluoranthene, and pyrene, whereas hard coal gasification wastewater was characterized by relatively higher concentrations of nitrites, As, Cr, Cu, benzene, toluene, xylene, benzo( The underground coal gasification (UCG) is considered to be a prospective technique of coal utilization for the purposes of energy generation and chemical synthesis substrates production. Its advantages over a traditional coal gasification process results from the utilization of coal in situ and includes avoidance of cost and/or risk generating operational steps, like involvement of manpower underground, coal transport and pre-processing at the surface, as well as surface gasification infrastructure. It also enables to utilize coal resources otherwise abandoned for technical or economical reasons. Therefore, it is of special interest to Poland with the energy sector based predominantly on coal Białecka .The first large-scale UCG trials date back to the 1930s of the twentieth century (former Soviet Union). Since then, a few dozen large-scale experiments and commercial applications of the UCG process have been accomplished  and the hierarchical clustering analysis (HCA), were used in effective exploration of the studied experimental data.The experiments of lignite and hard coal underground gasification were performed in the ex situ reactor. The in situ conditions of the UCG process were simulated both in terms of coal seam and the surrounding rock mass. The UCG ex situ reactor was designed in the shape of rectangular prism of the internal workspace dimensions of: 2.6 × 1.0 × 1.1 m. Lignite or hard coal blocks were placed inside the strata layers, composed of sand collected from the site of lignite and hard coal blocks extraction and grinded clay slates. The scheme of the ex situ reactor is presented in Fig. 3/h). Exothermic reactions of coal with oxygen enabled heating up the coal seam and accumulation of the sufficient amount of thermal energy for the endothermic gasification reactions. The amount of the gaseous product and its composition were continuously measured with the flow meter and the two-channel gas chromatograph Agilent 3000A, respectively. The wastewater was collected in the separator and sampled every 12 h of the experiments duration.Lignite and hard coal blocks were ignited with a specially designed gas burner fuelled with a propane–butane gas and oxygen as the oxidant. The ignition step was terminated after approximately 2 h, when the temperature in the gasification channel exceeded 400 °C. Next, the gas burner was replaced with the gasification agents feeding system and the gasification process was started. The duration of the gasification experiments presented in the study was 50 h. The process was carried out under ambient pressure with oxygen as the gasification agent (flow rate of 3–6 mThe block of lignite (BL) was supplied by the open cast Bełchatów (PGE KWB Bełchatów SA), and two blocks of hard coal, ZHC and BHC, were provided by Ziemowit and Bobrek coal mines (Kompania Węglowa SA), respectively. The samples were analyzed in the accredited Laboratory of Solid Fuels Quality Assessment of the Central Mining Institute in accordance with the relevant standards in force: PN-G-04511:1980 , PN-G-04560:1998 and PN-ISO 1171:2002 (ash), PN-G-04516:1998 and PN-ISO 562:2000 (volatiles), PN-G-04513:1981 , PN-G-04584:2001 and PN-ISO 334:1997 , PN-G-04571:1998 , PN-G-04516:1998  . The rows and the columns of the matrix X (12 × 46) represent the wastewater samples collected after the 12th, 24th, 36th, and 48th hour of the experiments of BL, ZHC and BHC gasification duration, and the 46 measured parameters, respectively. Since the measured parameters differed in their ranges, the studied data set X (12 × 46) was standardized, according to the formula:Sk denote the mean of the jth column and its standard deviation, respectively. The standardized data matrix is denoted as Xc (12 × 46).The studied UCG wastewater data set was organized in the matrix X (m × n), into matrices S (m × A), and D (A × n), where A denotes the number of significant factors:The simplest way of tracing the relationships between the analyzed samples is their visualization in the space of measured parameters. Since the analyzed wastewater data would require 46-dimension space (46 parameters measured for each wastewater sample), the reduction of the data dimensionality was needed. The PCA Joliffe  is one oS is called a score matrix and includes, in case of the data set considered, information on wastewater sampled during three ex situ UCG experiments performed on lignite  and hard coal  in the 12th, 24th, 36th, and 48th hour of each experiment duration. The matrix D, called a loading matrix, describes the measured parameters. Columns of matrix S and rows of matrix D are called principal components (PCs) or eigenvectors and are constructed in a way maximizing description of the data variance. The way of construction of consecutive PCs is enforced by their orthogonality and their arrangement according to decreasing data variance they describe. Since the principal components provide the information not only on the data structure but also on the experimental error, the optimal number of the principal components (A) need to be selected, enabling effective data modeling and simultaneously, partial elimination of experimental error. The matrix E includes the part of data, which has not been modeled by the constructed model with A PCs. The problem of ineffective data dimension reduction in PCA may be resolved with a use of the HCA fluoranthene contents (parameter nos. 17 and 45). The highest values of these parameters were observed for BHC gasification samples (objects nos. 9–12). The uniqueness of the sample collected after the 12th hour of BHC gasification test resulted from the highest chromium content (parameter no. 17) and the lowest anthracene content (parameter no. 39). The uniqueness of the sample collected after the 24th hour in BL gasification test stemmed from high anthracene content (parameter no. 39).The PC1 described 32.94 % of the total data variance. It showed the differences between the samples taken in the entire period of experiment duration. The most significant difference was observed between the wastewater sampled after the 24th hour of BL gasification experiment (object no. 2) and after the 12th hour of BHC gasification test (object no. 9). Along the PC1, three groups of wastewater sampled in BL (objects nos. 1–4), ZHC (objects nos. 5–8), and BHC (objects nos. 9–12) gasification experiments, respectively, may be distinguished. Based on the loading plots, a conclusion may be drawn that the differences between the samples collected in lignite and hard coal gasification experiments were caused mainly by the highest anthracene content (parameter no. 39) observed in lignite UCG experiment (objects nos. 1–4). The wastewater generated in hard coal gasification experiments was characterized by relatively higher chromium and benzo and after the 36th hour of BL and BHC gasification experiments (objects nos. 3 and 9), respectively. Based on the loading plot, a conclusion may be drawn that the sample collected after the 12th hour of ZHC gasification experiment was characterized by relatively high cadmium, lead, and naphthalene contents  and the highest cobalt content (parameter no. 21). Objects nos. 3 and 9 were characterized by relatively high pH, and high ammonia nitrogen and ammonium content  and low cadmium, cobalt, lead, and naphthalene contents . The highest ammonia nitrogen and ammonium contents (parameter nos. 3 and 13) and the lowest manganese content (parameter no. 22) were observed for the sample collected after the 36th hour of BL gasification experiment (object no. 3).Based on the PC3 (describing 16.26 % of the data variance) the uniqueness of wastewater sampled after the 48th hour of ZHC gasification test (object no. 8) was observed. The sample was characterized by the highest ethyl benzene content (parameter no. 33) and the lowest sulfates content (parameter no. 11). The PC4, which described 10.36 % of the total variance was constructed due to the difference between the samples produced after the 12th and 24th hour of BHC (objects nos. 11 and 12) and after the 12th and 36th hour of ZHC and BHC gasification experiments (objects nos. 5 and 9), respectively. The PC5 revealed the uniqueness of wastewater sampled after the 12th hour of BL gasification experiment and after the 24th, 36th, and 48th hour of ZHC gasification . Based on the loading plot, a conclusion may be drawn that the uniqueness of the samples produced after the 12th and 48th hour of BL and ZHC gasification experiments  was caused by the highest total amount of chloride and sulfate (parameter no. 12) when compared to all the remaining samples. The samples collected after the 24th and 36th hour of ZHC gasification (objects nos. 6 and 7) were characterized by the lowest total chlorides and sulfate amounts (parameter no. 12), the highest boron content (parameter no. 16) and relatively high conductivity and lead content (parameter nos. 1 and 26) when compared to the remaining samples. The uniqueness of the sample produced after the 36th hour of ZHC gasification (object no. 7) was caused by the highest lead content (parameter no. 26).a)anthracene, chrysene, and benzo(b)fluoranthene contents . Furthermore, a negative correlation was observed between ammonia nitrogen (parameter no. 3) and cobalt and naphthalene contents (parameter nos. 21 and 35), between pH (parameter no. 2) and lead and naphthalene contents (parameter nos. 26 and 35), between ammonium (parameter no. 13) and cobalt and naphthalene contents (parameter nos. 21 and 35) and between anthracene (parameter no. 39) and arsenic and benzo(a)pyrene contents (parameter nos. 15 and 46).Since the compression of the data using PCA was ineffective, a conclusion may be drawn that only weak correlation between the variables could be observed in the presented data set. The loading plots revealed a positive correlation between pH, ammonia nitrogen, and ammonium ; lead and naphthalene (parameter nos. 26 and 35); acenaphthene, phenanthrene, and fluoranthene ; and copper, benzene, toluene, xylene, benzo anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, and benzo(a)pyrene contents, respectively.Group E, including parameter nos. 15, 17, 23, 31, 32, 34, and 42–46, which represent contents of arsenic, chromium, copper, benzene, toluene, xylene, benzo and parameters are sorted according to the order of objects and parameters at dendrograms in Fig. The HCA completed with a color map of the standardized experimental data is a simple visualization method. Namely, the color map is constructed for standardized transposed data matrix k)fluoranthene  and lower contents of cadmium, mercury, and anthracene  than observed for the remaining samples.The analysis of the dendrogram of 12 objects in the space of the 46 measured parameters sorted according to the Ward linkage method with the color data map see Fig.  enables a) anthracene, chrysene, and benzo(b)fluoranthene , and the lowest content of titanium (parameter no. 29).Furthermore, the sample collected after the 12th hour of BHC gasification (object no. 9) was characterized by the highest contents of total and bound cyanides, arsenic, chromium, copper, benzene, toluene, xylene, benzo(All samples of ZHC gasification process grouped in the cluster B were characterized by relatively lower content of sulfates (parameter no. 11), and higher contents of phenols and boron (parameter nos. 10 and 16), than observed for the remaining samples of BHC and BL gasification experiments.The sample collected after the 48th hour of ZHC gasification test (object no. 8) differed from the remaining samples collected after the 12th, 24th, and 36th hour of process duration (objects nos. 5–7) due to the highest content of chlorides, total amount of chloride and sulfate, copper, molybdenum, selenium, and titanium . The uniqueness of the sample collected after 12th hour of ZHC gasification (object no. 5) was also observed resulting from high contents of free and bound cyanides, antimony, cadmium, and mercury , the highest contents of aluminum, cobalt and manganese  as well as the lowest contents of acenaphthene, phenanthrene, and fluoranthene .a) anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, and benzo(a)pyrene  and relatively higher contents of acenaphthene, phenanthrene, fluoranthene, and pyrene .All samples of BL experiments grouped in the cluster C differ from the samples of hard coal gasification experiments mainly due to relatively lower contents of nitrites, boron, chromium, aluminum, lead, benzene, toluene, xylene, naphthalene, fluorene, benzo(The sub-cluster C1 (objects nos. 2–4) was characterized by the highest contents of acenaphthene, phenanthrene, fluoranthene, and pyrene . The sample collected after the 36th hour of lignite gasification (object no.3) was characterized by the highest conductivity (parameter no.1) and the highest contents of ammonia nitrogen, suspended matter, ammonium, and anthracene , whereas the wastewater sampled after 48th hour of lignite gasification (object no. 4) was characterized by the highest content of acenaphthene (parameter no. 36) and the lowest contents of antimony, boron, zinc, and pyrene . The uniqueness of the sample collected after the 12th hour of lignite gasification (object no. 1) resulted from the highest contents of sulfates and iron (parameter nos. 11 and 30), relatively high contents of total amount of chloride and sulfate, antimony, cadmium, manganese and mercury  and the lowest contents of ammonia nitrogen, ammonium and chromium .In addition to water-soluble, polar phenolics, low soluble organic compounds such as BTEX (parameter nos. 31–34) as well as the PAHs (parameter nos. 35–46) constitute the major threat to the groundwater in terms of UCG induced contamination. PAHs are one of the most widespread organic pollutants which could be formed in underground coal gasification. The amount and type of PAHs as well as their homologs