Legionella, and to investigate the association between physicochemical parameters and Legionella colonization. Standardized hygiene inspection was conducted in 51 hotels, and 556 water samples were analyzed for Legionella spp. Free chlorine concentration, pH, hardness, conductivity, and trace metals were defined in cold water samples. The results of inspections and chemical analyses were associated with the microbiological results using univariate and logistic regression analysis. According to the score of the checklist used for the inspections, 17.6% of the hotels were classified as satisfactory, 15.7% as adequate, and 66.7% as unsatisfactory. Moreover, 74.5% of the hotels were colonized by Legionella spp. and 31.4% required remedial measures according to the European guidelines. Legionella spp. were isolated in 28% of the samples. Unsatisfactory results of inspections were associated with Legionella presence (relative risk (RR) = 7.67, p-value = 0.043). In hot-water systems, <50 °C temperatures increased the risk of Legionella colonization . In cold-water systems, free chlorine concentration <0.375 mg/L (odds ratio (OR) = 9.76, p-value = 0.001), pH ≥ 7.45 , and hardness ≥321 mgCaCO3/L  increased the risk, whereas copper pipes demonstrated a protective role . The majority of the hotels inspected were colonized with Legionella. Supplementary monitoring of the risk factors that were identified should be considered.This study aimed to assess the colonization of hotel water systems in central Greece and Corfu by  Legionella spp. are Gram-negative bacteria causing Legionnaires’ disease , and Pontiac fever  [Legionella infection [Legionella species and more than 70 serogroups of the microorganism have been identified [Legionella spp. is a common inhabitant of both natural and artificial aquatic environments [ humans) . The eldnfection . Currententified . Legioneronments .Legionnaires’ disease constitutes a significant public health issue worldwide. The majority of cases are sporadic and community-acquired 70%), followed by travel-associated (20%), and healthcare-associated (10%). The general case fatality rate of Legionnaires’ disease is high at approximately 10%. For hospital-acquired cases, the case fatality rate reaches and can exceed 25%, while travel-associated cases reach a 6% case fatality rate 0%, follo.In 2013, Greece had a total of 9,667 hotels, which included a total of 401,332 rooms . OftentiLegionella, one of the most frequent members of the water distribution system (WDS) microbiota [Legionella [Legionella were conducted in Greece from Alexiou et al. in 1989, Mouchtouri et al. in 2007, and finally, Fragou et al. in 2012; however, none of the aforementioned studies evaluated the influence of physical and chemical characteristics of water on Legionella presence. Similarly, the majority of the published data regarding hotel colonization with Legionella focus on hot-water systems, since these temperatures favor Legionella growth [The main reason for legionellosis in travelers can be attributed to the poor maintenance of hotel plumbing systems. The absence of regular cleaning and disinfection results in salt accumulation and biofilm formation and the subsequent colonization of crobiota ,13. Highgionella ,13. In Ggionella . Studiesa growth . A recena growth .Legionella growth. For this reason, the evaluation of cold-water systems is equally important. This study was designed in order to explore and assess associations between specific physicochemical parameters of cold-water distribution systems (WDS) and Legionella colonization. The overall aim was to identify potential risk factors and promote the implementation of preventive measures.In Greece, especially during the summer months, the water pipes in hotels are exposed to high temperatures, and the temperature of circulating water often reaches the optimum temperature for Hygiene inspections and water sampling were conducted in 51 hotels in central Greece and Corfu. Corfu was chosen because of the increase in travel-associated cases of Legionnaires’ disease as recorded by the European Legionnaires’ Disease Surveillance Network (ELDSNet) in 2011 . In the 2S2O3 × 5H2O), in order to neutralize the disinfectant action. The samples intended for physicochemical analysis were collected in sterilized 500-mL glass bottles. The sampling was representative of the water distribution system of each hotel, and included cold- and hot-water supply systems, swimming pools, and hot tubs (spas). For each sample an appropriate sampling form was filled out which denoted the date and time of the sampling, the hotel name, the sampling point, the disinfectant used, the free disinfectant concentration, the temperature of the water, and the pH measured. Cold-water samples were transferred to the laboratory under refrigeration at a maximum temperature of 6 °C, and hot-water samples at room temperature within 48 h after sampling.Overall, 556 water samples were collected between October 2011 and December 2012. In detail 496 samples were collected from showers, 36 samples from swimming pools, eight samples from taps, two samples from coolers, three samples from boilers, three samples from cold-water tanks, four samples from hot tubs, one sample from a fountain, and three water samples from cooling towers. The sampling was performed in accordance with International Organization for Standardization (ISO) 5667-5 . The samThe workflow of the methodology is demonstrated in Legionella colonization using a checklist as described by Hadjichristodoulou et al. [Hygiene inspections of each touristic accommodation were conducted in order to estimate the risk of u et al. . The iteu et al. .In the checklist, information concerning water tanks, pipe material, general information about the pressure, the filters, tank decontamination, cold- and hot-water distribution systems, boilers, water storage tanks, taps, and firefighting installations were recorded. Additional information included in the checkbook data of the accommodation, as well as information concerning measurements performed by the health personnel, were recorded (if available). A total of 39 checkpoints were evaluated. Each negative answer received a negative score, with the classification of the hotels as follows: satisfactory operation, total negative rating −7 and no critical check points were observed; adequate operation, total negative rating from −8 to −14; unsatisfactory operation, total negative rating less than −15. The checklist used in order to evaluate the hotels is included in the −1 and 10−2 dilutions were aseptically transferred in sterilized petri dishes of 92 mm. Melted medium plate count agar  at 45–50 °C were poured into the dishes. After solidification of the medium, the dishes were incubated at an ambient air temperature of 36 ± 2 °C for 44 ± 4 h; after the incubation period, all colonies from all dilutions were counted and the number of colony-forming units  and BCYE without cysteine (BCYE − cys) media. The dishes were incubated at 36 ± 1 °C for two days, and the colonies which grew on BCYE but failed to grow on BCYE − cys medium were regarded as Legionella spp. Both the species of Legionella and the serogroup were determined using a latex agglutination test (Oxoid Legionella Latex Test). The sensitivity of the method was 100 cfu/L.For all the samples collected, ications . BrieflyIn total, 158 cold-water samples were submitted to further physicochemical analysis. The following physicochemical parameters were defined for each sample: temperature, pH, free chlorine concentration, conductivity, hardness, and the content of Fe, Mg, and Zn ions.Temperature, pH, and free chlorine concentration were measured on site. The temperature of all samples collected was measured using a validated digital thermometer. The pH, as well as the free chlorine concentration, was defined using a spectrometer . Free chlorine concentration was expressed in mg/L.The conductivity of the samples was defined using an electrochemical method, and the measurements were performed with a calibrated Consort conductivity meter. The measurements were expressed in μS/cm.2+) and manganese (Mg2+). In order to define the hardness, 50 mL of the sample was placed in a conical bottle and 4 mL of NH3 solution, along with an indicator, was added. Afterwards, ethylenediaminetetraacetic acid (EDTA) solution was added until the color of the solution turned green. The volume of the EDTA solution needed was titrated and multiplied by 20 in order to calculate the value of the hardness in units of mg/L CaCO3. Respectively, to calculate the calcium concentration, 50 mL of each sample were placed in a conical bottle, together with 2 mL of NaOH solution and a Calcon indicator. Again, EDTA solution was added until the color of the solution turned blue. The volume of the EDTA solution needed was titrated and multiplied by 8.015 in order to calculate the value of calcium concentration in units of mg/L Ca units, and by 2.5 in order to calculate the value of calcium concentration in units of mg/L CaCO3.Both hardness and calcium concentration were determined using titration methods. Hardness expresses the content of the water in polyvalent cations, mainly calcium (Ca3 (0.2 mL HNO3 in 100 mL of each sample). Subsequently, each sample was absorbed and inserted as a cloud, either in a flame or in a graphite oven, where the solver is evaporated and the metals’ ions are atomized. When the atoms of the metal pass through their constant intensity beam and wave length, part of the radiation is absorbed and the reduction of the intensity is measured, with the proportionate concentration of the metal ion recorded in μg/L.Finally, in order to define the heavy metal concentration in the samples collected, atomic absorption spectroscopy  was used. Samples were filtered using a 47-mm nitrocellulose membrane with 0.45-µm pores , and acidification of the samples was performed until reaching a final concentration of 0.2% HNO®). Statistical analysis was performed using IBM SPSS Statistics software .All data collected from the study hotels (questionnaire and laboratory results) were entered into an Excel file . Qualitative variables are presented as frequencies with percentages. The receiver operating characteristic (ROC) curve analysis was conducted to determine the optimal cut-off values of the physicochemical parameters for Legionella colonization, calculating the relative risks (RRs) and the corresponding 95% confidence intervals (95% CIs). In cases where the relative risk could not be calculated due to zero frequencies, the Haldane correction was applied. In addition, Spearman’s correlation coefficient was calculated to explore any correlation between physicochemical parameters and Legionella concentration.In the univariate analysis, chi-square test or Fisher’s exact test was used to identify any association between categorical factors , conductivity, hardness, calcium, and pipe material) and Legionella colonization by calculating the odds ratios (ORs) and the corresponding 95% CIs. Legionella presence (≥100 cfu/L) was defined as the dependent variable, and the risk factors that were found to be statistically significant in the univariate analysis were defined as independent variables.In the multivariate analysis, multiple logistic regression analysis was performed using the backward conditional method to identify the independent risk factors for p-value of less than 0.05 was considered to be statistically significant.A result with a Legionella spp., while 16 hotels (31.4%) required intervention measures according to the European Working Group for Legionella Infections (EWGLI) criteria. While nine hotels (17.6%) were classified as “satisfactory operation” by the hygiene inspections and eight hotels (15.7%) were classified as “adequate operation”, 34 hotels (66.7%) were classified as “unsatisfactory operation”. The most frequent types of critical points recorded during inspection control included the following: no disinfection of the system occurred when it was not used for over a month; there was presence of stagnant water in the pipes for over a week; the flushing procedure was not applied in the case of stagnant water; there was presence of salts on the showers; regular water sampling was not performed at least every six months; the outgoing cold-water temperature was higher than 25 °C; and residual chlorine concentration was <0.2 mg/L. None of the hotels that were classified with satisfactory operation required corrective actions, whereas, in 16 out of 42 hotels (38.1%) that were classified with adequate or unsatisfactory operation, corrective measures were necessary. Specifically, an unsatisfactory result of the inspection was associated with Legionella colonization and the need for corrective intervention   were found to be colonized with = 0.043) .Legionella spp. were detected (≥100 CFU/L) in 160 out of 556 (28%) of the water samples analyzed. Specifically, 151 samples from showers, five from taps, one from a boiler, and three from hot tubs were positive for Legionella detection. The microorganism was detected in 100 out of 239 (41.8%) hot-water samples, in 57 out of 266 (21.4%) cold-water samples, and in three out of four (75%) hot-tub samples. The results of Legionella detection and the correlation with water temperature are presented in Legionella detection in temperature ranges are demonstrated in Legionella spp. detection, but the results were not statistically significant. Zn ion concentrations were similar in both positive and negative samples, as shown in Legionella spp. is also demonstrated.Concerning Fe and Mg ion concentrations, higher values were recorded in positive cold-water samples for Legionella spp. colonization of the cold-water systems, using univariate analysis. The optimal cut-off values of the physicochemical parameters which favor the colonization of the cold-water system with Legionella spp. were defined using the ROC curves. A positive association was found in samples with free chlorine concentration <0.375 mg/L, hardness ≥321 mgCaCO3/L, calcium concentration ≥150 mgCaCO3/L, TPC ≥2.5 × 104 cfu/mL, conductivity values ≥1775 μS/cm (25 °C), and finally, pH values ≥7.45. There was a negative association with colonization of Legionella spp. when the pipes of the hotel plumbing systems were made from copper.Legionella spp. regardless of other risk factors . Similarly, pH values ≥7.45 , TPC ≥2.5 × 104 cfu/mL , and hardness concentration ≥321 mgCaCO3/L  increased the risk of colonization of cold-water supply systems with Legionella. Finally, it is indicated that copper pipes demonstrate a protective role against colonization of the plumbing system with Legionella .The results from the univariate analysis mentioned above were confirmed by the multivariate analysis . SpecifiLegionella spp. were found to colonize 75% of the hotels evaluated. Previous studies in Greece demonstrated variations in Legionella colonization rates, with 86% [Legionella. Similar variations were recorded in studies conducted within other countries of the European Union. In Croatia, none of the hotels that remain open throughout the year were found to be colonized, whereas 40% of the hotels with periodic operation were colonized with the bacterium [Legionella spp. were detected in 26% of the hot-water networks of 121 residential buildings in Pisa during a three-year survey on Legionella presence [Legionella spp. [Legionella spp. concentration in water systems of hotel and health care facilities [In the present study, with 86% , 21% [14with 86%  of the hacterium . In Italpresence ,26,27,28presence , whereaspresence . Finallylla spp. . Despitecilities .Legionella spp. This implies the presence of risk factors that favor Legionella proliferation in cold-water systems as well. One of the main reasons for this could be the high environmental temperatures in the Mediterranean during the summer months, which leads to increases in the temperature of circulating water in pipes, which is optimum for Legionella growth. While the majority of studies highlighted above focus on hot-water networks, in our study, 21.4% of the cold-water samples were positive for Legionella colonization and underlines the importance of continuous record-keeping of water system data by the competent personnel [Legionella colonization and confirmed previous published data that supported the importance of maintaining disinfectant residual [4 cfu/mL, conductivity ≥1775 μS/cm (25 °C), hardness ≥321 mgCaCO3/L, and calcium concentrations ≥150 mgCaCO3/L increased the risk of colonization of cold-water supply systems with Legionella spp. Concerning the pH levels, prior studies reported that L. pneumophila has reduced viability and cultivability at higher pH levels [Legionella growth [Legionella colonization were suggested [In this study, 31.4% of the hotels evaluated needed intervention measures according to the EWGLI criteria. Concerning the hygiene inspection classification, the majority of the hotels’ WDS (66.7%) were classified as “unsatisfactory”. When a hotel was classified as adequate or unsatisfactory operation, it exhibited an almost eight times higher risk of requiring corrective intervention, whereas none of the hotels that were classified with satisfactory operation needed to undertake corrective measures. These results confirm that a satisfactory level of operation, as it is defined by the use of the checklist, could possibly be reassuring about ersonnel ,33. Concresidual ,14. Regaresidual ,36,37. OH levels ,38,39. Pa growth . Concerna growth , negativa growth , and no 75 μS/cm  °C, harduggested . AlthougLegionella, and finally, record-keeping of the aforementioned data by well-trained competent personnel is the basis of a water safety plan that every hotel must implement [Legionella. Thus, consideration of supplementary monitoring of these specific physicochemical parameters, which appear to influence the colonization and proliferation of Legionella in hotel water supply systems, could contribute to prevention of travel-associated Legionnaires’ disease.Our findings support that the implementation of control measures and the monitoring of the systems should be continuous. Checking for damage, corrosion, contamination, visible biofilm formation of the system and its parts, continuous monitoring of the water quality, periodic sampling for mplement ,34. The Legionella, further studies must be conducted in order to thoroughly comprehend and evaluate the various parameters affecting Legionella proliferation as part of the water microbiome. The present study offers a complete assessment of hotel water supply systems combining standardized inspections, risk assessment, physicochemical measurements, and microbiological analyses for Legionella of a large number of water samples. We propose consideration of supplementary monitoring of water hardness and evaluation of pipe material, which appear to influence the colonization and proliferation of Legionella in hotel WDS, in order to prevent travel-associated Legionnaires’ disease cases. The high case fatality ratio of hospitalized cases and the extensive publicity that travel-associated cases usually receive, in combination with the great importance of tourism not only to Greece but also internationally, should raise the awareness of both public health authorities and the hotel operators, in order to prevent and promptly identify cases.Although there is strong evidence that various physicochemical agents of the water influence the colonization of systems with


Current therapies for the mucopolysaccharidoses (MPS) do not effectively address skeletal and neurological manifestations. Pentosan polysulfate (PPS) is an alternative treatment strategy that has been shown to improve bone architecture, mobility, and neuroinflammation in MPS animals. The aims of this study were to a) primarily establish the safety of weekly PPS injections in attenuated MPS II, b) assess the efficacy of treatment on MPS pathology, and c) define appropriate clinical endpoints and biomarkers for future clinical trials. Subcutaneous injections were administered to three male Japanese patients for 12 weeks. Enzyme replacement therapy was continued in two of the patients while they received PPS and halted for two months in one patient before starting PPS. During treatment, one patient experienced an elevation of alanine transaminase, and another patient experienced convulsions; however, these incidences were non-cumulative and unrelated to PPS administration, respectively. Overall, the drug was well-tolerated in all patients, and no serious drug-related adverse events were noted. Generally, PPS treatment led to an increase in several parameters of shoulder range of motion and decrease of the inflammatory cytokines, MIF and TNF-α, which are potential clinical endpoints and biomarkers, respectively. Changes in urine and serum glycosaminoglycans were inconclusive. Overall, this study demonstrates the safety of using PPS in adults with MPS II and suggests the efficacy of PPS on MPS pathology with the identification of potential clinical endpoints and biomarkers. The mucopolysaccharidoses (MPS) are a group of inherited metabolic disorders caused by deficiencies of the individual lysosomal enzyme(s) needed to break down glycosaminoglycans (GAGs). GAGs are long chains of carbohydrates that serve as major components of proteoglycans present in the extracellular matrix (ECM). They function to regulate the movement of molecules through the ECM and to enhance sliding between adjacent tissue; therefore, GAGs are essential to construct all connective tissues, including bones, cartilage, tendons, and skin. However, in the MPS disorders, GAGs accumulate in cells and connective tissues over time, giving rise to progressive damage that affects physical appearance, motor abilities, organ function, and cognitive development . In totaMPS type II is an X-linked recessive disorder  caused bSeveral therapies to address the enzyme deficiencies characteristic of MPS have been developed, mainly, enzyme replacement therapy (ERT) and hematopoietic stem cell therapy (HSCT). ERT involves injecting a functional, recombinant form of the defective enzyme(s). It has been successful in alleviating some clinical manifestations of MPS  and is a®. Instead of addressing the defective enzyme, SRT aims to reduce the biosynthesis of the substrate, in this case, GAGs. Unlike ERT, SRT is theoretically non-immunogenic [Substrate reduction therapy (SRT) is an alternative approach to treating MPS, and several SRT agents are currently being assessed for therapeutic potential. These include genistein, rhodamine B, and Odiparcilunogenic . Additiounogenic ,42.Pentosan polysulfate (PPS) is a semisynthetic, heparin-like glucosaminoglycan of low molecular weight derived from beech trees that has anti-inflammatory and pro-chondrogenic properties. PPS is FDA-approved to treat interstitial cystitis. The mechanism by which treatment occurs in this disorder is not fully established, but PPS is largely thought to replace GAGs at sites of damage in the inner wall of the bladder . PPS canPPS is appealing as an agent to treat MPS since it has positive and extensive human safety data . It alsoBased on these observations, PPS treatment led to a lowering of the inflammatory cytokines, TNF-α, interleukin-8 (IL-8), and macrophage inflammatory protein-1α (MIP-1α) in several MPS animal models ,53,54, aThe primary goal of this study was to evaluate the safety of weekly injections in a small cohort of adult Japanese patients with MPS II. This study also aimed to assess the efficacy of PPS treatment on MPS pathology, specifically through the measurement of joint mobility, GAG accumulation, and levels of inflammatory cytokines.Informed consent was obtained for all individuals. The study was approved on (5 August 2014) by the ethics committee on human research at Gifu University  and followed the ethical principles of the Declaration of Helsinki. Three adult males with attenuated MPS II at 22, 34, and 37 years of age, who will be referred to as Patients 1, 2, and 3 respectively, received weekly injections of PPS for 12 weeks at an initial dosage of 0.5 mg/kg and all subsequent injections at a dosage of 1.0 mg/kg. During the trial, Patients 1 and 2 continued ERT, and for Patient 3, ERT was stopped for two months before the PPS trial. For Patients 2 and 3, injections were administered for 12 consecutive weeks; however, due to an unrelated convulsion experienced by Patient 1 after injection 6, PPS treatment was halted for two weeks and then resumed. Several assessments were performed before, during, and after the clinical trial to assess PPS injections for safety and efficacy. More details about the demographics of each patient are depicted in Blood tests were performed at the start of the clinical trial and continued weekly for 14 weeks (Week 0 to Week 14). Measurements were taken for white blood cell (WBC) count, hemoglobin, platelet count, aspartate aminotransferase (AST), alanine transaminase (ALT), prothrombin time, and activated partial thromboplastin time (APTT). Although the duration of the trial was only 3 months, the following tests were conducted before and after the clinical trial in order to assess if PPS alleviated skeletal symptoms: rating of knee joint pain as reported by the patient on a 100 mm visual analog scale of 0–10 (0 representing “no pain” and 10 representing “the most severe pain imaginable”), 6-min walk test (6MWT), 3-min stair climb test (3MSCT), knee joint cartilage ultrasonography, and shoulder range of motion (ROM) . Additionally, an electrocardiogram, echocardiogram, ultrasonic cardiography (UCG), brain magnetic resonance imaging (MRI), abdominal computer tomography (CT), auditory brainstem response (ABR) test, pulmonary function test, and visual test were conducted. In general, any adverse digestive, respiratory, cardiovascular, and neurologic symptoms were recorded if detected during the trial.Weekly urine and blood serum samples were collected from the start of the clinical trial, before the first injection, to four weeks after the last injection. The urine samples were collected at the same time of day. These blood and urine samples were then analyzed for keratan sulfate (KS), HS, and DS using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, as developed by Oguma and Tomatsu in 2007 ,61,62,63The following serum cytokines were measured