Survival and success rates of tooth transplantations even after long follow-up periods have been shown to be very high. Nevertheless, it is important to analyse factors potentially influencing these rates. The aim of this study was to assess the influence on success of potential factors.The research was based on a retrospective analysis of clinical and radiological data from a sample of 59 subjects (75 transplanted teeth). The follow-up period varied from 0.44 to 12.28 years (mean 3.95 years). Success rates were calculated and depicted with Kaplan-Meier plots. Log-rank tests were used to analyse the effect of root development stage, apex width, the use of enamel matrix proteins or the surgeon on success of transplantations.p = 0.001) and tooth type (p ≤ 0.001) were significantly associated with transplantation success. Use of enamel matrix proteins (p = 0.10), root development stage (p = 0.13), the recipient area (p = 0.48) and apex width (p = 0.59) were not significantly associated with success.Results for success of premolar transplantations were comparable with already published data, while molars performed worse than shown in other studies. The surgeon performing the transplantation (Molar transplantations were not as successful as premolar transplantations; however, success rates varied greatly depending on the surgeon’s experience. The use of enamel matrix proteins as well as root development stage, the recipient area and apex width did not show significant associations with success of tooth transplantations. Le Chirurgien Dentiste [The first documented case report of a homoplastic tooth transplantation was described by Fauchard in 1728 in his book Dentiste  and lateDentiste –8, the aDentiste . The advDentiste . In addiDentiste –14.Prior studies analysing transplantations have claimed three fourths root length as being the ideal development stage for transplantation , 15.Treatment with enamel matrix proteins like Emdogain®  has been shown to exhibit an activating effect on periodontal cells –20. PosiAlthough the published findings indicate very high success  and survival rates , it is important to identify all factors determining success and survival rates of tooth transplantations. These factors are the key to higher clinical success and to prevention of unnecessary failures.The aim of this study was to record success rates of tooth transplantations and to examine parameters which may be associated with success.This research project was reviewed and approved by the ethics committee of the canton of Zurich, Switzerland.All consecutive patients from the dental clinic of the University of Zürich who had undergone one or more tooth transplantations were included in order to minimize selection bias. The search was performed by a computer specialist on the patients’ database system, where all transplantations had been registered. The database was introduced in January 2000, and all registered transplantation cases up to 31 December 2012 were included. Before the year 2000, no complete patient database was available.The following information was extracted from the retrieved files: date of birth; date of transplantation; age at transplantation; numeric number of the transplanted tooth; recipient area; root length in millimetres (measured on single X-rays) and development stage at transplantation according to Moorrees et al. ; apex wiThe individual success period of each transplanted tooth was defined as the time period from the transplantation to the latest exam appointment where the tooth was in situ with no clinical signs of failure. Progressive root resorption, periodontal problems, ankylosis and crown to root ratio greater than 1.0 were considered to be failures , 28, wheThe radiographic evaluation was carried out by two researchers independently. Results were subsequently discussed until consensus was reached.x < 2 mm and x ≥ 2 mm. Results of statistical analyses with p values smaller than 0.05 were considered to be statistically significant.Data were coded in Excel  and analysed using STATA statistical software . Descriptive statistics such as mean, median, standard deviation and absolute and relative frequencies were computed. Kaplan-Meier plots were produced, and association between success and root development stages, application of enamel matrix proteins, apex width, recipient area and surgeon were examined using the log-rank test. The root development stage according to Moorrees  was group < 0.001) , use of enamel matrix proteins (p = 0.10), apex width (p = 0.59) or the recipient area (p = 0.48). However, strong associations between success and surgeon (p = 0.001) and success and tooth type (p < 0.001) were identified  were included. Data of the latter were analysed in this study. Fourteen patients had undergone a transplantation of molars (wisdom teeth) and 53 of premolars. Reasons for drop outs can be seen in Fig. 01) Fig. . No signed Table . Kaplan-ea p = 0.8. HoweveOur sample showed a vast range of observation periods. This can be explained by the fact that failures, when they happened, often occurred at an early stage after transplantation. In contrary, some teeth had been observed over a long period of time for protocol reasons (consecutive patients). Since only few successful transplantations had observation times shorter than 1 year and by analysing censored data using Kaplan-Meier and log-rank tests with failure as the event, results account for the wide range of observation periods.Several factors are likely to be associated with survival and success of tooth transplantations. We examined the effect of tooth type, root development stage, recipient area, surgical trauma  and use of intraoperative drugs on success of tooth transplantations.p = 0.001) when analysed with the log-rank test (Table Premolars with an estimated 10-year success rate of 81.6 % compared favourably to molars with an estimated 10-year success rate of 33.8 %. Thus, while our results for premolars were comparable with most published studies , molars st Table  with theThe use of enamel matrix proteins did not improve success in our sample. However, because of the small number of events, results should be interpreted with caution. Nevertheless, since prior studies without use of enamel matrix proteins performed equally well for premolars and better than our sample for molars , 12, 30,Our study found no significant associations between success and root development; however, it is a common clinical practice at our clinic to avoid transplantation of teeth with complete root formation, and thus, to reach a significant level of association as shown in two recent studies , 28 was Since the study was retrospective, it is susceptible to limitations interconnected with this type of investigation. By defining endodontic treatment as not being a failure, a potential bias may arise since endodontically treated teeth can develop complications which can lead to necessity for extraction or such rated as failure according to the applied grading system. Nevertheless, in a study by Andreasen , teeth rMolar transplantations were not as successful as premolar transplantations; however, success rates also varied greatly depending on the surgeon’s experience. In our sample, the use of enamel matrix proteins as well as root development stage, the recipient area and apex width did not show significant associations with success of tooth transplantations.


X) were significantly related to AR after adjusting for age and gender (p < 0.05). Because both pollutants are considered to be traffic emissions, this study suggests that traffic emissions in Taiwan need to be controlled to lower the prevalence of children’s AR.The occurrence of allergic rhinitis (AR) may be significantly influenced by air pollution. This study examined the association between the first occurrence of AR in preschool children and the pre-incident levels of air pollutants in Taiwan. We identified 9960 eligible subjects from a systematic sampling cohort database containing 400,000 insureds of the National Health Insurance from 2007 to 2011 and matched them with the environmental monitoring data from 2006 to 2011 according to the locations of their clinics. Pre-incident levels were determined using the average concentrations of air pollutants one or two weeks prior to the AR diagnoses. Logistic regression analyses were performed to determine any significant relationships between AR and specific air pollutants. The first AR incidence for Taiwanese preschool children, which increased with age, was 10.9% on average; boys appeared to have a higher percentage (14.2%) than girls (8.27%). Among the air pollutants, carbon monoxide (CO) and nitrogen oxides (NO Allergic rhinitis (AR) is a common respiratory disease occurring in individuals who breathe air with allergens that cause nose inflammation and swelling. A variety of allergens can trigger AR, including molds, pollens, animal dander, and dust mites . SeasonaX), ozone (O3), particulate matter (PM), and sulfur dioxide (SO2), several have been found to be related to AR; however, the relationships are not consistent. For example, NO2, PM10 (aerodynamic diameter ≤ 10 μm) and SO2 have been associated with AR in China [3 and PM2.5 (aerodynamic diameter ≤ 2.5 μm) are the related environmental factors [A number of studies have indicated that the increasing prevalence of AR is associated with air pollution severity ,10,11,12in China ,14, wher factors . Despite factors ,18,19,20Many studies have focused on the relationships between AR and the environment; however, not all age groups are studied evenly. There are relatively few data for preschool children, likely due to their low prevalence of AR or their limited ability to independently fill out research questionnaires. Nevertheless, preschool children’s health warrants additional attention and care, and this issue is relevant for discussion and investigations. Thus, we conducted this study in preschool children using Taiwan’s National Health Insurance Data, from which the AR subjects were identified by doctor’s diagnoses, and combining the environmental data from the subjects’ adjacent monitoring sites to determine the association between the first occurrence of AR and environmental pollutants.We used the systematic sampling cohort database of 400,000 insureds of the National Health Insurance of Taiwan from 2007 to 2011 and found 23,103 preschool children aged 0–6 years. After excluding those who did not have sufficient information or adjacent environmental