depend on the operating conditions of combustion/gasification process. The PAHs toxicity is structurally dependent  and the samples obtained after the 12th, 24th, and 36th hour of ZHC gasification experiments (objects nos. 5–7). The cluster B is composed of the samples collected after the 24th, 36th, and 48th hour of BHC gasification (objects nos. 10–12)  and the samples collected after the 36th and 48th hour of BHC gasification (objects nos. 11 and 12) included in the cluster A were characterized by relatively higher naphthalene and fluorene contents. The uniqueness of the objects nos. 6–7 of the cluster A could be also observed. These objects were characterized by high anthracene content, the highest naphthalene and fluorene contents and the lowest pyrene content.k)fluoranthene, the highest benzo(a)anthracene, chrysene, benzo(b)fluoranthene, and benzo(a)pyrene and the lowest anthracene contents when compared to the remaining wastewater samples.The samples collected after the 12th and 24th hour of BHC gasification (objects nos. 9 and 10) grouped in the cluster B were unique due to high benzo(k)fluoranthene contents and relatively higher acenaphthene, phenanthrene, anthracene, fluoranthene, and pyrene contents. The uniqueness of the wastewater sampled after the 24th and 48th hour of lignite gasification (object no. 2) was caused by the highest phenanthrene, fluoranthene, and pyrene contents and the highest acenaphthene content, respectively.The cluster C, composed of samples collected during the entire BL gasification experiment (objects nos. 1–4) was characterized by the lowest naphthalene, fluorene, and benzo components.Lignite ex situ UCG process was characterized by higher total amount of wastewater generated and higher coal consumption rate, when compared to the respective values reported for the ex situ UCG process on hard coal.Higher amount of wastewater generated in lignite gasification process resulted from significantly higher moisture content in lignite when compared to hard coal samples.The chemometric methods, such as the PCA and the HCA enabled to effectively explore environmental data concerning the amounts and composition of wastewater generated in lignite and hard coal ex situ UCG experiments.a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, and benzo(a)pyrene contents.The samples collected in lignite gasification experiment were characterized by the highest acenaphthene, phenanthrene, anthracene, fluoranthene, and pyrene contents, whereas the samples of hard coal gasification experiments were characterized by relatively higher nitrites, arsenic, chromium, copper, benzene, toluene, xylene, benzofluoranthene content, the highest benzo(a)anthracene, chrysene, benzo(b)fluoranthene, and benzo(a)pyrene contents and the lowest anthracene content.The samples collected after the 12th and 24th hour of BHC gasification were also characterized by high benzo(The uniqueness of the sample collected after the 48th hour of ZHC gasification resulted from the highest content of ethyl benzene and the uniqueness of the wastewater sampled after the 12th hour of BHC gasification could be attributed to the highest contents of benzene, toluene, and xylene.


Background. Neurofibromatosis type 1 is a multisystemic, progressive disease, with an estimated incidence of 1/3500-2500. Mitochondrial diseases are generally multisystemic and may be present at any age, and the global prevalence is 1/8500. The diagnosis of these disorders is complex because of its clinical and genetic heterogeneity. Case Report. We present a rare case of the association of these two different genetic diseases, in which a heterozygous missense mutation in the NF1 gene was identified which had not yet been described (p.M1149 V). Additionally, the patient is suspected of carrying an unspecified mutation causing respiratory chain complex I deficiency. Clinical presentation included hypotonia, global development delay, reduced growth rate, progressive microcephaly, and numerous café-au-lait spots. Discussion. To the best of our knowledge this is the first report of complex I deficiency in a patient with neurofibromatosis type 1. It is very important to maintain a high index of suspicion for the diagnosis of mitochondrial disorders. In this patient, both the laboratory screening and muscle histology were normal and only the biochemical study of muscle allowed us to confirm the diagnosis. It has he Iris) .Mitochondrial disorders are a heterogeneous group of diseases characterized by defects of mitochondrial structure and oxidative phosphorylation –8. TheseThe patient, a boy, was born after a normal pregnancy of 39 weeks, through an instrumented vaginal delivery. His birth weight was 2970 g. At birth, he had a low Apgar score , hypotonia, grunt, and respiratory depression, requiring noninvasive ventilation. During neonatal intensive care stay, he maintained hypotonia and feeding difficulties. Inborn errors of metabolism  and congenital hypothyroidism were not detected in the analysis of the Guthrie card. The metabolic study  and brain magnetic resonance (MRI) were normal. He was discharged from hospital at 28 days of life to continue followup as an outpatient and was referred to a developmental early intervention program.café-au-lait spots, negative for Leigh syndrome or mitochondrial dysfunction.Family history was notable for the mother, uncles, and grandmother having various café-au-lait spots, greater than 0.5 cm in diameter, (these appeared in the first year of life and increased progressively in number and size) and a systolic murmur.Regarding the milestones, he just reached the sitting position after twelve months. Gait and first words were present at twenty-four months. At three years of age, he maintained low weight and short stature for his age, hypotonia, and global development delay. Physical examination revealed progressive microcephaly, more than six polymerase chain reaction followed by direct sequencing of genes encoding subunits of complex I. The most common mutations and mitochondrial DNA deletions of large size   were sent to research. Then we studied the seven mitochondrial genes  and subsequently sequenced the eleven nuclear genes in which mutations have been so far described—NDUF1, NDUFS2, NDUFS3, NDUFS4, NDUFS6, NDUFS7, NDUFS8, NDUFV1, NDUFV2, and NDUFA1 NDUFA8. All the molecular study was negative. Cardiac evaluation detected a low grade pulmonary stenosis; cardiomyopathy and arrhythmia were not observed. Subclinical hypothyroidism  was also detected. The remaining studies  were normal. Ophthalmologic and audiologic evaluations were normal.Molecular study confirmed an heterozygous mutation in the NF1 gene -exon 26). Liver, renal, and hemopoietic function were normal along with plasma lactate, pyruvate, amino acids, and urinary organic acids profile. Magnetic resonance spectroscopy and deltoid muscle histology were normal. The analysis of enzymatic activity of mitochondrial respiratory chain complexes I–V in muscle (spectrophotometric assay) revealed partial (29%) deficit of complex I activity relative to citrate synthase . In the molecular research, we used the technique of 10 plus levothyroxine and maintained dietary supplementation as well as occupational and speech therapies.He started treatment with coenzyme QAt the age of six, the brain MRI revealed subthalamus lesions of probable hamartomatous nature. At this age, evaluation with the Ruth Griffiths scale revealed global developmental delay involving uniformly motor, verbal, and cognition skills . According to the classification of the Diagnostic and Statistical Manual of Mental Disorders Fifth Edition (DSM-V) and the Conner's Ratting Scales, the patient did not meet criteria for attention-deficit/hyperactivity disorder (ADHD).Currently, he is eight years old and is clinically well and asymptomatic. Nevertheless, cranial magnetic resonance imaging revealed a left optic nerve glioma in its prechiasmatic segment . After eThe authors present a child with NF1 in which a heterozygous missense mutation in the NF1 gene was identified that had not yet been described. Since NF1 is a very common disease , its assThese two diseases share important characteristics: they are both multisystemic and progressive , 7. It iThese diseases are frequently associated with neurocognitive deficits, including ADHD, autism spectrum disorders, behavioral abnormalities, and psychosocial issues , 14. We Optic nerve glioma developed in approximately 15% of patients with NF1 , 16. It It is very important to maintain a high index of suspicion for the diagnosis of mitochondrial disease, because most patients do not present easily recognizable disorders . Except Mitochondrial complex I deficiency is the most common defect of the oxidative phosphorylation system . Most caArun et al.  demonstr


Leishmania (V.) braziliensis are endemic in Paraguay. We performed a series of knowledge, attitudes, and practice (KAP) surveys simultaneously with individuals in endemic communities in San Pedro department (n = 463), health professionals (n = 25), and patients (n = 25). Results showed that communities were exposed to high risk factors for transmission of L. braziliensis. In logistic regression analysis, age was the only factor independently associated with having seen a CL/MCL lesion (P = 0.002). The pervasive attitude in communities was that CL was not a problem. Treatment seeking was often delayed, partly due to secondary costs, and inappropriate remedies were applied. Several important cost-effective measures are indicated that may improve control of CL. Community awareness could be enhanced through existing community structures. Free supply of specific drugs should continue but ancillary support could be considered. Health professionals require routine and standardised provision of diagnosis and treatment algorithms for CL and MCL. During treatment, all patients could be given simple information to increase awareness in the community.Cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL) due to  Leishmaniasis is an important cause of disability in 98 endemic countries and 3 territories with 350 million people living at risk of infection , 2. It iLeishmania (V.) braziliensis, initially by multilocus enzyme electrophoresis [Nyssomyia whitmani , Migonemyia migonei  and Nyssomyia neivai  [Oryzomys, Calomys, Oligoryzomys and Akodon, and marsupials of the genus Didelphis .CL is endemic in 22 Latin American countries. In Paraguay, CL has traditionally been endemic in most of the eastern region, especially in San Pedro, Canindeyú, and Alto Paraná departments , 15. Durphoresis  and, frophoresis . Sand flo, 1926) . The silHere we explore the knowledge, attitudes, and practices of endemic communities as well as patients and health professionals to derive recommendations for the improvement of prevention and care. Understanding the impact of leishmaniasis and human behaviour surrounding the disease is crucial to improving control and treatment. San Pedro department, located northeast of the capital Asunción, was chosen for the community survey because it has one of the highest levels of CL/MCL endemicity in Paraguay . The primary researchers participated in a 2-day workshop with researchers who were conducting parallel studies in Brazil, Venezuela, and Peru. A pilot study of 100 individuals from an endemic community, 10 health professionals, and 10 CL/MCL patients was performed in Paraguay to validate the questionnaires, and adjustments were made accordingly. All questionnaires included both closed and open-ended questions and were appropriate and available in Spanish and Guaraní, the national languages of Paraguay.knowledge of CL/MCL in terms of local names, transmission, symptoms, prevention, and information source; attitudes whether the disease was considered to be a problem and if so, what kind of problem and for whom; practices with regards to prevention, treatment seeking behavior, side effects of treatment, and economics of treatment. For the community KAP, interviews were conducted at the household level with one respondent per household and elicited responses concerning: ρ (rho) was calculated at 0.25, and a cluster size of 7 with a design effect of 2.5 was used. A cluster was defined as 7 households. The precision of confidence intervals was set to ±7.5% around an estimated proportion of 50%. A sample size of 448 with an added refusal rate of 3% achieved a final sample size of 460. All villages in the three selected districts were considered, with the sample divided amongst them proportional to the total reference population following the Expanded Programme for Immunization (EPI) methodology [Sample size for the community KAP was determined from a pilot survey in Venezuela in July 2006 . Intracluster correlation hodology . For eacThe patient KAP included open-ended questions on the patient's experience with CL/MCL, mechanisms of receiving information, treatment-seeking behavior, costs of treatment, barriers to treatment, management of side effects, perception of efficacy, and the disease impact on personal, social, and working life. Purposive sampling strategy was used for patient interviews. For inclusion, patients had to have had active CL and/or MCL within the last 2 years. A caretaker responded to the questionnaire on behalf of patients less than 14 years. Patients were identified through existing clinical records; 25 were selected, 14 from San Pedro and 11 from other endemic areas, who were interviewed at the IICS.The health professionals KAP included open-ended questions on: knowledge of current protocols for treatment, personal experience with treatment, specific training, opinions of patient experiences , what would happen if treatment was no longer offered freely, and main obstacles facing CL/MCL treatment. Purposive sampling strategy was also used for the 25 health professional interviews. Health professionals could be doctors, nurses, midwives, nursing assistants or health promoters, providing they had experience of assessing and treating CL/MCL patients. n = 250) for