before, during, and after the trial via enzyme-linked immunosorbent assay (ELISA): macrophage migration inhibitory factor (MIF), TNF-α, vascular endothelial growth factor (VEGF), IL-1β, IL-8, interleukins 10 and 18 , monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor receptor 1 (TNFR1). Measurements were taken at weeks 0, 4, 10, 14, and 17 for Patient 1 and at weeks 0, 4, 8, 12, and 16 for Patients 2 and 3. All measurements were performed in triplicates according to standard protocols using human antibody ELISA kits .Overall, PPS was well tolerated in the patients, and no serious drug-related adverse events were noted. Patient 1 experienced a convulsion between the sixth and seventh injections. Follow-up electroencephalography (EEG) was conducted and showed mild abnormality with paroxysmal discharge, indicating that the convulsion was likely due to epilepsy. This was the second convulsion that Patient 1 experienced in his life, the first of which occurred approximately five months before the clinical trial began. Thus, it was concluded that the convulsion was not PPS-related. Additionally, Patient 3 experienced a mild elevation of ALT from weeks 6 to 11. At week 6, ALT levels were detected slightly above the normal range (5 IU/L above a range of 5 to 40 IU/L); however, the levels decreased over time and were close to baseline by week 13.No other adverse effects were detected, except for mild pain and subcutaneous hemorrhage that came with injection. Such effects were temporary and disappeared within two to three days and one to two weeks, respectively. No vomiting, diarrhea, coughing, difficulty of breathing, tachycardia, or chest pain was detected within the duration of the experiment. There were no changes in electrocardiograms for any of the patients. Left atrial to aortic root ratio decreased slightly for Patient 1 and ejection fraction decreased slightly for Patients 1 and 2. According to pulmonary function test results, the overall severity of pulmonary impairment did not change with treatment. Additionally, there were no changes in hearing and visual tests, brain MRI, or abdominal CT scan with treatment. All three patients had retinitis pigmentosa and did not have hepatosplenomegaly before and after the trial. Further details of the tests assessed for adverse effects are depicted in the Although the main objective of this study was to assess the safety of PPS injections in adult MPS II patients over three months, some clinical/pathological parameters were monitored for changes. Patient 1 experienced knee pain and irregularity of knee joint cartilage (as evidenced by ultrasonography) before the initiation of PPS treatment. After the three-month trial, the pain rating decreased slightly from 3 to 1 , and cartilage irregularity decreased slightly, as indicated by ultrasound examination. Patients 2 and 3 did not experience any knee pain or cartilage irregularity before the trial, and this did not change with PPS treatment.Additionally, right and left shoulder flexion increased for Patients 1 and 2, and there was no change for Patient 3. Right and left shoulder extension decreased for Patient 2, and there was no change for Patients 1 and 3. For shoulder abduction, there was an increase observed for the right shoulder of Patient 1 and for both shoulders of Patient 2. There was also an increase in adduction for both shoulders for Patients 1 and 2. The degree of outer rotation decreased for both shoulders in Patient 1 and increased in the left shoulder for Patient 2. The degree of inner rotation increased for the left shoulder of Patient 1 and both shoulders for Patient 2. There was no improvement in ROM for any of these parameters in Patient 3, who was not under ERT during PPS administration. For the 6MWT and 3MSCT, there was a slight decrease in scores for Patients 1 and 2. Measurements could not be obtained for Patient 3 . Measures of these parameters are depicted in Overall, large ranges of values were observed for urinary GAGs. No clear trends were observed in all three patients. Additionally, it could not be concluded what kind of change, if any, was induced by PPS treatment, as urinary GAG levels underwent dramatic fluctuations before and after the period of treatment.Di-4S values had an average range of 246.3% (times baseline) across the three patients. For Patient 1, the change from before PPS treatment to one week after treatment was negative, and for Patients 2 and 3, Di-4S increased with treatment. Across the three patients, DiHS-0S and DiHS-NS values had an average range of 147% and 167.3%, respectively. Similar to Di-4S, HS disaccharides underwent a negative percent change for Patient 1 and a positive percent change for Patients 2 and 3. The average concentration range of monosulfated keratan sulfate (KS) and disulfated KS was 180.3% and 246.3%, respectively. During PPS treatment in Patient 1, disulfated KS underwent a negative percent change, and monosulfated KS underwent a positive percent change. In Patient 2, both monosulfated KS and disulfated KS increased with PPS treatment. In Patient 3, these disaccharides decreased with treatment. For all three patients, urinary GAGs underwent dramatic fluctuations after PPS was discontinued.For Patients 1 and 2, Di-4S was within mean + 2SD  at baseline; for Patient 3, Di-4S was above the normal range. These observations did not change with the PPS administration. In Patients 1 and 3, monosulfated KS stayed above and within the normal range, respectively, with treatment. Before, during, and after treatment, DiHS-NS and DiHS-0S was measured to be above the normal range in Patients 1 and 3. For Patient 2, DiHS-0S started within the normal range but was elevated with treatment . The valAs with urinary GAGs, large ranges of values were observed for serum GAGs. No clear trends were observed in all three patients. Additionally, it could not be concluded what kind of change, if any, was induced by PPS treatment, as serum GAG levels underwent dramatic fluctuations before and after the period of treatment.Di-4S values had an average range of 130.3% (times baseline) across the three patients. For Patient 1, the change from before PPS treatment to one week after treatment was positive, and for Patients 2 and 3, Di-4S decreased with treatment. Across the three patients, DiHS-0S and DiHS-NS values had an average range of 91% and 107.7%, respectively. In Patient 2, the percent change from before treatment to one week after the last PPS injection was positive for DiHS-0S and negative for DiHS-NS. HS disaccharides underwent a decrease in Patient 1 and an increase in Patient 3. The average concentration range of monosulfated KS and disulfated KS was 82.3% and 181%, respectively. In Patients 1 and 2, disulfated KS underwent a negative percent change, and monosulfated KS underwent a positive percent change with treatment. Conversely, in Patient 3, monosulfated KS underwent a positive percent change, and disulfated KS underwent a negative percent change. For all three patients, serum GAGs underwent dramatic fluctuations after PPS was discontinued. In general, serum GAGs were initially measured within the normal range, and this was kept during and after treatment. There was, however, an exception observed for monosulfated KS: monosulfated KS was measured above the normal range at baseline, and this did not change with treatment . Results3 pg/mL), a trend towards reduction was also noted in the patients with PPS treatment. Again, Patient 2 showed the clearest response. For all other inflammatory cytokines measured,  have been identified previously in attenuated MPS II [p.Arg443Ter is a nonsense mutation in a CpG hot-spot of exon 9, and it involves an arginine residue [p.Asp308Asn and p.Cys171Arg are missense mutations, the former is present in exon 7 [The primary aim of this study was to evaluate the safety of weekly subcutaneous PPS administration in adult MPS II patients and to define clinical endpoints and biomarkers for further clinical trials. All patients in the study had attenuated MPS II. As listed in d MPS II ,66,67,68 residue . Both p.n exon 7 ,66,67,68Importantly, as in the previous six-month clinical study of attenuated MPS I patients, this study demonstrated an excellent safety profile of PPS in MPS II. As PPS is mainly processed in the liver and can be a weak anti-thrombotic agent , potentiGiven the very short time frame of the study (three months), we did not expect to observe clinical or pathologic improvements in the patients, and patients were not enrolled in the study based on the extent of their clinical or pathologic disabilities. Nonetheless, we did monitor several of these parameters throughout the study. For example, several parameters of skeletal pathology were assessed to determine the efficacy of PPS. Patient 1 experienced decreased knee pain and cartilage irregularity. In Patients 1 and 2, shoulder flexion, abduction, adduction, and inner rotation increased by a range of 6° to 14°, 30° to 68°, 17° to 32°, and 41° to 75°, respectively. However, these changes went unnoticed by the patients themselves, and all other parameters of shoulder ROM did not change or improve. Additionally, there were no improvements in the other tests for mobility (6MWT and 3SCT). Therefore, the findings suggest that PPS had relatively mild efficacy on MPS skeletal pathology during this three-month period. The results also suggest that this efficacy is strengthened when combined with ERT, as Patient 3, who remained off of ERT during PPS administration, experienced less improvement than Patients 1 and 2. It must be cautioned, however, that this was a very short duration-study in only three patients, and more clinical studies are required to assess efficacy.Although there were mild improvements in skeletal pathology, changes in urine and serum glycosaminoglycans were inconclusive. No common trends were detected across the subjects of the study. Within both the period of treatment and recovery from treatment, patients experienced a great range of GAG levels with respect to baseline. Furthermore, there was often significant variation detected between consecutive time points. These two factors made it difficult to conclude whether a specific GAG (serum or urine) increased or decreased with treatment. This is a notable limitation of the study and is most likely due to the short time frame in which GAG levels were measured and/or individual variability that is particularly emphasized due to the small sample size. As the hallmark of MPS is GAG storage and excretion, and GAG tissue, blood, and urine levels have been used as a biomarker to assess therapeutic efficacy, future studies should utilize a longer treatment and recovery time to allow for the possible action of PPS to take full effect. In addition, a larger sample with wild-type controls should be used to conduct more in-depth statistical analysis when determining the effect of PPS on this parameter of MPS pathology.Hennermann et al. previously evaluated the effects of PPS injections (at 1 or 2 mg/kg) on four adult patients with MPS I treated for six months in combination with ERT . It was The anti-inflammatory effects of PPS have been extensively documented, and broad anti-inflammatory effects were previously studied in three MPS animal models ,53,54,56The mechanism by which inflammation occurs in MPS has not been fully elucidated, and a hierarchy of relevant cytokines has not yet been fully determined. Out of cytokines studied here, only TNF-α has been implicated in MPS. In the context of the disease, TNF-α contributes greatly to bone pathology . MIF is ®, but clinical trials are being conducted by Inventiva, testing the agent’s potential as a treatment for MPS VI. Perhaps the most prevalent of the available substrate reduction agents is genistein, which works by regulating expression of GAG synthesis genes [Currently, several other agents are being tested for the treatment of MPS, in addition to PPS and ERT. For example, treatment with rhodamine B led to an improvement in behavioral tests for MPS IIIA  and MPS is genes ,76; geniis genes . Similaris genes ,42.Among the various experimental therapies being studied in MPS, PPS is somewhat unique since it not only reduces GAG storage but also significantly reduces inflammatory cytokines in MPS animal models. It also appears to cross the blood–brain barrier, although this has only been studied in the context of one neurological MPS model (MPS IIIA) . There iIn conclusion, this study established the safety and analyzed the potential effectiveness of PPS treatment in three adult male patients with MPS II. There was no serious adverse effect noted throughout PPS treatment, indicating that weekly PPS treatment can be safely used in adults with MPS II. Furthermore, this study has shown that PPS treatment may decrease pro-inflammatory cytokines that are implicated in MPS physiology. Further research should be conducted to fully elucidate the mechanism by which PPS works to decrease inflammation and to confirm its treatment efficacy as a GAG-reducing agent. In the future, testing should be performed over a longer course of time and with a broader range of subjects, including in children, as early intervention is critical in MPS.