monitoring data, there were 9960 children, of which 4456 were male and 5504 were female. The diagnoses were made following the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM), and the codes 477.0, 477.1, 477.2, 477.8 and 477.9 were categorized as AR. Those who were assigned to any of the ICD-9CM codes were selected as study subjects, and only the first diagnoses were used to relate to the air pollution at one and two weeks prior to them. Each eligible subject must have had more than one AR-related visit and been confirmed to be AR-free for at least one year prior to the first AR diagnosis during the period; that is, the year of 2007 was considered a clearance period from which no AR records were retrieved. There were 7326 AR-related visits, of which 1088 children, 632 male and 456 female, were eligible and included in the study. Because of the secondary data with the removal of personal identification, the study was granted for exempt review by the Research Ethics Committee of Tzu Chi General Hospital .X, O3, PM10, and SO2) were available for download (in daily and monthly averages) from the Taiwan Air Quality Network (http://taqm.epa.gov.tw/taqm/en/default.aspx). To address possible latency in health effects for the cohort database covering 2007–2011, we retrieved the environmental monitoring data from one year ahead of the period (2006) to 2011.Environmental monitoring sites adjacent to or sharing the same district codes with the subjects’ clinics were used for the sources of environmental data, which came from 47 of the available 66 environmental monitoring sites in Taiwan. Data for the monitored air pollutants  in a logistic regression analysis to calculate the odds ratios (ORs) for AR occurrence and the levels of air pollution when adjusting variables that may have been effective in incidence . Because air pollution-induced respiratory symptoms were confirmed to be related to exposures for several days to weeks ,22 and tThe first AR incidence in Taiwanese preschool children for 2007–2011 was 10.9%, with the boys’ outcome significantly higher than the girls’  . The rat10 and SO2. The mean concentration of SO2 (4.81 ± 1.85 ppb) was far below its annual standard (30 ppb), showing the efficacy in emission control, whereas that of PM10 (56.8 ± 14.4 μg/m3) was close to the standard (65 μg/m3), suggesting a need for control efforts.The statistical results of the daily concentrations of the monitored air pollutants corresponding to the subjects are presented in X, a byproduct of combustion, was highly correlated with CO and SO2 (r = 0.851 and 0.809), indicating emission sources of motor vehicles and industry/power plants, respectively, because CO was primarily produced by motor vehicles and SO2 was a major combustion product of fuel [X was also highly correlated with O3 but in an opposite direction (r = −0.801), and a similar correlation was found between SO2 and O3 (r = −0.485). The negative association between NOX or SO2 and O3 suggests the consumption of either precursor of the photochemical smog reaction that generates O3. PM10 was not significantly associated with any other variables with the exception of SO2 (r = 0.531), indicating that a certain quantity of PM10 in Taiwan was generated from fuel combustion.The correlation between the air pollutants is given in  of fuel . NOX wasX  with the low level (Level 1) either one or two weeks prior to the occurrence , the level differences did not significantly affect AR occurrence. We also performed multivariate logistic regression analyses to determine the association between AR and all air pollutants together but excluded CO from the analyses because of its collinearity with NOX (r = 0.851). NOX is the only variable that was significantly related to AR with ORs as high as 1.94 (95% CI = 1.41–2.71) for one week before and 1.50 (95% CI = 1.19–2.03) for two weeks before. The pre-incident levels one week before occurrence resulted in higher ORs than that two weeks before, suggesting that AR induced by air pollution should occur within a week. In summary, the relatively high pre-incident levels of traffic-related pollutants (NOX coupled with CO) were likely to lead to AR within the following week.Age and gender both affected the AR incidence  , which h3 and NOX [3 is a secondary product derived from the photochemical smog reaction, which consumes NOX and hydrocarbons in the presence of sunlight to form O3 [3. By integrating these three Taiwanese studies, we conclude that the correlation between air pollutants has been consistent since 1994 and the association between AR and outdoor concentrations of CO and NOX has been identically significant for preschool children, middle school children and adolescents.Other than the association between AR and traffic-related air pollutants, our results also resembled those of previous Taiwanese studies by demonstrating a negative association between O and NOX ,10. This form O3 ; additio3 in Taiwan had gradually increased over the years (reflecting climate change), which resulted in higher temperatures in favor of O3 production [3 in this study, O3 is a threat to respiratory health due to its reactivity and irritating effect. However, studies have found a significant association between O3 and respiratory allergies [3 should not be overlooked.In comparing our data with the environmental data of both previous Taiwanese studies ,10, we foduction . Despitellergies ,9,12, su2.5, one of the major pollutants of traffic emissions, was not included in this study because such data were not measured at all environmental monitoring sites. We expect associations between PM2.5 and respiratory symptoms to be assessed in the future in Taiwan when PM2.5 becomes a regular measure for all environmental monitoring sites.There were several limitations to this study. First, we did not have access to personal information and thus could not assess direct AR-triggering effects; using the identification-free cohort database, however, we were able to determine the impacts of outdoor air pollution on AR. Secondly, environmental monitoring data were derived based on the locations of the clinics and not where the patients actually lived because of a lack of access to personal information. Fortunately, this did not appear to present an issue because most Taiwanese who lived in urban areas were believed to go to nearby clinics for minor respiratory diseases, such as colds and AR. Thirdly, PMX, which are considered to be traffic-related pollutants, were found to be significantly associated with AR in preschool children during the assessed period in Taiwan. The association is consistent with those derived from previous Taiwanese studies focusing on middle school children, suggesting that traffic emissions in Taiwan need to be more effectively controlled to lower the likelihood of AR occurrence.The first AR incidence for preschool children in Taiwan was higher in males than females for 2007–2011. CO and NO


Analysis across rehabilitation revealed SICI was reduced for the contralateral M1 at first walk compared to discharge (p = 0.003). For the ipsilateral M1 both short and long-latency intracortical inhibition were reduced at admission (p < 0.05) and prosthetic casting (p < 0.02). Analysis of the neurophysiology and gait function revealed several interesting relationships. For the contralateral M1, reduced inhibition at admission (p = 0.04) and first walk (p = 0.05) was associated with better gait function. For the ipsilateral M1, reduced inhibition at discharge (p = 0.05) was associated with poor gait function. This study characterized intracortical excitability in rehabilitating amputees. A dichotomous relationship between reduced intracortical inhibition for each M1 and gait function was observed at different times. Intracortical inhibition may be an appropriate cortical biomarker of gait function in lower limb amputees during rehabilitation, but requires further investigation. Understanding M1 intracortical excitability of amputees undertaking prosthetic rehabilitation provides insight into brain reorganization in the sub-acute post-amputation period and may guide future studies seeking to improve rehabilitation outcomes.Reorganization of primary motor cortex (M1) is well-described in long-term lower limb amputees. In contrast cortical reorganization during the rehabilitation period after amputation is poorly understood. Thirteen transtibial amputees and 13 gender matched control participants of similar age were recruited. Transcranial magnetic stimulation was used to assess corticomotor and intracortical excitability of M1 bilaterally. Neurophysiological assessments were conducted at admission, prosthetic casting, first walk and discharge. Gait variability at discharge was assessed as a functional measure. Compared to controls, amputees had reduced short-latency intracortical inhibition (SICI) for the ipsilateral M1 at admission ( Transtibial amputation of a lower limb has a significant effect on human function and requires extensive rehabilitation to restore mobility using a prosthetic limb. Despite lengthy rehabilitation , many amN-methyl-D-aspartate (NMDA) receptor activity in upper-limb amputees has been observed  years] admitted for prosthetic rehabilitation at one regional rehabilitation hospital in Adelaide, SA, Australia  were recTo assess changes in brain neurophysiology transtibial amputee participants attended one afternoon laboratory session at each of four pre-selected key time points during their rehabilitation. Assessments were conducted at time of admission to rehabilitation, prosthetic casting, first walk, and discharge from rehabilitation, individualized for each amputee . During Surface EMG was recorded from each RF using 10 mm-diameter Ag/AgCl electrodes . Electrodes were placed 2 cm apart over the muscle bellies, with the distal electrode positioned ∼12 cm superior to the patella. A 20 mm-diameter reference Ag/AgCl electrode  was placed over the patella. Prior to affixing the electrodes the skin was prepared for optimal contact by shaving to remove hair, lightly abrading and cleaning with alcohol. EMG signals were sampled at 2000 Hz , amplified , band-pass filtered (20–1000 Hz) and stored for oﬄine analysis .2 unit . Both single and paired-pulse TMS were delivered using a flat 70 mm wing diameter, figure eight coil. The coil was held tangentially over the scalp with the handle pointing 30° posterior-medially in the transverse plane positioned over the contralateral cortex 1 cm