the categories of knowledge, attitudes, and practices as well as a composite KAP score . These scores were analyzed descriptively. Data from the community KAP were double-entered into EpiData. Validation of the datasets was performed, and the data were cleaned for inconsistencies and then transferred to STATA 8.0 for further analysis. Descriptive, bivariate, and logistic regression analyses were performed. Adjusted odds ratios were calculated to evaluate the independent associations between sex, age, income, education or location, and specific knowledge, attitudes, and practices, adjusting for the clustered survey design . The adjEthical approvals were from the Ethical-Scientific Committee at the IICS-UNA in Paraguay and the ethical committee of the London School of Hygiene and Tropical Medicine. All respondents participated by informed consent form and were assured of confidentiality and anonymity. After each interview, all respondents received printed materials with information about CL/MCL, provided by the Ministry of Public Health.A total of 463 people were interviewed: 69% (321/463) from rural areas, 25% (116/463) from peri-urban, and 6% (26/463) from urban areas. Respondents were aged between 14 and 87 years with a mean of 40 years . The agrRodents, marsupials, and dogs, possible reservoirs for CL/MCL, were reported to live within 20 paces of 81% (376/463: dogs) and 40% (184/463: rats) of households. Just over 80% (374/463) owned a dog, and for 7% (25/374) had their dog sleep inside the house. Five percent (22/462) of respondents lived within 20 paces of rubbish piles. Whilst most of the population slept in beds indoors , bed net use every night was low , with 86% (397/463) never having used a bed net. Llaga in Spanish and Kuruvai in Guaraní). From both questions, 213 respondents had neither seen a CL/MCL lesion nor heard about CL/MCL and were, therefore, excluded from dependent questions concerning CL/MCL; questions relating specifically to CL/MCL were pursued with the remaining 250. For these respondents knowledge of CL/MCL was moderate: 70% (176/250) understood that everyone can get CL/MCL but 38% (94/250) believed direct transmission of CL/MCL from person to person was possible. Most respondents believed that dogs 36% (91/250), sand flies 29% (72/250), mosquitoes 8% (20/250), or other insect bites 35% (87/250) were responsible for transmission (more than one answer possible). The mean KAP score for knowledge was 5.6 , and the range was 1–12  of respondents had previously seen a CL/MCL lesion, while half  had heard of CL/MCL  named skin lesions as a symptom of CL/MCL and 14% (36/250) cited lesions of the nose and mouth. Thirty percent (76/250) did not know any signs or symptoms of the disease. Only a quarter of the sample (62/250) knew that one could have CL/MCL and be asymptomatic. Most respondents heard about CL/MCL from their family, friend, or neighbour . Other sources of awareness and information included: television , radio , community health worker , school , and brochure or poster  (more than one response considered). The majority of respondents believed that CL/MCL could be prevented  . KAP scores for attitude were moderate, and the mean for 250 respondents was 3.1 (a perfect score was 7) see . RespondFifteen people out of 250 (6%) reported that member(s) of their families had been infected with CL/MCL within the previous two years. Fourteen infected family members sought help for CL/MCL, primarily from the hospital (8/14) followed by the pharmacy (4/14), local health centre (2/14), family, friend, or neighbour (2/14), and traditional healers (2/14) (more than one answer possible). Twelve community respondents reported being personally infected with CL/MCL, with time of onset of symptoms and treatment seeking between 1–6 months  . Eleven P = 0.002) adjusting for sex, income, location of house, and education. The odds of having seen a CL/MCL lesion increased with age, relative to those aged 14–24, such that those aged 35–44 years had OR 3.4 (95% CI: 1.6–7.1), those aged 45–59 years had OR 4.2 (95% CI: 1.9–8.9), and those aged 60 years and above had OR 4.1 (95% CI: 1.7–9.7). Factors independently associated with having heard of CL/MCL were age, education, location of house, and income , environmental clean-up , indoor residual spraying , treated dog collars , and insecticide treated bed nets  . Twenty-five patients or their carers were interviewed . Most . When asked specific questions about CL/MCL, only 6/25 patients identified sand flies as responsible for CL/MCL transmission. Nearly three quarters (18/25) knew that CL/MCL could be prevented. Twenty-eight percent (7/25) knew that one could be asymptomatic with CL/MCL. Nearly all patients  reported having seen a CL/MCL lesion when shown a photograph while 76% (9/25) had heard the term for cutaneous leishmaniasis  reported that CL/MCL was a problem in their area. When asked why it was a problem, the range of responses included associations with CL/MCL cases they had seen, new settlements, indigenous settlements, the interior of the country, and some named specific localities. When asked what would happen without treatment 7/25 believed they would have become increasingly sick, 1/25 that they would have been scarred, and 13/25 that they would have died. Time between onset of symptoms and treatment-seeking ranged from less than one week (2/24) to more than one year (4/24), with most patients waiting between 1–6 months (11/24) . Two patThe cost of treatment borne by the patient  varied from no costs (5) to 20,000–700,000 Paraguayan Guaraní (PYG) (13) to over 10,000,000 PYG  (3)—nearly half of the average per capita income . Mean total cost of treatment was 1,970,476 PYG ($465 USD), and median total cost was 300,000 PYG ($71) .Most of the 25 health professionals were nurses or auxiliary nurses (13/25); 11/25 were doctors; 8 were from Asunción and the Central department; 10/25 had access to the Internet, and 15/25 had access to medical journals; 24/25 would have liked more information about leishmaniasis . Nearly all of the health professionals when shown a photo of a lesion had seen CL/MCL before (23/25). Most described it as an illness transmitted by sand flies (21/25), dogs (3/25), and mosquitoes (3/25). There was a fairly consistent understanding that lesions appeared with impact on the skin, in particular the nose and mouth. Some professionals (2/25) reported that leishmaniasis was transmitted from person to person. Most respondents (17) reported that without proper treatment the patient's condition would worsen and five stated that it could be fatal. When asked if CL/MCL is a problem in the area, 10 respondents answered yes, 12 said no, and 3 did not know; those agreeing that CL/MCL was a problem cited patient-focused reasons such as economic barriers (diagnosis and treatment are expensive) and that people were not treated. Other reasons given included deforestation and the need for fumigation to eliminate leishmaniasis. All 25 respondents were asked to describe the