ETS2 and ZNF408 genes, respectively. Compound heterozygosity was identified in CENPF and TTN genes. Existing knowledge on the genes and bioinformatics analyses suggest that EST2, ZNF408, and CENPF might be promising candidates for ID causative genes.Although it has been a half-century since dioxin-contaminated herbicides were used to defoliate the landscape during the Vietnam War, dioxin contamination “hotspots” still remain in Vietnam. Environmental and health impacts of these hotspots need to be evaluated. Intellectual disability (ID) is one of the diseases found in the children of people exposed to the herbicides. This study aims to identify genetic alterations of a patient whose family lived in a dioxin hotspot. The patient’s father had a highly elevated dioxin concentration. He was affected with undiagnosed moderate ID. To analyze de novo mutations and genetic variations, and to identify causal gene(s) for ID, we performed whole genome sequencing (WGS) of the proband and his parents. Two de novo missense mutations were detected, each one in  From 1962 to 1971, about 19 million gallons of Agent Orange (AO) and other toxic chemicals contaminated with very high concentrations of dioxin were sprayed in South Vietnam [Although it has been a half-century since the end of the Vietnam War, very high dioxin concentrations still exist in the areas of former military airbases, such as Bien Hoa, Da Nang, and Phu Cat, where the toxic chemicals were stored and used for sprays ,4. TheseDioxin is one of the most toxic compounds that can cause reproductive and developmental problems as well as cancers. In Vietnam and the United States, birth defects were observed in children of persons exposed to dioxins , suggestDe novo mutations (DNM) occur as single nucleotide variants (SNVs), short insertions/deletions (indel), copy number variations (CNVs), or structural variations (SVs), and are shown to contribute significantly to sporadic genetic disorders, such as ID  and autiOver the last few years, many genetic investigations of ID have been carried out using next-generation sequencing approaches ,18. WGS We performed WGS of a Vietnamese family living in a dioxin contamination hotspot, where the father had a highly elevated dioxin concentration and the son was affected with an undiagnosed moderate ID, in order to identify variants which might be contributing factors in the development of the ID.A family from Bien Hoa city, Dong Nai province, Vietnam, one of dioxin contamination hotspots, was recruited through Vietnam Military Medical University and the Institute of Genome Research after obtaining informed consent. The couple lived in this location since 1975. The father was detected to have a highly elevated dioxin concentration, but the family was generally healthy, although their son, born in 1989, was affected with moderate ID. They had no other relatives with cognitive defects. The patient’s symptoms of cognitive impairment were recognized at 11 months of age. He went to school for a few years but never learned to read and write. His level of ID was in the moderate (IQ < 50) range. He was independent in self-care, such as eating, drinking, and toileting, but was unsociable. He had to be supervised when he went out. He had other signs and symptoms of health problems, such as an asymmetric body, hypotonia, and moderate twitching.Neurological and medical assessment of the affected member was performed by clinicians of Vietnam Military Medical University. All biological samples in this study were approved by the Institutional Review Board (IRB) of Hanoi Medical University, Hanoi, Vietnam (no. 123/HMU IRB), and RIKEN, Japan. The blood samples, ~30 mL from the father and ~1–2 mL from the mother and son, were collected, frozen, and stored at −80 °C until used. For the dioxin content analysis of the father’s blood serum, potassium dichromate was added to a ~15 mL blood sample, just before delivery to the ERGO Laboratory, Hamburg, Germany, to perform high resolution mass spectrometry (HR-MS) analyses. The sample was tested for seven PCDDs and ten PCDFs according to the protocol of Schecter et al. .Five hundred base pair (bp) insert libraries were prepared according to the protocol provided by Illumina and sequenced on the HiSeq2000 platform  with paired reads of 101 bp.BWA.v0.59  was usedIn addition to de novo mutations, we also searched for mutations consistent with autosomal recessive model by focusing on non-synonymous SNVs, coding indels, and variants in splice sites. Prediction of the possible impact of the amino acid substitution on the structure and function was performed using PolyPhen-2 , ProveanIn this study, the total toxic equivalency value (TEQ) of dioxin and dioxin-like compounds in the serum of the father was measured as ~115 ppt while the TCDD and PeCDD were ~87 and ~12 ppt, respectively. These two very toxic congeners consisted of ~86% of the total TEQ. All of the other 15 remaining congeners constituted only ~16 ppt, or ~14% of TEQ . The whole genomes of the three individuals  were sequenced with average ≥ 30× coverage. The total number of SNVs identified in the father, the mother, and the son were 3,436,963, 3,698,813, and 3,569,445, respectively . MeanwhiAfter filtering for SNVs present in the dbSNP version 138 and the 1000 Genomes database , the remETS2 and ZNF480 genes (ETS2: NM_001256295.1:c.485C>T (p.T22I) and ZNF480: NM_144684.2:c.1504C>T (p.R502W)) (Ninety-three de novo heterozygous variants were identified in the proband . Of thes.R502W)) , Table 3CENPF (p.P791A and p.C1307Y) (TTN (p.P13274S and p.K8548N) genes   and TTN N) genes . In addieletions .TCDD and PeCDD are two of the most toxic chemicals of PCDDs. TCDD is widely known to come from 2,4,5-trichlorophenoxyacetic acid  herbicide, while PeCDD is believed to derive from 2,4-dichlorophenoxyacetic acid  herbicide, produced in the middle of 20th century . From 19The TCDD level of the father’s blood in this study was 87 ppt and contributed to 75% of the total TEQ, which was in the range of these estimations and much higher than those of reported normal blood TCDD levels in the South Vietnam [−8 variants per nucleotide per generation which is consistent with previous reports, ranging from 1.1 × 10−8 to 3.8 × 10−8 variant per nucleotide per generation [Our analysis detected 93 de novo heterozygous variants in the proband. The number of variants corresponds to a rate of 1.42 × 10neration ,31,32,33neration .ETS2 and ZNF480 genes. Compound heterozygous variants were detected in CENPF and TTN genes. The homozygous CNVs, which were hemizygous in the parents, contained NBPF25P, KLRC1, and KLRC2 genes. Variants in the TTN gene were found with low frequency in East Asian population (0.0015 and 5.8 × 10−5) reported in ExAC database, while NBPF25P is a pseudogene. The KLRC1 and KLRC2 genes encode killer cell lectin-like receptor C1/2. Therefore, we considered that they are unlikely to be causal gene(s) of ID, and focused on ETS2, ZNF480, and CENPF genes.In an attempt to identify possible causal gene(s) for ID, we analyzed genes with de novo variants, CNVs, and germline variations. De novo non-synonymous variants were found in ETS2 gene encodes a transcription factor that regulates numerous genes and is overexpressed in the brain and fibroblasts of Down syndrome (DS) individuals. ETS2 can trans-activate the APP gene via specific Ets-binding sites in the APP promoter, resulting in the increase of beta-amyloid production in patients with DS [ETS2 transactivation domain located in the N-terminal part of the protein has been shown to affect its activity [ETS2, resulting in p.T22I at the N-terminus of the protein, which might alter protein translocation. The  with DS . Mutatioactivity . Our stuZNF480 gene belongs to a large family of transcription factors [ZNF480 in schizophrenia patients, and variants in other C2H2-type zinc finger proteins have been reported to be associated with ID [ZNF480 is very limited, the results suggest that ZNF480 can be a candidate for a causal gene for ID in the patient.The  factors , which a factors . We iden with ID ,38. AlthCENPF, which encodes the kinetochore protein expressed during the cell cycle [CENPF, leading to truncated proteins, in patients with ciliopathy and microcephaly phenotypes [CENPF gene (−6 in the Exome Aggregation Consortium database. The variation p.C1307Y is predicted to have an effect on protein function by Polyphen-2 (score = 1.00) and Provean (score = −4.22) programs. The very low frequencies in the general populations and the predicted functional effect of the variants suggest that the CENPF gene is a promising candidate gene for ID.Applying a recessive model, we identified a compound heterozygous variant in ll cycle  and in denotypes . In our NPF gene . The NM_ETS2 and ZNF480 genes and the compound heterozygous variations in CENPF in the son with an undiagnosed ID. Considering the functional roles of these genes and previous studies on mental diseases, they might be promising candidates for further studies performing functional analyses.In this study, we presented our comprehensive analysis of genetic variations and de novo variants in a family whose father had up to 115 TEQ ppt in his serum as a result of living in a dioxin hotspot. We identified de novo variants in


Oryza longistaminata, written by Kanako Bessho-Uehara, Jovano Erris Nugroho, Hirono Kondo, Rosalyn B. Angeles-Shim, Motoyuki Ashikari, was originally published electronically on the publisher’s internet portal (currently SpringerLink) on 8 May 2018 without open access.The article Sucrose affects the developmental transition of rhizomes in With the author(s)’ decision to opt for Open Choice the copyright of the article changed on 5 May 2019 to © The Author(s) 2019 and the article is forthwith distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, duplication, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.The original article was corrected.