posterior and 1.5 cm lateral to the vertex (CON) and M1 ipsilateral to the amputated side (M1IPSI) by systematically moving the coil over a 1 cm grid from this location and marked on the scalp. Active motor threshold (AMT) was determined separately for each M1 as the minimum stimulus intensity eliciting a 100 μV MEP in five of ten stimuli in the contralateral RF , long latency intracortical inhibition (LICI), and intracortical facilitation (ICF). For each measure, 16 non-conditioned and 16 conditioned MEPs were evoked in randomized order. The test stimulus was set to produce a half maximum MEP (50% MEPmax), ensuring the test MEP was evoked from the linear portion of the stimulus response curve for each individual . This meFor both single and paired-pulse TMS, MEPs where pre-stimulus root mean square EMG (rmsEMG) were two standard deviation (SD) above or below the mean were removed prior to averaging to ensure consistency of MEP responses (number of responses removed ranged between 0 and 3). From the retained traces MEPs were measured peak-to-peak and conditioned MEPs were normalized to non-conditioned MEPs to determine a ratio of facilitation or inhibition.Spatial-temporal gait variability was assessed at discharge from rehabilitation as a measure of gait function. Previous studies demonstrate that increased levels of gait variability are associated with falls histories in transtibial amputees  and it wCON and M1IPSI were log transformed to achieve normality. Significance level was set at p ≤ 0.05 and SPSS software was used for all statistical analyses .The normality of data was checked with a Shapiro–Wilk normality test. ICF assessed in M1t-test. Descriptive statistics were used to report stump-length and time since amputation for the key rehabilitation milestones.Age was compared between amputees and controls with an independent t-tests to confirm presence of inhibition (SICI and LICI) and facilitation (ICF). SICI, LICI, ICF for each M1 and the LI were separately compared between controls and amputees at admission and discharge with independent t-tests. For amputees, AMT, SICI, LICI, ICF for each M1 and LI were individually assessed with a 4 SESSION repeated measures ANOVA (rmANOVA). Post hoc tests explored significant effects and were corrected for multiple comparisons using a modified Bonferroni test  for each hemisphere. For controls, pre-trigger rmsEMG for conditioned and non-conditioned MEPs for SICI, LICI, ICF were individually compared with paired t-tests for each hemisphere. For amputees, paired-pulse test MEP amplitude was compared between sessions with a 4 SESSION rmANOVA for SICI, LICI, ICF for each hemisphere.The optimal conditioning stimulus intensity responsible for producing maximal SICI for each participant was determined at admission and used in off-line analysis of SICI for comparison across rehabilitation time points. This optimal conditioning stimulus determined at admission was kept constant throughout rehabilitation. Similarly, the optimal inter-stimulus-interval responsible for producing maximal ICF for each participant was determined at admission and analyzed across rehabilitation time points. This optimal inter-stimulus-interval determined at admission was kept constant throughout rehabilitation. For SICI, LICI, and ICF in each M1, conditioned and non-conditioned MEPs were compared at admission to rehabilitation with paired oni test . The corThe association between discharge gait function  and neurophysiology measures across rehabilitation time points  were assessed for each test session with linear regression analyses. Linear regression models were controlled for age and stump-length as these factors can affect gait function . Indepenp = 0.49). Mean stump-length of amputees was 18.1 (SD 3.4) cm. Amputees were admitted to rehabilitation a median 14 (range 7–18) days following amputation surgery. Median time to prosthetic casting was 32 (range 14–41) days, to first walk was 50 (range 32–75) days and to discharge from rehabilitation was 87 (range 47–99) days. There were no interruptions to the rehabilitation program or transfers back to acute care for illness for any amputees during the study.There was no difference in age between amputees  and controls , 80% AMT (n = 2) and 90% AMT (n = 4); and for M1IPSI were 70% AMT (n = 3), 80% AMT (n = 4) and 90% AMT (n = 6). The optimal ISI for evoking ICF in M1CON were 10 ms (n = 4) and 15 ms (n = 9); and for M1IPSI were 10 ms (n = 7) and 15 ms (n = 6). For control subjects, the optimal conditioning stimulus intensities for evoking SICI in M1CON were 70% AMT (n = 4), 80% AMT (n = 4) and 90% AMT (n = 5); and for M1IPSI were 70% AMT (n = 7), 80% AMT (n = 1) and 90% AMT (n = 5). The optimal ISI for evoking ICF in M1CON were 10 ms (n = 5) and 15 ms (n = 8); and for M1IPSI were 10 ms (n = 8) and 15 ms (n = 5). At admission to rehabilitation analysis of M1CON found the conditioned MEP was significantly smaller than the non-conditioned MEP for SICI  and LICI , and larger for ICF . For M1IPSI, the conditioned MEP was significantly smaller than the non-conditioned MEP for LICI . However, for M1IPSI there was no significant difference between conditioned and non-conditioned MEPs for both SICI  and ICF  indicating modulation of intracortical excitability in this hemisphere early following amputation.For amputees, the optimal conditioning stimulus intensities for evoking SICI in M1IPSI in amputees at admission  compared to controls . There was no difference in the other neurophysiological measures between amputees at admission and controls . There was no difference between amputees at discharge and controls for any neurophysiological measure .There was reduced SICI in M1p = 0.48; see Figure 1). AMT did not vary significantly across session for M1CON (p = 0.85) or M1IPSI (p = 0.39). For M1CON, there was significant modulation of SICI , but not LICI  or ICF . Post hoc analysis for SICI found there was disinhibition at first walk compared to discharge . For M1IPSI, there was significant modulation of SICI , and LICI , but not ICF . Post hoc analysis for SICI found there was disinhibition at admission  and prosthetic casting  compared to discharge. Post hoc analysis for LICI found there was disinhibition at admission  and prosthetic casting  compared to first walk. A summary of neurophysiological data is provided in Table 1.The LI did not change across rehabilitation (p ≥ 0.21) or controls . For amputees, the average test MEP to assess paired-pulse measures was 0.40 mV (SD 0.35) for M1CON and 0.54 mV (SD 0.39) for M1IPSI. For controls, the average test MEP to assess paired-pulse measures was 0.69 mV (SD 0.32) for M1CON and 0.75 mV (SD 0.75) for M1IPSI. The test MEP amplitude did not vary across sessions for amputees .There were no significant main effects or interactions for pre-trigger rmsEMG for amputees . There was a negative relationship between SICI assessed in M1CON at first walk and step-width variability at discharge . There was a negative relationship between SICI assessed in M1IPSI at discharge and step-length variability at discharge . For all three regression models, no other independent variables controlled for were significant (p ≥ 0.10). There were no other significant associations between neurophysiological measures and gait variability measures .For amputees, normalized step-length variability was 4.2% (SD 1.8), normalized step-time variability was 9.4% (SD 3.7) and normalized step-width variability was 13.7% (SD 3.6). Linear regression models controlling for age and stump-length found three significant relationships between neurophysiological measures and gait function characterized by gait variability measures. There was a negative relationship between SICI assessed in M1CON at first walk and was restored by discharge. SICI and LICI were both reduced in M1IPSI at admission and prosthetic casting. Disinhibition of M1CON at admission and first walk was associated with better gait function at discharge, while disinhibition of M1IPSI at discharge was associated with poorer gait function at discharge. These findings contribute to the understanding of human amputee neurophysiology and may be relevant for future studies seeking to improve rehabilitation outcomes.The main findings of this study were that there was longitudinal modulation of intracortical inhibition in M1 bilaterally during prosthetic rehabilitation, which was associated with gait function in this group of transtibial amputees. SICI was reduced in M1CON for amputees was similar to controls. However there was significant disinhibition at time of first walk, which was restored by discharge. Pharmacology studies indicate SICI reflects GABAA receptor mediated inhibition, as the administration of GABAA receptor agonists lorazepam . It is unlikely that reduced inhibition is related to the loss of afferent input from the amputation as it was not observed at admission, but at first walk, 50 days post-amputation. We suggest the reduction in inhibition is most likely a neuroplastic response partly driven by the process of prosthetic rehabilitation. Reduced GABAergic inhibition is thought to promote conditions conducive to facilitating the induction of synaptic plasticity (At admission to rehabilitation, SICI in M1orazepam  and diazorazepam  increaseasticity  and is aasticity . AlthougCON. There was an even greater reduction in SICI and a reduction in LICI in M1IPSI following amputation across the early phases of rehabilitation. Similar to the contralateral M1, the reduction in SICI indicates GABAA receptor mediated inhibition likely underpins cortical reorganization in M1IPSI. Furthermore, we observed a reduction in LICI at admission and prosthetic casting. Cortical interneurons responsible for LICI directly inhibit corticospinal output via post-synaptic GABAB receptors and pre-synaptic GABAB receptors on inhibitory interneurons responsible for SICI (IPSI at admission and prosthetic casting and this concurrent reduction likely reflects that LICI was attenuated in the early phase of rehabilitation via reduced post-synaptic GABAB receptor activity. However, at time of first walk we observed reduced SICI and increased LICI in M1IPSI, reflecting increased excitability of pre-synaptic GABAB receptors leading to auto-inhibition.Interestingly modulation of intracortical excitability following amputation was not limited to M1for SICI . In the B receptor activity in post-acute amputees is consistent with a previous lower limb amputee case study which also reported