protocol for diagnosis and treatment of CL/MCL. Most began with an examination of the ulcer itself; 14 who had the capacity would use the Montenegro skin test; 4 would use biopsy smears for diagnosis. Two mentioned, the immunofluorescence test and two the PCR test, although not always available. Four respondents said that they did not know how to do the diagnosis. Over half (13) of the health professionals have had previous experience treating leishmaniasis. These 13 form the denominator for the remaining questions on treatment. These health staff treated primarily CL/MCL (10), with 1 treating VL only and 2 treating both CL and VL. The 13 experienced respondents offered drugs for free and concluded that otherwise people would not follow the treatment because they could not afford medication. Four respondents referred patients to the hospital or health centre for treatment and drugs mentioned to treat leishmaniasis included Glucantime, Pentostam, antimonials in general, and Amphotericin B for MCL. When asked what costs patients incur, most (10) cited transportation costs to and from the hospital and several others mentioned maintenance and accommodation, consultation, other medications, or loss of income. This research provides an extensive detailed analysis of perceptions and behavior surrounding prevention, treatment, and knowledge of CL and MCL in San Pedro department, Paraguay. Although the data collection was concentrated primarily in one department, results and analyses can be extrapolated to other parts of the country with similar socioeconomic characteristics. This is the first study in Paraguay to assess the KAP of endemic communities, patients, and medical professionals simultaneously, with a substantial community sample. Due to the security situation, interviewers could not conduct their research at night: a sampling limitation for the community survey was, therefore, biased towards female respondents (296 : 167). Leishmania (Viannia) braziliensis in Paraguay but they have been implicated elsewhere [Leishmaniasis is spread by some 30 different sand fly species and affects a wide variety of hosts including humans, dogs, horses, and foxes , 24. Rodlsewhere , 26. Othlsewhere  and soillsewhere ; around lsewhere . In Paralsewhere , 30. EnvDespite the risk factors, regular use of bed nets was low among both patient, and community respondents . Recent studies from Iran and Venezuela indicate that consistent use of insecticide treated bed nets and curtains provide some personal protection against sand fly bites and transmission of zoonotic CL . Low useIn all three surveys, respondents consistently mentioned costs associated with treatment of leishmaniasis. Although antimonial drugs are offered at no cost in Paraguay, not all costs associated with treatment are included in current government policy. Among the additional costs patients reported were, consultations, related medical supplies and antibiotic therapy for secondary infections. Collateral costs such as loss of income, travel, and maintenance costs during treatment also impose an economic burden and may affect followup of cases. Patients may weigh the financial risk of seeking treatment with the impact on their vulnerable household economy. This may well affect the number of cases recorded, increase time between onset of symptoms and treatment, and increase the number of mucosal cases. Our findings suggest that a holistic policy approach to treatment needs to be explored, which includes cost recovery mechanisms to ease the financial burden that treatment can bring to disadvantaged populations. For example, enlisting a specific agency or company to transport patients by motorbike to their closest health post could ease the transportation burden for some patients, proving to be a highly cost effective measure. A holistic policy approach to treatment would save the enormous costs associated with complex treatment and clinical management of late cases. One clear finding of this research is the need for increased and consistent awareness building activities in endemic communities. At the community level, just over 50% were aware of CL/MCL and specific knowledge of the disease and its transmission, and prevention was moderate. Some misinformation was apparent, the concept that leishmaniasis can be passed from person to person, an echo of a pilot patient and health professional KAP in Paraguay . This misconception may augment stigma and hinder CL and MCL sufferers from accessing assistance and care.Furthermore, across the three surveys, less than half of respondents (in the community survey only 10%) agreed that leishmaniasis was a problem in their area. Awareness activities need to describe frequency of disease, the cost and duration of treatment, and severity of metastatic disease (MCL). Those living in endemic communities could also be better informed on transmission by sand flies, prevention methods, personal risk, how to identify a skin, lesion and where and when to seek treatment. Older people may be useful to incorporate in educational activities as they have a higher awareness level than their younger peers and may command a degree of respect in community outreach. Education could acknowledge differential risks for men and women, their roles and responsibilities for health. For example, messages to women might include treatment seeking information and environmental clean up, which could be undertaken at the household level, whereas men could learn about occupational exposure, prevention of sand fly bites and recognition of a skin lesion. Based on our data, radio is the preferred means for mass media campaigns, because TV ownership is moderate and newspaper readership is low. Other avenues for education and awareness activities include churches and elementary schools. Most community members and patients received their information from family members, friends, or neighbours, confirming earlier findings . Community mobilization programs need to incorporate and activate such existing social networks to maximize information dissemination. Most patients did not receive information at time of treatment. Health professionals are recommended to take advantage of consultation to inform and assist patients to understand their condition, treatment, and how to prevent it in the future. Simple posters are recommended to assist medical personnel in the clinics, and simple brochures with pictures for patients to take home to families and neighbours. It is also suggested that the National Control Programme follow-up on the extent of distribution of printed materials for patients, health professionals, and communities. Commonly materials are produced but may not actually reach their target audiences due to distribution constraints, turnover of staff, lack of funds, and low priority of prevention . Another means of communication to consider is the use of mobile phone technology to remind patients of follow-up appointments. Incomplete treatment is a serious hazard for the development of drug resistance. Increased