Internet- and mobile-based interventions are effective for the treatment of chronic pain. However, little is known about patients’ willingness to engage with these types of interventions and how the uptake of such interventions can be improved.The aim of this study was to identify people’s acceptance, uptake, and adherence (primary outcomes) with regard to an internet- and mobile-based intervention for chronic pain and the influence of an information video as an acceptance-facilitating intervention (AFI).In this randomized controlled trial with a parallel design, we invited 489 individuals with chronic pain to participate in a Web-based survey assessing the acceptance of internet- and mobile-based interventions with the offer to receive an unguided internet- and mobile-based intervention for chronic pain after completion. Two versions of the Web-based survey (with and without AFI) were randomly sent to two groups: one with AFI (n=245) and one without AFI (n=244). Participants who completed the Web-based survey with or without AFI entered the intervention group or the control group, respectively. In the survey, the individuals’ acceptance of pain interventions, measured with a 4-item scale (sum score ranging from 4 to 20), predictors of acceptance, sociodemographic and pain-related variables, and physical and emotional functioning were assessed. Uptake rates (log in to the intervention) and adherence (number of completed modules) to the intervention was assessed 4 months after intervention access. To examine which factors influence acceptance, uptake rate, and adherence in the internet- and mobile-based interventions, we conducted additional exploratory subgroup analyses.P=.64, P=.56, P=.75, respectively). Most participants reported moderate  to high  acceptance, with 9.6% (11/115) showing low acceptance . Further, 67%  and 62%  had logged into the intervention. In both groups, an average of 1.04 (SD 1.51) and 1.14 (SD 1.90) modules were completed, respectively.In total, 57 (intervention group) and 58 (control group) participants in each group completed the survey and were included in the analyses. The groups did not differ with regard to acceptance, uptake rate, or adherence  Chronic pain as a disease in its own right is a major global health problem -3. In thd=0.58 at posttreatment and follow-up, respectively )We used the Patient Health Questionnaire depression scale PHQ-8 -64) for  for 64])PHQ-8 assesses all Diagnostic and Statistical Manual of Mental Disorders, fifth edition (DSM-V) symptoms of major depression with the exception of suicidal or self-injurious thoughts during the last 2 weeks. Ratings are given on a 4-point Likert scale ranging from 0 ‘‘not at all’’ to 3 ‘‘nearly every day.’’ The scores for each item are summed up to produce a total score between 0 and 24 points. A cutoff score of 5-9 represents mild depressive symptoms; 10-14, moderate; 15-19, moderately severe; and 20-24, severe . The CroGAD-7 consists of 7 core symptoms of the DSM-V diagnostic criteria A, B, and C for generalized anxiety . The iteWe measured internet usage using the question ‘‘How often do you surf the internet?’’ Answers are rated on a 5-point Likert scale ranging from 1 ‘‘seldom or never’’ to 5 ‘‘multiple times per day.”AFI consisted of a 3-minute introductory and information video to ACTonPain with screenshots of the program in order to improve patients’ acceptance. After completing the questionnaire, participants were invited to receive ACTonPain treatment in an unguided version and without short message service (SMS) text messages (SMS Coach). This version of ACTonPain was provided without any human support and should be therefore of special interest for public health services due to its high scalability and low costs.ACTonPain consists of an introduction and 7 consecutive modules. The intervention targets core change processes proposed by Hayes et al  and is dThe effectiveness of ACTonPain was investigated in a three-armed RCT with a total of 302 participants who were randomly assigned to either ACTonPain guided, ACTonPain unguided, or waitlist control. Guidance was given by trained eCoaches (psychologists) who provided individualized standardized feedback after each module and reminded the participants to keep to the schedule of the treatment and set up deadlines. Additionally, participants could receive supportive SMS text messages (SMS Coach).The primary outcomes were acceptance, uptake, and adherence.We operationalized acceptance on the basis of the well-established unified theory of acceptance and use of technology UTAUT ,68). Thi. Thi68])The items of the UTAUT acceptance were developed based on previous studies ,47. The We operationalized uptake as log-in (yes or no) to IMI assessed 4 months after intervention access. The period of 4 months was chosen, as this should have been enough time for the participants to start with the intervention and work through all 8 modules. We assumed that 4 months after intervention access is a reasonable time to assess uptake and adherence.We operationalized adherence as the number of completed modules of the intervention assessed 4 months after intervention access.The secondary outcomes were the predictors of acceptance according to UTAUT as well as internet anxiety.According to the UTAUT model, there are 4 key predictors of either the behavioral intention or usage behavior of IT: performance expectancy, effort expectancy, social influence, and facilitating conditions . The iteTwo items for internet anxiety were adapted from Venkatesh et al  (1) “Thet-tests for independent samples. The descriptive statistics were based on nonimputed data, while all following analyses were conducted after multiple imputations with 20 imputations using the imputation algorithm implemented in SPSS .We conducted data analysis using SPSS Statistics 22 . As only those participants who completed the Web-based survey were included in the analysis, there were no missing data in this study. Descriptive statistics are provided for sociodemographic data and functioning to describe the sample. To test for randomization imbalance between IG and CG, we employed chi-square tests and t-tests for independent samples and chi-square tests. In case of significant group differences, standardized mean differences (Cohen d) with a 95% CI were computed to quantify the effect. As this study includes multiple primary outcomes, we used a Bonferroni adjustment for the P values of .02 . This procedure resulted in sufficient statistical power with the sample to detect differences between the two conditions that were larger than Cohen d=0.65.To detect differences between IG and CG regarding acceptance, uptake, adherence, and the predictors of acceptance as well as internet anxiety, we conducted t-tests, and chi-square test). For this purpose, variables were dichotomized using defined cutoffs  or a median split . Note that the results of the subgroup analyses and analysis on secondary outcomes are exploratory and underpowered; adjusting for multiple testing would not be meaningful [To examine potential subgroup differences  regarding acceptance, uptake, and adherence, exploratory analyses are provided .Of 489 persons, 141 (28.8%) responded to the invitation. After we excluded those who did not provide informed consent (n=22) or did not fulfill the inclusion criteria (n=4), we included 57 and 58 participants in IG and CG, respectively. The missing value was between 0% and 5.7% per variable, and Little’s Missing Completely at Random test indicated that the data were missing at random  of the participants were female. Ages ranged from 18 to 76 years with a mean age of 50.42 (SD 13.67) years. The majority of the sample  reported a pain duration of longer than 2 years, with 57.4% (66/115) suffering for more than 5 years. In addition, 86.1% (99/115) and 75.7% (87/115) of the participants reported at least mild symptomatology of depression and anxiety, respectively. P=.64, P=.56, P=.75, respectively). Among the total sample, 8.7% (10/115) showed a low, 59.1% (68/115) a moderate, and 31.3% (36/115) a high level of acceptance, with an average sum score of 13.76 (SD 3.54). There was no significant difference between IG and CG with regard to acceptance, uptake, or adherence (Note that 9% (5/57) and 10% (6/58) of participants in IG and CG, respectively, did not complete the survey and therefore did not indicate whether they wanted to receive the intervention. Then, 7% (4/57) and 3% (2/58) of participants in IG and CG, respectively, did not want to receive the intervention, and 84% (48/57) and 86% (50/58) of participants in IG and CG, respectively, signed up to receive the intervention. Four months after receiving access to ACTonPain, 65.2% (75/115) of the sample had logged in. This represents an uptake rate of 68%  and 62% . With regard to adherence, the participants completed 1.09 (SD 1.72) modules on average. That is, the average participant only completed the introduction module. The results showed that 5.2% (6/115) participants did not complete any modules after log-in and 3.5% (4/115) completed all the modules in the study. Hence, the treatment dropout rate was at 96.5% (111/115). P=.88, P=.16, P=.96, P=.69, P=.68, respectively; There was no significant difference between IG and CG with regard to performance expectancy, effort expectancy, social influence, facilitating conditions, or internet anxiety  with no group differences between IG and CG. This acceptance level is higher than the levels examined in equivalent previous studies ,46,47. IThe comparatively high acceptance in both groups of this study is potentially due to selective sampling. We recruited the participants from a pool of persons who had already expressed interest in participating in a previous study on ACTonPain. After the end of recruitment for the main study, we invited all persons who were not randomized in the study to participate in this study and to receive ACTonPain as an incentive after completion of the survey. Hence, the participants in this study expressed their interest for ACTonPain twice. Therefore, the level of acceptance most likely reflects the acceptance and uptake in many IMI efficacy studies consisting of a population that is considerably more interested and open to IMIs than the general population . TherefoDespite the high level of acceptance, the uptake rate was only moderate at 65.2%. In comparison, the uptake rate in the main evaluation of ACTonPain ,56 was aIn conclusion, AFI had no effects in a sample with high initial acceptance. This is in line with the assumptions of the Health Action Process Approach (HAPA ), which Applying the assumptions of HAPA to our sample, an AFI might be the wrong means to increase intervention uptake and adherence. The participants showed high intentions to use an IMI, which means they are classified as intenders in the sense of HAPA. Instead of an AFI, which provides information on efficacy, data security, etc, our participants might have profited more from concrete action and coping planning, action control, and social support. This assumption is supported by a recent study of Zarski et al , where pWith most participants only completing the introduction module, adherence is substantially lower in this study than in the evaluation study of the exact same intervention ,56. TherThe different findings concerning adherence and dropout not only highlight the influence of guidance and SMS but also the importance of the setting in which participants receive IMIs. Guidance and prompts  are two of the most investigated adherence facilitating factors in the research on IMIs, ultimately increasing the effect of the respective IMI ,82-87. HIn addition to the abovementioned high baseline acceptance, the rather general content of AFI might also explain why AFI was not effective in this study. As discussed in a previous study on acceptance of IMIs in patients with diabetes , AFIs taSeveral limitations in this study are noteworthy. First, the recruiting strategy might have influenced the way the participants filled out the survey, and their answers might have been more socially desirable. Consequently, the results on acceptance and uptake might not be representative for the population of patients with chronic pain, but they are likely to be representative for the population of patients with chronic pain in previous efficacy trials on IMIs for chronic pain. Hence, this study provides information on participants’ acceptance in efficacy studies that can be useful for the interpretation of their respective results. This is especially the case regarding their generalizability to routine clinical practice given that most of these studies are conducted under ideal circumstances with highly specified inclusion and exclusion criteria .In connection with the abovementioned lack of implementation facilitating factors in our AFI, a further limitation of this study is that it is only based on the UTAUT model. The UTAUT model and other equivalent models on the acceptance of IMIs as evaluated in a previous study , as wellFinally, the reliability of the acceptance scale was relatively low at .71 compared with previous studies . HowOverall, this study yields evidence that patients’ uptake and adherence to an IMI for chronic pain is low, despite high acceptance. The first main contribution of this study is that it shows how acceptance rates can differ between a sample participating in an IMI efficacy trial as represented in this study and a sample collected from a routine health care setting, represented in our previous studies ,47,66. TThis study also indicates that high acceptance does not guarantee sustainable use of IMIs. Further models, such as the HAPA model, need to be used in order to develop strategies to increase adherence in IMIs. Equivalent to the discussion on acceptance rates, the different settings of efficacy trials and routine health care settings appear to play a crucial role for adherence in IMIs. This might explain the high discrepancy between adherence in this study and the evaluation study of the same intervention ,56, as wIn conclusion, this study shows that acceptance can be much higher in a sample participating in an IMI efficacy trial than in the target population in routine health care settings. Therefore, future research should be conducted within naturalistic settings with more representative samples. Further, strategies to increase adherence in IMIs need to be developed involving IMI users, developers, and providers.