a reduction in GABAB receptor activity over the entire rehabilitation period in M1IPSI (B receptor activity is more evident in the early phase of rehabilitation as opposed to the later rehabilitation period. Reduced GABAA and GABAB receptor activity in M1IPSI provide additional evidence of bilateral cortical reorganization following unilateral amputation (A and GABAB receptor activity in the ipsilateral M1. The specific timing of modulation of intracortical inhibition also suggests the critical period for reorganization within M1IPSI is early in the rehabilitation phase prior to receiving an interim prosthesis. Further attention to this pre-prosthetic rehabilitation period may improve rehabilitation outcomes and should be further investigated.The finding of reduced GABAn M1IPSI . Howeverputation . Our finIPSI may reflect a cortical contribution to driving the residual muscles of the amputated lower limb via up-regulation of ipsilateral pathways to the spinal cord. A second, and equally probable explanation, is that disinhibition of M1IPSI represents a use-dependent cortical response to greater reliance by the amputee on the non-amputated limb during their rehabilitation. Use-dependent plasticity may occur prior to provision of a prosthesis due to increased dependence on the non-amputated leg. This hypothesis may explain the earlier reorganization observed for the ipsilateral M1 compared to the contralateral M1. We propose that it is unlikely interhemispheric projections contributed to the reorganization of the ipsilateral M1 observed here. This is supported by the observation that the ipsilateral M1 appears to reorganize earlier than the contralateral M1, and furthermore, our previous work in long-term lower limb amputees (Interestingly our results indicate that the ipsilateral M1 reorganizes earlier than the contralateral M1 and this may reflect functional tasks performed in rehabilitation. The underlying mechanisms leading to the reduction in inhibition of M1 ipsilateral to the amputated limb are difficult to determine from this study. Previous studies indicate lower limb motor control relies on the bilateral cortical contribution from the primary motor cortices . We suggamputees  suggestsA receptor mediated inhibition within contralateral M1 at admission and first walk are adaptive neurophysiological responses, as they were associated with reduced gait variability. Therefore, reduced GABAergic inhibition at admission and first walk may be an appropriate neurophysiological biomarker of function at discharge. Conversely, an ongoing reduction in GABAA receptor mediated inhibition of the ipsilateral M1 may be a maladaptive neurophysiological response as it was associated with increased gait variability at discharge. Our results indicate normal levels of SICI within ipsilateral M1 should be restored following prosthetic casting and prior to prosthetic mobility to drive optimal recovery of function. Ongoing reorganization of M1IPSI at discharge may interfere with the normal pattern of bilateral cortical motor control of the amputated limb leading to degraded gait function as we demonstrated previously in long-term amputees (IPSI, which potentially impair control of prosthetic gait, may assist prosthetic rehabilitation.An interesting outcome of this study was the association between neurophysiology and gait function. The relationship between cortical neurophysiology and gait function at discharge indicate reduced GABAamputees . InterveThere are limitations to this study which should be acknowledged. First, it is difficult to evoke MEPs of reasonable amplitude from stimulation of the lower limb motor cortex given the location of the representations relative to the surface of the head. To address this we pre-activated the RF to a standardized and controlled level of activity while performing neurophysiological assessments. Although there was no significant difference in pre-trigger rmsEMG across sessions, muscle strength may have increased over rehabilitation which may have had a minor influence over the neurophysiological assessments. Second, relatively low levels of SICI were observed in this study compared to cortical representations in other muscles, particularly those of the upper-limb. Differences observed between muscle representations are likely the result of functional specificity  and the A receptor mediated inhibition at first walk may indicate mechanisms underlying adaptive cortical reorganization, leading to improved gait function. The period from first walk to discharge represents a critical time where the conditions for cortical reorganization are facilitated in the contralateral M1. For the ipsilateral M1, both GABAA and GABAB receptor mediated inhibition were reduced earlier in rehabilitation at admission and prosthetic casting. This response may reflect bilateral cortical control of the lower limbs or increased use of the non-amputated limb in the early, pre-prosthetic, rehabilitation phase. Poor gait function was observed when reduced intracortical inhibition in the ipsilateral M1 extends to discharge from rehabilitation. Future studies are required to further investigate intracortical inhibition as a biomarker using non-invasive brain stimulation interventions to ‘normalize’ intracortical inhibition and determine if this improves gait function at discharge.We demonstrated bilateral modulation of intracortical inhibition within M1 at specific time points during prosthetic rehabilitation in post-acute transtibial amputees. For the contralateral M1, reduced GABAThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Childhood vaccination remains a primary mechanism for reducing the burden of infectious disease. In the United Kingdom, as in many countries, a sustained effort is required to ensure that vaccination targets are met to afford protection to the whole population from vaccine preventable disease. The Celebrate and Protect programme is a collaborative partnership developed to improve the uptake of childhood vaccination across a number of boroughs within London through the use of a celebration card to encourage attendance for vaccination and enhance relationships between general practices and the parents/carers of children.This study was undertaken to assess the suitability, feasibility and acceptability of the Celebrate and Protect programme across nine boroughs in London. Data were collected either from telephone interviews (n = 24) or from focus groups (n = 31). A total of 55 key informants were included in the study, representing strategic, commissioning or policy leads, healthcare professionals and primary care teams delivering vaccinations and parents/carers of children under five.The analysis of data identified that whilst parents/carers saw the celebration card positively this raised the issue of ‘vaccine hesitancy’ and the lack of information that parents/carers have to make informed decisions about vaccination. Similarly, healthcare professionals viewed the programme positively and felt that it was deliverable within existing resources although they raised wider questions about on-going sustainability and about quantitative data collection. In relation to the collaboration between primary care and a pharmaceutical company in developing the Celebrate and Protect programme, it was generally felt that, provided appropriate governance is in place, it was a pragmatic approach in which the benefits outweighed any perceived disadvantages.The Celebrate and Protect programme was seen as an innovative collaborative programme to engage with parents and carers of children in order to improve relationships between service users and providers and subsequently increase vaccination uptake. The analysis demonstrates that that the celebration card is suitable for its purpose, acceptable to both healthcare professionals and to parents/carers of children and the Celebrate and Protect programme has been able to deliver its aims.Whilst the delivery of the ‘celebration card’ intervention in primary met its objectives there are some outstanding issues in terms of the sustainability of the initiative and the ability to demonstrate quantitative improvements in vaccination uptake rates. Vaccination is a public health intervention that has demonstrably reduced the global burden of infectious disease and which can, through comprehensive coverage, reduce health inequalities . There aCurrent estimates suggest that childhood vaccination coverage needs to reach 95 % to ensure herd immunity, a standard adopted by the World Health Organisation (WHO) . These dDespite the emphasis of both national and local public health programmes on ensuring the delivery of vaccines there is a wide geographical variation in uptake with some regions of England achieving less than 80 % coverage and other areas achieving 95 % or above  against meningitis…” “…I think …explain what some of the diseases that immunisation is trying to protect against…” (Group 3-parents/carers).Focus group participants made suggestions on how the cards could be improved in a way that could potentially sway the minds of those opposed to immunisation:“…link, with the added recommendation that you can call your healthcare professional via telephone on the card....”