patient education and financial facilities to aid disadvantaged patients will reduce the rate of patients not completing treatment.Health professionals displayed sufficient general information about leishmaniasis. However, it appears from their responses that there is a need for a streamlined approach to diagnosis of CL/MCL in Paraguay, paying particular attention to the level of health care facility, for example, what may be feasible at the primary health care centre may not be at a smaller auxiliary health post. In this respect, training of nurses and health promoters may also maximize the diagnosis and treatment of CL/MCL; algorithms for diagnosis and treatment could be made widely available. Methods of communication  with health professionals could also be enhanced to keep them updated and demonstrate the importance of leishmaniasis as a growing concern in their communities. As both CL and MCL are increasing in many countries globally, including Paraguay, the policy implications and recommendations discussed here are relevant in a wider context. Specifically, attention could be given to implementing cost effective measures that are likely to improve the timely treatment of CL and MCL patients, thus avoiding higher costs associated with complex treatment and clinical management of late cases. Government and private agencies need to be aware of the cost implications of treatment, even when antimonials are offered at no cost and could make cost recovery programs a priority to improve treatment seeking behavior and compliance with treatment regimens. Communities affected by CL and MCL could be prioritized for appropriate awareness campaigns, including lesion recognition, prevention, and information on treatment. Awareness campaigns could utilize the existing structures within the community, for example, schools, churches, and civil organizations and include older people and past patients in educational activities. By implementing the cost effective measures discussed in this paper, prevention of new cases, better case recognition, improved treatment-seeking behavior, and sustained compliance with treatment can be an attainable goal.


Pasteuria ramosa, a bacterium evolving in naturally, strongly, female-biased populations of its host Daphnia magna.Males and females differ in many ways and might present different opportunities and challenges to their parasites. In the same way that parasites adapt to the most common host type, they may adapt to the characteristics of the host sex they encounter most often. To explore this hypothesis, we characterized host sex-specific effects of the parasite We show that the parasite proliferates more successfully in female hosts than in male hosts, even though males and females are genetically identical. In addition, when exposure occurred when hosts expressed a sexual dimorphism, females were more infected. In both host sexes, the parasite causes a similar reduction in longevity and leads to some level of castration. However, only in females does parasite-induced castration result in the gigantism that increases the carrying capacity for the proliferating parasite.Daphnia represent different environments and reveal one parasite-induced symptom (host castration), which leads to increased carrying capacity for parasite proliferation in female but not male hosts. We propose that parasite induced host castration is a property of parasites that evolved as an adaptation to specifically exploit female hosts.We show that mature male and female  Males and females of the same species typically differ in many traits, so much so that the most striking differences among individuals of the same species are usually those between sexes. A given species may have differences in gametes, primary and secondary sexual characters, quality and quantity of hormones, but also differences in behavior, somatic structures, immune response and gene expression -4. TheseDaphnia magna and its natural bacterial parasite, Pasteuria ramosa, as a model system for host-parasite interactions. In this system, infection success depends on the combination of host and parasite genotypes [D. magna are strongly female biased. As such, parasites of D. magna will typically encounter females considerably more often than males and might be adapted to exploit that particular host type.We use the cyclically parthenogenetic crustacean enotypes . Host suenotypes . Even thenotypes ), there enotypes  and, as Daphnia focus exclusively on female hosts . Those  species -14 and hia model ,16. HoweD. magna, are also observed in infected males. If these symptoms are not seen in males, it is consistent with our hypothesis that the parasite adapted to exploit female hosts.The first aim of this study was to investigate if the two host sexes represented different environments for the parasite. For several genetically distinct host clones, we recorded the differences between host sexes of the same host clone  in parasite infectivity, virulence, proliferation and fitness. The second aim of our study was to test if parasite-induced castration and gigantism, described for infected female D. magna and P. ramosa, its bacterial parasite, we investigated if parasites exposed to male versus female host individuals differ in the likelihood of successful infection (Experiments 1 and 2), in parasite fitness, in the rate of proliferation within the host (Experiments 2 and 3), and in induced disease symptoms .In a series of experiments . Parasite density increased in males during the later phase of parasite proliferation . Individuals dead before Day 14 were excluded from further analysis.Over all the experiments, we found no significant difference in mortality before Day 14 (the earliest it is possible to reliably check for infection status) between male and female hosts, and between host clones ): "Host clone", df = 10, F = 2.5, Daphnia of both sexes lived longer than their infected counterparts  = 7.41, Z = 7.67, P < 0.00001, factor "Infection status × Sex", Exp(coef) = 1.37, Z = 1.035, P = 0.3). The median lifespan was reduced by about 50% in both sexes  and showed that control P. ramosa induces gigantism in its hosts in two experiments (Experiments 2 and 4). We found that infected females were larger than uninfected females  by looking for and counting spermatozoa. All adult males had spermatozoa, but infected males had significantly lower counts  with sexual size dimorphism developing from the third instar onwards. The developmental timing of the size sexual dimorphism may explain why females were infected at a higher rate than male hosts when encountering the parasite at adult stage, while infection rates were equal for exposures at an early juvenile stage. Daphnia passively capture P. ramosa spores from the water by filter feeding, and larger animals have higher filtration rates. Thus, as females grow faster and to a larger size, they may take up more spores with their food than males. A bias in parasite exposure due to sex size dimorphism has been proposed to explain