Large-scale distributed watershed models are data-intensive, and preparing them consumes most of the research resources. We prepared high-resolution global databases of soil, landuse, actual evapotranspiration (AET), and historical and future weather databases that could serve as standard inputs in Soil and Water Assessment Tool (SWAT) models. The data include two global soil maps and their associated databases calculated with a large number of pedotransfer functions, two landuse maps and their correspondence with SWAT’s database, historical and future daily temperature and precipitation data from five IPCC models with four scenarios; and finally, global monthly AET data. Weather data are 0.5° global grids text-formatted for direct use in SWAT models. The AET data is formatted for use in SWAT-CUP  for calibration of SWAT models. The use of these global databases for SWAT models can speed up the model building by 75–80% and are extremely valuable in areas with limited or no physical data. Furthermore, they can facilitate the comparison of model results in different parts of the world. Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.10043090 SWAT is a continuous-time, semi-distributed, and process-based model, which includes coupled upland and river processes. The land phase of SWAT includes hydrology, soil erosion, crop growth, nutrient cycling, algae transport, pesticide fate and transport, crop management, water transfer, snowfall and snowmelt, and soil temperature. The routing phase in the channels and rivers include processes such as flood routing, sediment routing, nutrient routing, pesticide routing, and routing in the reservoirs. The model is now being upgraded and restructured to SWAT+, and also coupled to glacier melt, heavy metal fate and transport, and other watershed-related processes.Soil and Water Assessment Tool (SWAT)2. SWAT models are used in a variety of applications, including quantification of water resources availability10, the impact of climate and landuse changes16, soil erosion19, water quality25, and ecosystem services28. SWAT also contains a modified version of the Environmental Policy Integrated Climate (EPIC) model30 for crop yield simulation31. In total, more than 4,500 ISI publications can be found using SWAT on various watershed-related issues and ecosystem services around the world, which is by far the most extensive collection of such literature in the world with an average of 550 peer-reviewed publications per year in the last 4 years .SWAT is the most widely used hydrological and water quality model in the world32. Next to model uncertainty, we have previously used the term conditionality34 to describe another constraint to a so-called calibrated model. All calibrated model parameters are uniquely conditioned on model assumptions, model structure, input data, as well as calibration data, calibration routines, and objective function definition. A calibration program, SWAT-CUP 35, was developed for the calibration of SWAT models. SWAT-CUP provides five different calibration routines and the option of choosing between 11 different objective functions. We have previously shown that the choice of different routines and objective functions lead to different parameters while producing equally acceptable calibration results37. It would be desirable to always obtain unconditional model parameters independent of calibration procedures and objective functions. For this reason, in the new version of the program, we have provided an option for multi-objective calibration, which provides an option of choosing any combination of the objective functions.Lack of data in many parts of the world is a severe impediment to hydrologic modeling. At the same time, much data generated on the global and local scales is also posing a modeling problem creating an additional source of uncertainty. Previous works have shown that the use of different databases for the same region leads to different model outputs and, consequently, different water resources estimates and different estimates of ecosystem variablesFurthermore, data processing and formatting of data for different applications are highly time-consuming and prone to errors, resulting in much of the research time to be spent on data preparation instead of modeling application and analyses. For this reason, we have put together global soil, landuse, and historical and future weather databases for use in SWAT and other similar watershed models Table  as descr38. These functions estimate parameters that are difficult to measure using easily measured soil properties such as texture, color, and structure, that are routinely recorded by soil surveyors39.There is a general lack of reliable soil information for many parts of the world, which has significantly disadvantaged evaluation of soil erosion, land degradation, environmental impact studies, and sustainable land management programs. Two highly-used global soil maps are the FAO/UNESCO Soil Map of the World and Harmonized World Soil Database (HWSD_v121). Both maps provide a limited description of parameters, which are not directly useful for hydrologic models. We have, therefore, used pedotransfer functions developed from soils around the world to create the needed parameters such as hydraulic conductivity, available water capacity, and bulk density. Pedotransfer functions “translate data we have into data we need”usersoil table in the main SWAT database “SWAT2012.mdb”.The FAO/UNESCO soil map of the world was prepared using the topographic map series of the American Geographical Society of New York at a nominal scale of 1:5,000,000 consisting of a 30 cm topsoil layer, and a 70 cm subsoil layer  and FAO Soil-ID  to give Af14-3C-1. Soil hydrologic groups were determined according to SWAT Manual40 based on the criteria in Supplementary Table 40. The potential or maximum crack volume of the soil profile (SOL_CRK) expressed as a fraction of the total soil volume was set to zero as there was no information available to evaluate this parameter. Other soil properties have initially been calculated10 using the program ROSETTA41. In the current study, we have updated this database using a large number of pedotransfer functions, as described below.Initially, in 2004, the first author created the soil database for the FAO/UNESCO 1995 soil map for quantification of water availability and quality in AfricaThe Food and Agriculture Organization of the United Nations (FAO) and the International Institute for Applied Systems Analysis (IIASA) combined the available regional and national soil information with the data already contained within the 1:5,000,000 scale FAO-UNESCO map, into a new comprehensive Harmonized World Soil Database (HWSD_v121). This map has a resolution of about 1 km (30 arc seconds) and consists of a 30-cm topsoil layer, and a 70-cm subsoil layer , organic carbon, pH, and electrical conductivity (EC). However, from a hydrological point of view, we require parameters such as bulk density, water storage capacity, and hydraulic conductivity for different soil layers, which we used pedotransfer functions to estimate. We estimated soil bulk density  database in Supplementary Table The GLCC from USGS is a landuse and land cover classification dataset based primarily on the unsupervised classification of the 1-km AVHRR (Advanced Very High-Resolution Radiometer) 10-day NDVI  composites  database in Supplementary Table The GlobCover is a European Space Agency initiative to develop global composites and land cover maps using observations from the 300-m MERIS sensor onboard the ENVISAT satellite mission  in the SWAT2012.mdb database and the lookup tables “Lookup_Landuse_GlobCover.txt” and Lookup_Landuse_USGS.txt. However, similar to the soil parameters, landuse parameters must be calibrated for a given location.The databases for the above two global landuse maps are supported by the table  reanalysis temperature and precipitation data from the Research Unit East Anglia (CRU TS 3.1)44. These daily data cover the period of 1950–2099 and have a resolution of 0.5°. Similar to CRU, they have been reformatted from NetCDF into SWAT-formatted text files.We provide five global climate models (GCM), each with four carbon evolution scenarios supported by ISI-MIP5 45. The 0.5° grid WATCH Forcing Data46 for the period of January 1, 1960, to December 31, 1999 (the reference period) was used as observation data to downscale the five GCMs44. WATCH is a combination of the ERA-40 daily data, the 40-year reanalysis of the European Centre for Medium-Range Weather Forecasts, and the Climate Research Unit TS2.1 dataset (CRU)43. The WATCH Forcing Data data combines the daily statistics of ERA-40 with the monthly mean characteristics of CRU and Global Precipitation Climatology Centre (GPCC) datasets and represents a complete gridded observational dataset for bias correction of global climate data44.The five GCM models include HadGEM2-ES, IPSL-CM5A-LR, MIROC-ESM-CHEM, GFDL-ESM2M, and NorESM1-M  from the earth’s land surface is collected by NASA using satellite data from 1982 to 200352. It is crucial to have a measure of AET when calibrating a SWAT model with river discharge data. Using river discharge alone, we can confidently estimate runoff and infiltration. However, components of the infiltrated water cannot be estimated with any degree of confidence. These components include soil moisture (S), aquifer recharge (AR), and actual evapotranspiration (AET) 54 are deposited at Pangaea and www.2w2e.com sites. There are 16,328 soil records in this data set.The Harmonized World Soil map and associated SWAT data files (Lookup Table and SWAT2012.mdb)55 are deposited at Pangaea and www.2w2e.com sites. There are 24 landcover types in this database.The Global Land Cover Characterization (GLCC) map from USGS and SWAT data file (Lookup Table and SWAT2012.mdb)56 are deposited at Pangaea and www.2w2e.com sites. There are 23 landcover types in this database.The GlobCover from the European Space Agency and associated data files (Lookup Table and SWAT2012.mdb)57 are deposited at Pangaea and www.2w2e.com.The historical CRU and future GCM weather data58 in text format is deposited at Pangaea and www.2w2e.com.Finally, the Global Actual Evapotranspiration Data62. Validation of these maps, which are based on satellite observations, are offered by ground-truth observations conducted by the map developers and also in various literature64.The global soil and landuse databases have been successfully used in many SWAT applications around the worldThere is a significant variation in the reported values of soil parameters in the literature and by various agencies. In this research, we used a large number of pedotransfer functions and soil samples from around the world to estimate the textural-based soil parameters. In Table usersoil) tables are in the SWAT2012.mdb database. The field (Name) is concatenated by using the fields SU-SYM74, SU-SYM90, MU_GLOBAL, and ISSOL as given in the original HWSD database. SU-SYM74 is the soil unit symbol according to the FAO-74 soil classification, SU-SYM90 is the soil unit symbol according to the FAO-90 soil classification, MU_GLOBAL is the Global Mapping Unit identifier, which provides the link between the GIS soil units and the attribute database, and ISSOL is a field indicating if the soil mapping unit is a soil (1) or a non-soil (0). All maps provided have the World WGS-84 Spatial Reference without any projection. The users will have to project these maps as needed before using it in the ArcSWAT or QSWAT models.There are 4,931 soil records in the FAO/UNESCO database, and 16,328 records in the HWSD soil map. Both (conditionality of their calibrated models. Calibrated model parameters are always conditioned on the input data, meaning one could obtain a different set of parameters if one had used a different set of available data. This is probably the most disappointing aspect of calibration.Different soil and landuse maps are provided to emphasize the fact that often more than one database is available for building and calibrating a model, and also to encourage the users to use different databases to realize the unconditionality, the calibrated parameters must be integrated over all conditioning factors. Hence, we recommend using different physical inputs and multi-objective calibration procedures.A calibrated model is, therefore, always: non-unique, subjective, conditional, and subsequently limited on the scope of its use. To achieve Supplementary Information.