. Or add that “…If you have any questions or concerns please come along or give us a ring…” because “…just think it gives leeway for people who feel a bit insecure and nervous, sort of isolated; it just makes it more robust…the information… it’s not just a reminder…you know, if you have any concerns, we’ll be happy to speak with you…” (Group 3-parents/carers).Participants felt adding a link to credible website would be invaluable:“… although I always had the intention to keep up with all the immunisation, I was always a little bit sceptical and worried about immunisation…I would like to be more informed about the [unclear] side effects of the immunisation…” (Group 3-parents/carers).And as one of the parents/carers mentioned:Impressive…’ …12 boroughs [PCTs] signed up.... 175 practices registered…definitely a starting point…can only go up.... parents more aware of imm[unisations]…” (Group 1-policymakers).Aside from comments on the contents of the card the focus groups discussions also addressed the desirability or potential demand for the cards, indicating that if they missed out on the celebration card many of the parents/carers would feel offended or excluded. From the provider perspective, the cards also seemed to be very popular, evidenced by the ability to recruit additional PCTs for subsequent waves of the Celebrate and Protect programme: ‘[Celebrate and Protect] “…there are practice variances - with some with total buy in and some that do not want to know/do not want any extra workload” (Group 1-policymakers).Also retention rates of the PCTs in subsequent phases were suggestive of the perceived effectiveness and popularity of the programme with two thirds continuing to be involved in the second phase, which covered an additional three PCTs. Nevertheless, responses from some strategic leads indicated that at a practice level there were notable variations in engagement:“…Celebrate and Protect supports communication…it makes things easier…” Moreover, in their experiences, “…mothers are reluctant to go to practices (for vaccination) unless invited as they feel surgeries are busy” and “do not like letters that sound threatening” (Group 1-policymakers).Understanding the perceived added value of the Celebrate and Protect programme was also important to encourage future engagement. Some participants, especially from the strategic leads and providers groups expressed the opinion that celebration cards played a necessary role in helping to communicate an issue that is otherwise delicate or difficult to broker with some parents/carers. They felt that celebration cards offered a suitable alternative:“…we were in bottom 10, since Celebrate and Protect, we are now in top 10…” (Group 2-practitioners).Additionally some providers felt that the Celebrate and Protect programme added value to the call/recall system and was of particular value to practices without a robust call/recall system:“… get [Celebrate and Protect] people up to date with vaccinations… going fine, actually…asked a couple of parents.... those asked said…reminded me to come…very happy…”“…Celebrate and Protect good for ‘impetus’ to start thinking about immunisation but need other basic processes in place, e.g. good call and recall system in practice and at PCT level; follow ups of defaulters; need for more ‘support processes…” (Group 2-practitioners).Practices that had an established call/recall system also noted the added value:“…Celebrate and Protect.... birthday cards have lessened my workload…don’t have to make phone calls.... surgery does not have to pay for postage.... reduced workload as do not have to speak to address concerns…” (Group 2-practitioners).Some providers however did not perceive the Celebrate and Protect programme as adjunct to a call/recall system but a substitute:“…Well I…I don’t really need because I have …the Red Book…my doctor rang me and sent a text, so I get reminded all the time”. “I still think it’s a really good idea… (but)… a letter would be better I wouldn’t need a pretty card” (Group 3-parents/carers).Some parents/carers who had good existing relationships with their practices didn’t see any added value to the card:“… I think you can’t take away from people, like face to face or call…”“…and if then they (mothers) say no then they can actually talk to them about the reason, so you’re addressing…any other issues that they might have…” (Group 3-parents/carers).Others who didn’t have existing relationships with their practice felt that the card would be more helpful but were keen to stress that the programme should not be an alternative to the current call/recall system:“… (Leads) were able to secure commitment from GP practices - as they have already established trust/good relationship with GP practices, so much easier to get GP buy-in to the project…” (Group 1-policymakers).Responses from the strategic leads indicated that the Celebrate and Protect programme was made possible by the involvement of immunisation leads linked to PCTs that had historically established trust with GP practices. The leads were encouraged to facilitate implementation through communication and provision of training and training materials to GP practices.not needed any support from lead PCT for Celebrate and Protect all very straightforward…with information pack…useful guide on how to do labels....” “.... working fine…not much extra work per month....” (Group 2-practitioners).“[we did] Some providers explained that implementation of the Celebrate and Protect programme didn’t require additional resources:“…before that (Celebrate and Protect) it was just me … Celebrate and Protect added boost…” “…it’s not a big job to do…an hour a month…” (Group 2-practitioners).If anything, some practices felt the Celebrate and Protect programme gave them the additional resources to support their work:“…Celebrate and Protect is driven by power of personality, not supported by system....” (Group 1-policymakers).Strategically however, there was a perceived need to secure commitment from an organisation to take a lead role (e.g. NHS London), because, thus far, the programme was largely driven by individuals, with a risk to its sustainability in the face of the imminent NHS structural changes.Many of the strategic leads felt that the Celebrate and Protect programme was a low cost initiative with an implementation strategy that could allow roll-out across other areas on both a local and national scale. Participants felt the programme was financially viable, especially for practices falling short of their target of 95 % coverage.Particular challenges around the ability to develop the evidence of the impact of the Celebrate and Protect programme were clear from responses from providers and strategic leads. Some respondents cited historical problems around the lack of robust performance management data systems, with inconsistencies in how data were captured and communicated across the different data systems used in the immunisation programme. Some felt that there were on-going inconsistencies on how data were recorded and that these issues may make it difficult to demonstrate the effect of the Celebrate and Protect programme.“… we are doing a lot better than we were…imms uptake has improved…no negative feedback from patients or staff…” “.... percentage of children coming in has risen … imms uptake [has increased].... our take up for 6 week check…good…anyway…” (Group 2-practitioners)However, direct feedback from some of the providers suggested that the Celebrate and Protect programme had improved uptake of immunisation:“…Cards were (in a box) waiting for me when I returned from Maternity leave” “…difficult in our practice…” “…takes time…writing names and address on envelope.... do not generate labels…” (Group 2-practitioners).Whilst the responses were positive in terms of the perceived increase in immunisation it would be difficult to attribute causality solely to the celebration cards. There were of course also challenges to implementation that were identified, especially relating to staffing:“…fantastic but…time consuming [especially] mail merge…have to ensure search is correct’.... one[issue]....’can miss new registrants in between monthly searches.... uses Open Exeter…” “…takes time…writing names and address on envelope.... do not generate labels…” (Group 2-practitioners).Some operational challenges cited included the time taken to generate address labels. It was also felt that the Celebrate and Protect programme needed a lot of attention to detail for fear of missing any children:doesn’t cover new parents/carers … they do not see us … see health visitor…health visitors remind them but [the] call has not come from [the] surgery so mothers forget…” “some parents take time to register their new-born” (Group 2-practitioners).“…(Celebrate and Protect) Some providers suggested expanding the remit of the Celebrate and Protect programme to include those children and their parents/carers not registered with a GP through engaging with health visitors and liaising with maternity units to co-ordinate from birth:“…do you see what I mean? You need to get your child to the clinic. You need to get them immunised. This [celebration card] is like; it is more of a positive reinforcement. The letter is more; you have been told off. This is more like… it is colourful…” “…good idea… a good reminder…because you have a little baby, sleepless nights, sometimes you might forget to make an appointment”, “Oh, even I missed the first MMR when he was two because I forgot, I’m involved in health and I forgot to get him immunised… (the card would have) without a doubt (helped)…” (Group 3-parents/carers).Whilst the idea of receiving celebration cards was rated highly by all categories of respondents, less than 10 % of the parents/carers attending the focus group had received one. Much of their response to the cards was framed as a comparison to more traditional call/recall mechanisms, especially practice letters.This was echoed by the providers who felt it was more acceptable to send celebration cards, as it would enhance the call/recall systems and improve relationships with patients.“…personally.... supportive of this stance.... embrace new ways of working.... learn from partners....”(Group 1-policymakers); “couple of years ago…probably would have had more reservations…but now as long as ethical issues are covered as required by DH policy document…we need to get used to working with private providers” “Personally ‘don’t have an issue”, because the “reputation of pharmaceuticals is changing”… (Group 2-practitioners).One particular aspect of the Celebrate and Protect programme—working closely with a pharmaceutical company—was seen as particularly novel but it was recognised that this could have some inherent risks. All categories of respondents expressed opinions related to the decision to work in collaboration with a pharmaceutical company and most responses were positive:“…growing reality…cannot afford purely a PH project as high costs.... three times costs for distribution and procurement” “…in the new world …you want to continue [working] with Sanofi.... they will make a corporate social responsibility contribution....” (Group 2-practitioners).The financial benefits of pharmaceutical industry involvement were recognised by some:“…uncomfortable territory for some…”(Group 2-practitioners); “…we.... met a lot of resistance … whether it was appropriate to work with a pharmaceutical company…[despite] DH guidelines [to].... support partnership working with industry…. [there is] local level resistance to engaging with this philosophy…” (Group 1-policymakers).Others expressed concerns about how this was represented:…If the NHS have approved, then I am okay with it…” (Group 3-parents/carers).