part of the sex bias in infection rates in other animals [Daphnia [As in many organisms, differences in morphology and physiology between male and female  animals  and betw[Daphnia ,19. HoweD. magna lived about 1.5 times longer (median) than infected males, but the cost of the parasite on host lifespan was approximately the same in both host sexes . While these results are not direct evidence for adaptation, the observed conditions theoretically favor parasite adaptation to the female host environment [Infected female ironment .Daphnia's longer lifespan  recorded in female hosts may be explained by a combination of female  Figures  and 4, l Figures  and high Figures . Larger  Figures . Our resDaphnia have been repeatedly reported to exhibit parasite-induced gigantism, that is, enhanced body growth, and castration upon infection with P. ramosa [for example, [Daphnia-Pasteuria system, gigantism has been proposed as a parasite adaptation, and not as a host response, to increase the parasite's lifetime reproductive success, that is, the number of spores produced until host death [P. ramosa increases the host carrying capacity, which allows parasites to reach higher spore counts. A higher carrying capacity may also decrease the parasite density temporarily  but are still strongly female-biased. This allowed us to have male and female host individuals from mothers raised in the same laboratory conditions. Host clones were kept in the laboratory in standardized medium , before the differentiation of major sexual dimorphic traits. Other experiments used D. magna individuals that were three-days old, an age at which sexual dimorphism  starts to be obvious. When applicable, body length of adult Daphnia individuals was measured as the distance from the top of the head to the base of the apical spine under a dissecting microscope.We used 10 different genotypes (clones) of aturally . D. magnum (ADaM ) at 20°CP. ramosa, we used clone C19, which was originally sampled from infected D. magna females in a population in Gaarzerfeld, Germany [D. magna in 500 μL of water. Spores were then counted under phase contrast microscopy  with a hemocytometer (Neubauer improved) and diluted to the desired concentration for host exposure (see below). As control, we used placebo suspensions obtained by homogenizing uninfected Daphnia. Particular host and parasite clones used in the experiments were not coevolving, which allowed us to specifically test for the factor "sex".For the bacterial parasite  Germany . This paD. magna individuals, exposure took place in 100-mL jars filled with ADaM and lasted 11 days (4 days in 20 mL followed by 7 days in 80 mL medium) before individuals were transferred to 80 mL of clean medium. For the smaller, one-day-old individuals, exposure took place in wells of 24-well plates containing 1 mL of ADaM and lasted two days before transfer to jars with fresh medium. The infection status of D. magna at the end of the experiments was assessed by checking, with phase-contrast microscopy, single individuals homogenized in 500 μL of medium. Individuals that died before Day 14 of the experiments, largely due to handling during sorting, were excluded because detection of P. ramosa infection is less reliable during early stages of infection. An overview of the experiments carried out is given in Table Infections were performed by exposing single host individuals to suspensions of parasite spores. For the larger and sexually dimorphic three-day-old Daphnia hosts in two experiments  of parasite spores: control (placebo obtained as described above), 5,000, 12,500, 31,300 or 78,100 parasite spores per jar. At exposure, hosts were three-days old. Eleven days after exposure, Daphnia were transferred to fresh medium and 21 days after that, we inspected all individuals  for the presence of infection with the naked eye. P. ramosa infections produce very clear symptoms visible by eye. By Day 20 post infection 100% of the infected hosts show these symptoms. For Experiment 2, we exposed very young (one-day-old) animals, which do not yet show sex differences in traits, such as body size. We used 20 males and 20 females of each of 7 D. magna clones  for exposure to each of 2 doses of parasite spores: 5,000 or 20,000 spores per well. As control, we used 14 control animals per clone and sex exposed to a placebo parasite suspension. Individuals dying during the experiment were recorded daily and stored for later analysis. We stopped the experiment 120 days after exposure  and checked infection status of every individual .We tested for a difference in infection likelihood between female and male D. magna clones. All individuals dead before Day 14 of the experiment were removed from the analysis, and the six control females that were still alive at Day 120 of the experiment were censored. To estimate parasite fitness, we counted the number of spores at death for two of the seven host clones (n = 49 for Kela 08-10 and n = 46 for Kela 20-13 in Experiment 2).To measure the parasite's effect on lifespan of male and female hosts, we used longevity data collected in Experiment 2 (details above and in Table Daphnia (clone SP1-2-3) exposed to 20,000 spores when one-day-old were killed, measured and homogenized for counting parasite spores (as described above) at Day 20  or at Day 27  of the experiment. We stopped the experiment at Day 27 because approximately 50% of the males were dead after that period . Note tD. magna clones  were exposed to 30,000 P. ramosa spores for 11 days. As controls, we used 13 males and 13 females per clone exposed to a placebo suspension. Twenty-one days after exposure, we measured the body length of all individuals still alive (n = 184) and recorded their infection status.To test for parasite-induced gigantism, we measured body length of 21-day-old live infected and non-infected individuals from Experiment 2 (clones Kela 08-10 and Kela 20-13) and from an extra dedicated experiment  or to a placebo (control) suspension (n = 25 per group of the same age). The number of spermatozoa was estimated by homogenizing individuals in 50 μL of medium, and counting a subsample of this suspension using a hemocytometer (Neubauer improved). We estimated the number of spermatozoa in control and infected males at ages 13 , 16, 19, 22, 24 and 26 days. Individual males were exposed to 50 μL of 2.5% nicotine (15 minutes in the dark), which stimulates muscle contractions and results in the release of mature spermatozoa. Spermatozoa counts were performed in a total of 120 infected and 110 uninfected hosts  with a binomial error distribution, and logit link .All analyses were performed with R . To compDaphnia medium; ANOVA: Analysis of variance; GLM: generalized linear model.ADaM: Artificial The authors declare that they have no competing interests.DD, DE and PL designed the study. DE provided the materials, while DD, PL and LD performed the experiments. DD analyzed the data. DD and DE wrote the manuscript. All authors have read and approved the manuscript for publication.