Obesity and certain nutritional deficiencies are global health problems that emerge in systems of interdependent individual biological and historical factors and social environmental determinants of health. Nutrition security is a framework that assumes stable access to sufficient innocuous and nutritious food , health care, and sanitation, and information that in conjunction allows self-care-oriented behavior for health protection. To understand the social environment of nutrition insecurity, the object of study was the food distribution and consumption system of a marginalized community in Hermosillo, Mexico. We assessed the distribution of food establishments by social marginalization level in basic geo-statistical areas and the nutrition security status of women in underserved neighborhoods. We found that in Hermosillo >90% of food establishments included for analysis  were distributed outside of areas with high levels of social marginalization. The nutrition security assessment suggests that low intakes of fruit and vegetables and high intakes of fat and sugar may be associated with food accessibility and acceptability factors in individual decision-making processes. Future research should take into account the variability of food system environments and address the particular needs of communities in terms of food and nutrition security. This hed height . WorldwiEven though undernutrition declined since 1990 , certain energy and nutrient deficiencies persist . Hunger 2) [The energy content and nutritional quality of diets demonstrate trends in food choice interrelated in obesity and nutrient deficiencies ,7. In 202) ,9,10. An2) . obesogenic environment and toxic environment to describe characteristics in cities and patterns in behaviors that explain the emergence of the obesity epidemic [In response, public nutrition and health research produced ecological models of obesity ,13,14,15epidemic ,13. The epidemic ,17,18,19epidemic . This scepidemic . According to the U.S. Institute of Medicine, a food system, commonly represented as a supply chain, is configured by interdependent and dynamic processes , heterogeneous autonomous agents , and shared structures between agents  . Given tNutrition security is a framework based on community studies aimed to identify the determinants of nutrition ,22. It aThe food environment refers to “the food available to people in their surroundings as they go about in their daily lives and the nutritional quality, safety, price, convenience, labelling and promotion of these foods” . ResearcThe limitation of geographical analysis in environmental research and health behavior leads to other proposals focused on individual activity patterns aimed to find key interactions between structural features and consumer food choice ,29. For From a systems science perspective, an innovative computational technique that can be used to generate simulations of social processes involved in the dynamics of malnutrition is Agent-Based Modeling (ABM) . Agent-bMexico is challenged by the double burden of malnutrition. On the one hand, overweight and obesity are estimated to affect 70% of adults aged ≥20 and 34% of school-age children ,32. In aMaize, wheat, rice, and other cereals are basic foods that provide ~32% of the total energy in the average diet. However, 26% is attributed to the consumption of sweet bread, deep-fried corn snacks, sugary drinks and other non-essential foods, while 6% comes from vegetables and fruits . This tyOver the last 70 years, the food distribution system transitioned from “traditional” markets to the expansion of supermarkets, convenience stores, fast food chains and specialty stores. Changes in the retail environment occurred especially after 1980 ,39. An iSeveral studies suggest that limited access to fruits and vegetables, stable product availability and food acceptability are important system drivers of consumption patterns for low-income populations ,42,43,44Furthermore, there are regional differences in nutrition and dietary patterns across the country. In northern states  the highest prevalence of obesity in adults (37.8%) is reported compared to central (29.4%) and southern (34.7%) regions  Figure . The higIn particular, in the northern state of Sonora, known for agricultural region Yaqui Valley, the home of the Green Revolution for wheat , it was In this case study, datasets from Mexican agency INEGI  were used to examine the distribution of food establishments in Hermosillo . We repoFrom April to June 2018, fieldwork followed in two neighborhoods with a very high degree of social marginalization to assess measures of nutrition security: dietary patterns, nutrition status, household food security and perceived food environment and behavior. In-person questionnaires and anthropometric data were collected by trained personnel. In accordance with the Declaration of Helsinki, the study protocol was approved by the institutional review board at Centro de Investigación en Alimentación y Desarrollo (CE/004/2018) and the informed consent procedure followed the Mexican norm for human subjects research (NOM-012-SSA3-2012).n = 760). Specifically, we included grocery stores from regional food chains and supermarkets, convenience stores and beer stores  from national food chains. Food chains operated nationwide were previously identified in research on the evolution of the commercial food distribution system in Mexico [Food establishment information was obtained from INEGI geo-statistical system DENUE , last updated March 2018 . From a n Mexico . SpatialWomen present the highest prevalence of obesity among the total population in the country  and a hiIn-person questionnaires included (1) a single 24-hour dietary recall, (2) a household food security scale, and (3) a perceived food environment and behavior survey, described below. (1) The 24-hour dietary recall (24-HDR) technique involved an interviewer asking the participant to describe all food and drink consumed in the 24-hour period before the interview, including portion size (assisted by food models) and recipes (when prepared at home) following the five-step multi-pass method . Portion(2) We used a household food security scale developed and validated for the northern region of Mexico, similar in format to the Household Food Insecurity Access Scale and the Latin American and Caribbean Food Security Scale (ELCSA), to capture household and individual access to food, as well as emotional perceptions streaming from food deprivation (food anxiety and lack of choice) ,60. The (3) An adaptation of the Perceived Nutrition Environment Measures Survey was used to study the perception of the local food environment and food shopping behavior. Glanz and colleagues designed four instruments to measure structural factors in defined scenarios  and a non-observational survey to capture the individual’s perspective on the food environment (NEMS-P) ,61. We u2) in reference to WHO: low weight <18.5, normal weight >18.49 and <25, overweight >24.9 and <30, obesity >29.9 [Anthropometry was measured to estimate body composition and nutrition status. The equipment used was a standing electronic scale (capacity 0–200 kg ± 0.05 kg), a portable stadiometer (capacity 30–205 cm ± 1 mm) and a measuring tape (0–205 cm ± 1 mm). During measurement, participants wore light clothes, an examiner helped position the participant and an assistant recorded weight, height and waist circumference (WC). Nutrition status was assessed estimating BMI , access to social security, time of residence in neighborhood, and participation on food assistance programs.Descriptive individual and household characteristics were analyzed in NCSS: Statistical Software 12.0.9. To analyze dietary patterns, total energy, protein, fat, carbohydrate and fiber were estimated. A list of 210 food items were categorized in one of 10 food groups, using the macronutrient content informed by an educational guideline for Mexican nutritionists  . Thirteen = 140) compared to supermarkets operated by national chains Walmart, Soriana and Casa Ley (n = 45), even when the latter have expanded their consumer markets since 2008 with the introduction of low-price neighborhood stores in Mexico  [Out of the 760 food establishments, 90% of grocery stores, 93% of supermarkets, 92% of convenience stores, and 98% of beer deposits were distributed in areas with low or medium levels of social marginalization . None ofExpress) . Other studies provide evidence for disparities in food distribution systems within and between regions ,66. A coAn example of contradictory analytical results in current food environment studies was reported by Barrera et al. . In theiIn the assessment of dietary patterns, estimated average total energy intake was 1320.1 ± 724.1 kcal/participant. Energy derived from the mean intake of 45.5 ± 33.8 g of protein, 48.3 ± 33.4 g of fat, and 181.8 ± 97.7 g of carbohydrates, while fiber consumption was 18.3 ± 14.9 g. In reference to the nutrition guidelines on the macronutrient composition of diets for healthy adults, the contribution of fat to total energy is slightly high at 31.7% (adequate intake is 25–30%), while carbohydrate and protein are within the recommended intervals at 55.2% and 13.1%, respectively (data not shown) . TortillThe dietary analysis showed a high consumption of non-essential foods. The combined proportion of sugar, sugar-sweetened beverages, and high added fat and sugar products  contributed 24.5% of total energy. In addition, the foods most frequently reported were flour tortillas, maize tortillas, sugar-sweetened beverages, beans, and table sugar. According to Bojorquez et al., the foods in this type of dietary pattern provide high energy at low economic cost for low-income women in Tijuana, Baja California . HoweverIn agreement with the dietary analysis, other studies show that fiber consumption of women in Mexico is lower than the recommendation set at 25 g/d . In 20122. However, 58% of the participants have access to limited primary health care. The nutrition assessment based on WC indicates that 20.7% of the participants are at lower risk of developing disease related to body visceral adipose tissue . HoweverIn addition, 18% of the participants perceived having an adequate access to the food supply, while 82.1% perceived food insecurity, an indicator of the availability, accessibility, and acceptability of foods consumed at home . In contThe results of the perceived food environment and behavior survey indicate that physical access to supermarkets, grocery stores, convenience stores, and beer stores affects consumer behavior because 49% reported walking for grocery shopping (which affects the frequency of food shopping and may limit the quantity of food carried back home)  Table 3Table 3. Since most of the participants reported shopping in neighborhood corner stores frequently, more than in commercial food retailers, food access, in terms of distance and transportation, is an important environmental factor involved in food decision-making processes of marginalized populations. The walkability factor was also identified in a review by Pitt et al. . Based oThis study had several limitations. Specifically, food establishment data obtained from INEGI were not physically validated. Field observations indicate that the location/operation of economic units may not be up-to-date in geo-statistical system DENUE. We suggest corroborating the data with other datasets or direct field observations. In attention to this problem, a research strategy used in a study by Sushil et al. was to validate food establishment data with the Google Geocoding application and by directly contacting a subsample of retailers to verify the address and physical location . Future In Hermosillo, the majority of grocery stores, supermarkets, convenience stores, and beer deposits operated by commercial regional and national food networks were distributed in areas with low marginalization levels. The variability in the food supply may be interrelated with accessibility and acceptability factors that explain dietary patterns high in fat and sugar and low in fruits, vegetables, and other antioxidant- and fiber-rich food sources. An overview of the food environment emphasizes the need to identify key features susceptible to change and with the capacity to influence populations on a massive level without producing unintended effects, beyond the unique fiscal policies already implemented in Mexico and other countries. Furthermore, the turn of public nutrition towards food labeling and other information-based programs may be overlooking the needs of underserved populations. In this context, unequal access to food and health supplies underlying the obesity epidemic and nutritional deficiencies should be accounted for in nutrition security research.