“Asked on their views about the NHS working with a pharmaceutical company, parents/carers were generally accepting of the concept provided that necessary approvals and authorisations were undertaken:“…I saw on the telly about price fixing with pharmaceutical companies, where they offer GPs incentives to prescribe their product....” (Group 3-parents/carers).Others however were quite sceptical in view of previously televised broadcasts on unethical activities by some pharmaceutical companies:“… (it would make one) feel warmer towards your GP surgery”, “…If you received a card from GP you would feel cared for…” (Group 3-parents/carers)Despite this suggestion, the aim of the Celebrate and Protect programme was primarily to increase vaccination and to improve relationships between parents/carers and primary care practices. Parents/carers identified with this aspect of the programme:“No. I don’t think it would make a difference for me because my practice is always grumpy. I don’t think it’ll change that” (Group 3-parents/carers).However, this was not shared by all:The Celebrate and Protect programme has demonstrated innovative partnership working bringing together strategic leads from the NHS and local government alongside partners from the pharmaceutical industry, Sanofi Pasteur MSD. The celebration card, which formed the main intervention delivered within the programme, was co-designed with parents/carers, who were able to influence the design and content of the card, providing some insight about why parents/carers may or may not choose to have their children immunised.Whilst this analysis did aim to assess the effectiveness of the Celebrate and protect programme in improving vaccination rates amongst different typologies of parents/carers, the programme is unlikely to have any significant effect on vaccine ‘rejecters’ and most likely to impact ‘vaccine hesitant’ parents/carers . FurtherThe Feasibility, Suitability and Acceptability framework is used to assess the relative success or failure of a strategic option.Following the qualitative evaluation, it would seem that the Celebrate and Protect programme could be judged as a low risk and low cost strategy that could be delivered feasibly within the resources and competencies that were available during wave one, although significant organisational and structural changes have occurred since then. The specific intervention (i.e. the celebration card and vaccine schedule) used within the programme has been shown to be suitable for achieving the objectives of the programme, although demonstrating the effectiveness of the intervention in terms of its impact on vaccination uptake remains a challenge. Whilst the intervention has been shown to be acceptable both to those delivering it (i.e. primary care staff) and to the intended recipients (i.e. parents/carers of children under the age of 5), a number of recommendations were identified from the focus groups that resulted in changes to the intervention for subsequent waves. This iterative development demonstrates the flexibility and adaptability of the celebration card.Acceptability of the programme at a strategic level was demonstrated throughout the evaluation, although due to the structural reorganisation of the NHS and changes in the roles and responsibilities of those initially involved in the programme, it should be recognised that there are important risks in terms of managing the relationships crucial to the feasibility of the Celebrate and Protect programme in the longer term.Collaboration with ‘industry’ can provide economies of scale along with strategic expertise and an in-depth understanding of logistics, which were fundamental to the delivery of the Celebrate and Protect programme. In addition it was observed that an important role of the industry partner, especially during the time of NHS transition experienced during the programme, was the consistency and continuity offered to the programme. As the Celebrate and Protect programme was initiated in conjunction with the industry partner, a unique collaborative framework was provided through which many different organisations voluntarily co-operated to develop a ‘bottom-up’ potential solution to an intractable problem. This benefit of course needs to be measured against any potential conflicts of interest that may be represented by such collaborations. In this case, as childhood vaccine is procured nationally through a tendering process involving multiple pharmaceutical companies, there appears to be no immediate conflict of interest in working with SPMSD given that the partnership abides by the DH/Association of British Pharmaceutical Industries Framework on partnership with pharmaceutical companies .Whilst the sampling strategy identified a broad range of parents/carers, for pragmatic reasons it did not specifically target the intended beneficiaries of the Celebrate and Protect programme in that the parents/carers attending the focus groups were not necessarily registered with GP practices participating in the first wave of the programme. In addition, the recruitment of primary care staff coincided with a busy influenza vaccination season resulting in a lower than expected participation.This paper reports on the findings of a study to assess the feasibility, suitability and acceptability of the Celebrate and Protect programme. The programme is innovative in a number of ways, having been established collaboratively by a range of partners including PCTs, GP practices, local authorities and a pharmaceutical company producing childhood vaccines. It uses a novel method (the celebration card) to engage with parents and carers of children in order to increase vaccination uptake and to improve relationships between service users and providers. The card was designed with input from all the relevant stakeholders, including parents/carers, and the programme was developed iteratively, with on-going review and evaluation so that potential improvements could be tested rapidly and incorporated without delay.The analysis presented here focuses on the suitability, acceptability and feasibility of the programme, drawing on qualitative data collected through interviews and focus groups as part of a wider evaluation process, which also involved a quantitative analysis of vaccination uptake rates. The analysis identified that many of the factors that have been proposed in the conceptual model of vaccine acceptance/rejection are indeed important to parents/carers .From this qualitative analysis, it is clear that the celebration card is suitable for purpose and acceptable both to healthcare professionals and to parents/carers of children. In terms of feasibility, the Celebrate and Protect programme has thus far been able to deliver its process aims (i.e. recruitment of practices and delivery of the celebration cards) within the allocated resources although several issues of on-going sustainability were raised, mainly in relation to the changing primary care infrastructure. In addition, the question of whether the very positive views expressed about the Celebrate and Protect programme can be linked to more quantitative outputs and outcomes (such as vaccination uptake rates) was not within the scope of this analysis but remains an important line of further inquiry.BME, black and minority ethnic; COVER, Cover of Vaccination Evaluated Rapidly; DH, Department of Health; GP, General Practitioner; MMR, measles, mumps and rubella; PCT, Primary Care Trust; PHE, Public Health England; SPMSD, Sanofi Pasteur MSD; TB, tuberculosis; WHO, World Health Organisation


This assay is highly rated amongst other similar probes thanks to its superior sensitivity and stability. However, we report here that Amplex Red is readily converted to resorufin by a carboxylesterase without requiring H2O2, horseradish peroxidase or oxygen: this reaction is seen in various tissue samples such as liver and kidney as well as in cultured cells, causing a serious distortion of H2O2 measurements. The reaction can be inhibited by Phenylmethyl sulfonyl fluoride (PMSF) at concentrations which do not disturb mitochondrial function nor the ability of the Amplex Red-HRP system to detect H2O2.In vitro experiments and in silico docking simulations indicate that carboxylesterases 1 and 2 recognise Amplex Red with the same kinetics as carboxylesterase-containing mitochondria. We propose two different approaches to correct for this problem and re-evaluate the commonly performed experimental procedure for the detection of H2O2 release from isolated liver mitochondria. Our results call for a serious re-examination of previous data.Amplex Red is a fluorescent probe that is widely used to detect hydrogen peroxide (H  •2O2 probe Amplex Red crosses the mitochondrial membrane.The H•Carboxylesterase catalyses HRP-independent oxidation of Amplex Red.•2O2 measurements in many tissues.This effect needs to be blocked to enable correct H•PMSF inhibits HRP-independent conversion of Amplex Red. They are generated within biological systems and play critical roles in most of them. For instance, there is a wealth of evidence showing that, by causing damage to macromolecules, ROS can contribute to aging processes and to the pathogenesis of multiple diseases 2O2- and HRP-independent oxidation of AR to resorufin has been described . This results in the raw quantitative values from liver mitochondria being much higher than those from other tissue mitochondria in similar experimental conditions and with similar oxygen consumption rates. This phenomenon has been discussed in the community but no explanation has been put forward so far. Frequently, it has simply been ignored One caveat of the AR assay that has been experimentally examined is its photosensitivity  as an enzyme that converts AR to resorufin without requiring either oxygen, hydrogen peroxide or a peroxidase. We show that contrary to widely held beliefs, mitochondrial membranes are permeable to AR and that AR is converted to resorufin by CES in the matrix of mitochondria from tissues with high CES expression. CES can be inhibited by Phenylmethyl sulfonyl fluoride (PMSF) at doses that do not interfere with either mitochondrial function or the kinetics of the HRP-catalysed oxidation of AR by H22.1C57Bl/6 male mice were purchased from Harlan . ICRFa are a substrain of C57Bl/6 kept as a long-established ageing colony at Newcastle 2.2g for 10 min at 4 °C. To obtain inner membranes, 100 mM NaCO3 was added to shaved mitoplasts and left on ice for 30 min, and centrifuged at 100,000g for 15 min at 4 °C. Protein concentration was assessed by BioRad Dc protein assay kit with BSA as standard.Mitochondria from liver, brain and skeletal muscle were isolated as described The specificity of the subfractions was confirmed by western blots using Apotosis inducing factor (AIF) as a marker for intermembrane space, NDUFA9, a subunit of the electron chain transport complex I that is localized in the inner membrane, and glutamate dehydrogenase (GDH), which resides in the mitochondrial matrix.2.32O2 release by isolated mitochondria was measured in the assay buffer containing 115 mM KCl, 10 mM KH2PO4, 2 mM MgCl2, 3 mM Hepes, 1 mM EGTA, 0.2% fatty acid free BSA, pH 7.2 at 37 °C, in the presence of exogenous superoxide dismutase (75 U/ml), horseradish peroxidase (HRP) (2 U/ml) and Amplex Red (50 µM) at 37 °C. The fluorescent intensity of resorufin, the oxidised product of AR, was monitored kinetically in a plate reader  at excitation 544 nm and emission 590 nm. The experiments were protected from light.H2O2. Accordingly, the H2O2 generating system (i.e. the concentration of mitochondria) was also lowered so that it did not exceed the capacity of H2O2 detection at the given HRP concentration.For the experiments to test catalase sensitivity to resorufin, the concentration of HRP was lowered to 0.05 U/ml in order for catalase to compete for H2O2 release). The fluorescent intensities of the experiments were calibrated against that obtained by the addition of a known amount of H2O2 to the experimental media in the presence of AR and HRP.Typically, the basal rate  was measured for 8–10 min, then respiratory substrates  were added to initiate respiration  was added to the experimental medium immediately prior to the or during the measurement. Controls received ethanol only.In the Homovanillic acid (HVA) assay, AR was replaced with HVA (4 mM) while all the other experimental conditions remained identical to the AR assay, and the fluorescent intensity was read at excitation 310 nm and emission 430 nm.2.4To monitor the conversion of AR to resorufin by different tissue samples, various organs were washed to remove blood and homogenized in PBS, and 0.4 mg/ml samples were used in PBS containing 50 μM AR. The conversion of AR to resorufin by Ces1b enzyme was monitored in double distilled water. To create anaerobic conditions, the reaction was carried out in an anaerobic chamber , with oxygen concentration <5 ppm.2.5Oxygen consumption rates (OCR) by isolated mitochondria were measured in a Seahorse XF24 analyzer (Seahorse Biosciences) as described The OCR during conversion of AR to resorufin by Ces1b enzyme was monitored in a high-resolution respirometer, Oxygraph-2k (Oroboros Instruments).2.6AR was purchased from Life Technologies. All other chemicals were from Sigma. Carboxylesterase 1 isoform b human was also from Sigma (E0287); according to the manufacturer, the enzyme was formulated in 0.1 M Potassium phosphate buffer. The antibodies used in western blot experiments were as follows: NDUFA9 , AIF , GDH , CES1 .2.7http://imagej.nih.gov/ij).3D images of purified mitochondria and mitoplasts stained with AR and mitotracker green  were acquired using an LSM510 confocal  and deconvolved using Huygens software . Subsequently, images were analysed using average line profiles through the central plane of the stack using ImageJ  0.1% formic acid in MS grade water, (B) 0.1% formic acid in acetonitrile) to 95% B over 2 min on a Waters Acquity UPLC BFM C18 column . The eluant was monitored using a Waters Xevo G2XS QqTOF mass spectrometer operating at a scan frequency of 10 Hz either in MS mode or in MSMS mode scanning for the precursors 214.0 and 258.0 with a collision energy ramp from 25 to 40 eV. Data were acquired in the range of m/z 50 to m/z 400 at a resolution of 22,000. Using MassLynx 4.1 extracted ion chromatograms were generated for AR (m/z 258.0766), RT (1.13 min) and for resorufin (two peaks were observed at m/z 214.0514 with RTs 0.99 min (unchanged averaged peak area over the whole time course of the experiment) and RT 0.96 min (occurring only after addition of CES1b and increasing over time to saturation)). Scans were averaged across the peaks for each molecule.2.9The structures of AR, resorufin and 3,7-dihydroxyphenoxazine were optimized by using the B3LYP functional and the standard 6-31G basis set as implemented in Gamess 33.12O2 generation. Oxygen consumption and H2O2 generation greatly increase as mitochondria are fuelled with substrate and the electron transport chain activity increases (state 2). Accordingly, the rates of AR oxidation by H2O2 to resorufin were close to zero during state 1 but significantly increased after the addition of substrate, as seen in kinetic measurements from brain or muscle mitochondria  is minimal and there is very little Hchondria a and b. chondria a and b. chondria a. Furthechondria c. Howevechondria c. The sa3.22O2 release 2O2  of liver mitochondria treated with different concentrations of PMSF in states 2, 3, 4 and after complete uncoupling using FCCP . HVA is another commonly used HRP-dependent fluorescent probe to monitor H2O2 generation. With this probe, there was no HRP-independent HVA oxidation in liver mitochondria  in liver mitochondria should represent AR oxidation rates measured from PMSF treated mitochondria in the presence of HRP, that is the AR oxidation specific to mitochondrial Hilar see a. To tesgreement a. Moreovchondria b. H2O2 rusing AR b and c. R method c. Brain R method d and e, ng liver f–i.2O2 release. It should be noted that the cell lines rank differently in terms of PMSF-dependent and –independent resorufin production rate in accordance with primary fibroblasts being more dependent on oxidative phosphorylation (and thus producing more H2O2) than immortalized and tumour cells.Frequently, the AR method is used to measure hydrogen peroxide release from cultured cells. We measured resorufin fluorescence generated with and without HRP, PMSF and catalase a and b f3.3To identify the source of the HRP-independent AR conversion to resorufin by liver mitochondria, we first assessed the possibility of microsomal contamination. However, our mitochondrial preparations were pure; fewer than 5% of the total of the >600 detected proteins in purified liver mitochondria were of microsomal origin In order to determine the submitochondrial localisation of the site of AR conversion to resorufin, mitoplasts (mitochondria without the outer membrane), ‘shaved’ mitoplasts , and inner mitochondrial membranes were prepared from purified liver mitochondria c. All th2O2 (49 pmole) was then added. In this setting, mitochondria are the only source of AR. Only if the washed mitochondria retain AR and release it into the assay medium, can resorufin be generated by HRP-catalysed oxidation by H2O2. This happened for both brain and liver mitochondria , confirming that AR but not HRP was able to permeate the mitochondrial membrane. Together, these data indicate that AR can transverse the mitochondrial membrane in both directions (either by diffusion or by a transporter-mediated mechanism).To see whether AR can cross mitochondrial membranes in both directions, liver and brain mitochondria were incubated with AR for 10 min at 37 °C, washed twice and then incubated in fresh assay medium with HRP but not AR. External Hchondria f, albeitchondria f. Quantietection g. Conver3.4To identify enzymes that could perform the HRP-independent, PMSF-inhibitable conversion of AR to resorufin, we searched lists of proteins specific to highly purified mouse liver mitochondria e.g., gut and lung) might be due to cross-reactivities and/or different isoforms being expressed. Within liver mitochondria, CES1 is located in the matrix  converts AR to resorufin in the absence of Hin vitro d. The rein vitro e. This rubstrate f. Followubstrate . The UV ubstrate . The reaubstrate a. Importubstrate , and conubstrate . Loperamubstrate . These de.g. Val254, Leu255, Leu318, Ile359, Ile388 and Met425). Notably, Leu255 and Ala222 also contact the methyl group of the acetyl moiety. In contrast, the two hydroxyl functions were involved in not more than weak H-bonds with backbone atoms (e.g. the 3-hydroxyl approaches the backbone carbonyl group of His468). Together, the molecular docking simulations confirmed the overall stability of the computed complex and revealed the key role played by Van der Waals and hydrophobic interactions in both complex stabilization and undocking processes To understand the potential reactions between carboxylesterases and AR, we performed docking simulations based on the resolved structure of CES1 in complex with a heroin analogue (PDB Id: 1MX9) and the published homology model of CES2 Using these models together with published correlative data in vitro conversion of AR to resorufin was observed to be as efficient in an anaerobic chamber (oxygen concentration <5 ppm) using degassed water as it was in air  compared with brain or muscle mitochondria, whereas they are equally sensitive to reverse flow mediated H2O2 release (i.e. ∼50% reduction in the rate of H2O2 release when complex II linked substrate was used).It had previously been claimed that liver mitochondria produced more ROS than mitochondria from other tissues under comparable experimental conditionssues see f–i. Our 2O2 still holds, as long as inhibitors of HRP-independent AR conversion are added to the experiments.We verified that membranes of intact mitochondria are permeable to AR, but not to HRP. Therefore, the notion that the AR method quantitatively detects extramitochondrial release of Hex vivo, this conversion of AR to resorufin changes with the physiological state of the tissue, for instance, it increases with donor age and decreases under dietary restriction (Supplementary Second, our data provide a rationale for a novel carboxylesterase assay by measuring the PMSF-sensitive and HRP-independent conversion of AR to resorufin. In vitro, CES converts AR quantitatively to resorufin, following strict Michaelis–Menten kinetics f. In livFinally, our data support the speculation that mitochondria could function as sites of xenobiotic drug metabolism. If AR crosses mitochondrial membranes other molecules may have the same ability. Enzymatic reactions involving CES will frequently produce free radical species, either directly or indirectly. A localisation of carboxylesterases in the mitochondrial matrix with its high concentration of antioxidants might therefore be advantageous for the cell.