The study of European rabbit immunoglobulin genes has contributed decisively to the current knowledge on antibody structure and diversification. The European rabbit has also been increasingly used as an animal model for the study of many human diseases, such as syphilis, tuberculosis, and AIDS. As such, the study of its immune system genes is of crucial relevance, but the study of rabbit immunoglobulins has focused only on the IgG and IgA antibodies. In this study, we added to the knowledge of the rabbit immune system by investigating the genetic diversity of two antibodies, IgM and IgE, in wild and domestic rabbits as well as other rabbit close species. With the data obtained in this study, we showed a high similarity between the different rabbit close species studied and we pointed out important genetic differences in the wild and domestic rabbits. Our findings are a valuable tool for the management of rabbit wild populations and domestic breeds and may contribute to the identification of immunoglobulins genetic variants with greater efficiency against pathogens.Oryctolagus cuniculus) has been an important model for immunological studies but the study of its immunoglobulins (Ig) has been restricted to its unique IgA and IgG. Here, we studied the genetic diversity of IgM and IgE in several species of leporids and performed population genetics studies on European rabbit wild populations and domestic breeds. The leporids sequencing showed that these Ig are well conserved (98% sequence similarity among leporids), For IgM the Cµ1 and Cµ4 were the most diverse and most conserved domains, respectively, while for IgE the Cε1 was the most diverse domain and Cε2 and Cε3 the most conserved domains. The differences in the pattern of most conserved and most diverse domain between the Ig isotypes are most likely related to each isotype function. The genetic population data showed contrasting results for IgM and IgE. For both Ig, as expected, a greater diversity was observed in the original species range, the Iberian Peninsula. However, unexpectedly the genetic diversity found for IgE in the domestic animals is higher than that for the French wild populations. These results will increase knowledge of the genetic diversity of leporids and wild and domestic rabbit populations and are important tools for the management of wild populations and rabbitries.The European rabbit ( Immunoglobulins (Igs) are one of the most emblematic components of the jawed vertebrate immune system. These heterodimeric glycoproteins act as a critical part of the immune system by recognizing and binding to antigens and subsequently triggering mechanisms that lead to its destruction . In euthOryctolagus cuniculus) has been used as an animal model for diseases and immunological studies . One ofgermline ,4. IgM abclasses . For IgAbclasses ,7 being bclasses ,9,10,11)IgM, in its transmembrane form, is the first isotype to be expressed during B-cell development. As such the VH and VL regions associated with this Ig have not undergone much antigenic driven diversification being able to recognize a wider range of antigens than other isotypes, although showing low affinity . In mammO. cuniculus algirus in the south-west area of the IP and O. cuniculus cuniculus in the north east area. After the last glacial peak, O. c. cuniculus expanded its range north, across the Pyrennes, towards France [Brachylagus, Bunolagus, Caprolagus, Lepus, Nesolagus, Oryctolagus, Pentalagus, Poelagus, Pronolagus, Romerolagus and Sylvilagus) [Lepus, a polytypic and cosmopolitan genus, and Nesolagus, Pronolagus and Sylvilagus, which comprise two, four and more than 16 species, respectively [Pronolagus, and that Romerolagus is at the second basal position [The European rabbit originated on the Iberian Peninsula (IP) , where ts France , from whs France ,16. It bvilagus) ,18. Mostectively ,23,24,25position ,27,28. IGHM and IGHE genes for six additional extant leporid genera: Bunolagus, Brachylagus, Lepus, Pentalagus, Romerolagus and Sylvilagus. Additionally, we compared the genetic diversity of these Igs between wild rabbit populations of O. c. algirus and wild and domestic populations of O. c. cuniculus.In this study, we extend the knowledge on IgM and IgE isotypes in leporids by sequencing the complete IGHE and IGHM evolution in leporids, we analysed specimens of Bunolagus, Brachylagus, Lepus, Pentalagus, Romerolagus and Sylvilagus genera. To assess the genetic diversity of European rabbit we analysed 10 individuals for each of three wild populations: O. c. algirus collected in Portugal and O. c. cuniculus collected in Spain and in France  [PCR amplifications of the four Y386696) . A fragmSequences obtained in this study were edited and aligned using CLUSTAL W  as impleSequence nucleotide diversity was estimated using DnaSP version 5.10 . HaplotySequences obtained in this study were deposited in GenBank (Accession Numbers: MN650919-MN651079).IGHM and IGHE genes for six leporid genera: Bunolagus, Brachylagus, Lepus, Pentalagus, Romerolagus and Sylvilagus. The gene structure is similar to the European rabbit IGHM and IGHE and we thus inferred the intron-exon organization from published rabbit IGHM and IGHE sequences. Splicing signals are present at the intron boundaries and therefore we assumed that all studied leporids share the same IGHM and IGHE exon organization as the European rabbit.We newly sequenced the IGHM domains with 24 amino acid variable positions, the majority of which involve one substitution and 11 are conservative regarding the amino acid properties. For the Cµ1 domain some genera specific residues are observed: Arg 117 for the European rabbit, Asn 3 and Ala 122 for Pentalagus and Asn 115, Gln 117 and Asn 118 for Romerolagus ; the remaining haplotypes are rare or uncommon (0.01–0.10 frequencies). Our results show a greater diversity of IGHE in the IP populations: of the 26 SNPs observed, 16 were found for the uniculus . Surprisuniculus . The nucuniculus . We recoO. c. algirus: M and F at CH2 residues 81 and 116, respectively; Spanish wild O. c. cuniculus: I and M at CH1 residues 4 and 84, respectively, and K at CH4 residue 82; and for the domestic breeds: K, A and T at CH1 residues 12, 44 and 45, respectively, G at CH2 residue 1.7 and V at CH3 residue 85 . The raIGHM and IGHE have been fairly conserved (each Ig has 98% sequence similarity among leporids), similarly to what has been found for IGHG [IGHM constant domains diversity was observed, with the Cµ1 domain being the most diverse and the Cµ4 the most conserved. This gradient in domain diversity was not observed for IGHE, the Cε1 is the most diverse domain but Cε4 shows greater diversity than Cε2 or Cε3 domains, the most conserved domains. For leporid IGHG a gradient in the constant domains diversity was described from the most conserved Cγ1 domain to the most diverse Cγ3 domain [The results obtained in this study show that throughout Leporid evolution for IGHG . A gradi3 domain  and for 3 domain ,7. The d3 domain ,38,39 an3 domain ,41,42, t3 domain ,44, agai3 domain , the mos3 domain ,7 and du3 domain . As for 3 domain ,47,48 a 3 domain . Thus, Ith century the rabbit was domesticated from French wild European rabbit populations [O. c. cuniculus [O.c. algirus populations, in agreement with the previously described pattern of highest genetic diversity in IP populations. Interestingly, the genetic diversity we found for the domestic animals IgE is higher than that for the French wild O. c. cuniculus  involve non-synonymous substitutions, in spite of the observed 31 SNPs only seven polymorphic amino acid positions were detected. IgM is the most ancient Ig isotype being present with the same function in all gnathostomes [The results we obtained for IgM, on the other hand, revealed a higher nucleotide and haplotype diversity for the iversity . Considehostomes . As suchIGHM and IGHE have been fairly conserved. Despite this, variable amino acid residues were identified for IgE which may improve the binding of IgE to its Fc receptors. The observed pattern of genetic diversity of IGHM and IGHE along the wild rabbit populations from Iberia and France is contradictory, being, as expected, higher for the Iberian populations for IGHE but surprisingly higher for French populations for IGHM. This is the first time that these two immunoglobulin isotypes were fully characterized for Leporids and for European rabbit wild populations and domestic breeds. These findings will be useful for the management of rabbit populations and for the understanding of the rabbit adaptive immunity against pathogens.Our findings show that throughout Leporid evolution