The ethical quality of animal research is important for many reasons, including for maintaining public support. We aimed to determine the reported attention to the ethical dimensions of the 3Rs  in critical care animal research published in 2012.2 statistic, with statistical significance accepted at P < 0.05.A data-collection form and instruction manual were created based on published recommendations, and completed for all consecutive critical care animal research  publications from January to June 2012 in three critical care journals. Predefined subgroups were by journal, sepsis model, and animal age, compared by using the χP = 0.023). No other statistically significant differences were found in reporting of any criterion by animal age, sepsis model, or journal.In total, 77 consecutive animal research publications were reviewed. Most studies did not report monitoring the level of anesthesia during invasive procedures, even when muscle paralytics were used, nor monitoring or treatment of expected pain. When euthanasia was used, the method was often not stated, and when stated, most methods were not appropriate for the species. A sample-size calculation was rarely used, and animal numbers were often poorly described. No studies performed a systematic review to ensure that the animal research would be useful and not simple repetition. Seventeen (22%) publications met the composite outcome of, if indicated, using anesthesia and pain control, and stating the method of euthanasia. Most studies were funded with public funds (foundation or government funding). Sepsis models less often met the composite outcome of, if indicated, using anesthesia and pain control, and stating the method of euthanasia (2 (7%) of 27 versus 15 (30%) of 50; Reported  ethical quality of animal research in three high-impact critical care journals during 6 months of 2012 was poor. This has important implications for the practice of critical care animal research. Biomedical animal research (AR) has an ethical dimension because it can cause harm, including pain, suffering, and early death, to sentient research subjects . To addrSeveral reasons exist to report and adhere to strict animal-welfare ethical guidelines in performing AR. First, animal pain and distress introduce uncontrolled confounding variables to research, violating the assumption of normal physiology and behavior , 5–10. PSecond, consideration of the interests of sentient animals is ethically appropriate, and the methods to monitor for and to treat animal distress should be described to ensure animal welfare –14.Third, poor treatment of experimental animals may lead to loss of public support. Recent surveys suggest that the public places high priority on animal welfare in science –17.The importance of ethical attention to animal welfare has been recognized before. Attempts have been made to address improving animal welfare in intensive care research , 18, 19.Of concern, the literature has shown a poor translation rate of AR to human medicine. This is true in critical care, for example, in the fields of sepsis –26, trauWe aimed to determine the reported attention to the ethical dimensions of the 3Rs in critical care AR published during 6 months of the year 2012. Of concern, we found that the reported ethical quality of AR in three high-impact critical care journals during the year 2012 was poor in several areas. during 6 months of the year 2012 to determine the reporting of a priori determined ethical-quality factors. These journals were chosen for convenience (they are available at our library) and familiarity (to the authors). No restrictions were made other than the study reported an AR experiment, defined as a procedure for collecting scientific data on the response to an intervention in a systematic way to maximize the chance of answering a question correctly or to provide material for the generation of new hypotheses . No other statistically significant differences were found in reporting of any criterion  of 27 versus 15 30%) of 50; 0% of 50;Reported  ethical quality of AR in three high impact critical care journals during 6 months of 2012 was poor. This is important for several reasons. First, pain and suffering cause changes in physiology, immunology, and behavior that confound interpretation and extrapolation of experimental results –10. ThusP value, power, and minimally important difference, were calculated, hence markedly improving the quality of an experimental result, we believe the authors would be likely to report this. A systematic review of the AR in a specific area also, we believe, would be likely to be reported as done, as these are time consuming, and strengthen the importance and validity of a study; few systematic reviews are reported in the AR literature, most of poor quality [It is important to point out that it could be argued that what was not reported was actually done. For example, it is possible that monitoring and treatment of postoperative pain was done, but not reported; or that explicit consideration of alternatives to animal models was often done but not reported because of space restrictions. Thus, it is possible that the ethical quality of the AR was good, and only the reporting was poor. Conversely, many of the poor-quality items might have been expected to be reported if they were indeed performed. For example, if a sample-size calculation for a primary outcome, including a  quality , 41. Mor quality , 39.We also argue that better reporting of appropriate monitoring of anesthesia and pain control could only improve the quality, perceived reliability, and public perception of the published AR. For example, if a scoring system to monitor and then treat pain were used, specific criteria were used to titrate anesthesia, or specific enrichment of animal husbandry were used, we believe that space limitations are not a good reason to omit any reference to these high-quality items.In addition, it is important to report consideration of alternatives to animal models, as these are being developed in new subject fields that animal researchers may not be aware of, and progress in alternative methods requires drawing attention to it , 42, 43.Poor methodologic quality of AR has been reported before , 30, 33.The findings of this study are concerning. The ARRIVE guidelines, supported by many high-impact journals, and other national guidelines, suggest inclusion in publications of many of the factors we found to be poorly reported, including: monitoring of the level of anesthesia, monitoring of pain by using a validated scale, titration of anesthesia and pain control to stated goals, optimal methods of euthanasia, details of animal husbandry, sample-size calculation, clear animal numbers stated in methods and all results, systematic review of the animal literature to justify the current study, and consideration of all possible alternatives to use of animals , 23, 37.Limitations of this study include the small sample size of publications reviewed, the limited scope to critical care AR, and the low power to detect differences between subgroups, particularly given multiple comparisons. Nevertheless, this study is the first to focus on the ethical dimension of AR in critical care, and reviewed a relatively large series of consecutive publications in three high-impact critical care journals by using an objective data-collection form and instruction manual. Whether our findings from this small critical care AR cohort generalize to most AR is unknown; however, we believe this is likely because critical care experiments are more invasive than most other AR.We believe that it is time for a serious debate about the methods of AR in critical care. Better attention to, and reporting of, ethical factors in AR can only improve the research quality, distress of the animals, and public perception of AR. Journal editors and reviewers, and funding agencies should use their influence to improve quality reporting of AR they publish and support , 53.We found that reported  ethical quality of AR in three high-impact critical care journals during 6 months of the year 2012 was poor. These findings warrant the attention of clinicians, researchers, journal editors and reviewers, and funding agencies. Improved attention to the 3Rs by these groups can only improve AR quality, animal comfort, and the public perception of AR.Reported ethical quality of animal research in three high-impact critical care journals during 6 months of 2012 was found to be poor.Better reporting of ethical factors in AR may improve the research report and improve public perception of AR.These publications are, arguably, the public face of science using mostly public funds; unless the ethical quality of animal research reporting improves, animal research may be in jeopardy of losing public support.


Pediatric health care workers (HCW) often perform, promote, and advocate use of public funds for animal research (AR). We aim to determine whether HCW consider common arguments (and counterarguments) in support (or not) of AR convincing.P < .05 considered significant.After development and validation, an e-mail survey was sent to all pediatricians and pediatric intensive care unit nurses and respiratory therapists (RTs) affiliated with a Canadian University. We presented questions about demographics, support for AR, and common arguments (with their counterarguments) to justify the moral permissibility (or not) of AR. Responses are reported using standard tabulations. Responses of pediatricians and nurses/RTs were compared using Chi-square, with Response rate was 53/115(46%) (pediatricians), and 73/120(61%) (nurses/RTs). Pediatricians and nurses/RTs are supportive of AR. Most considered ‘benefits arguments’ sufficient to justify AR; however, most acknowledged that counterarguments suggesting alternative research methods may be available, or that it is unclear why the same ‘benefits arguments’ do not apply to using humans in research, significantly weakened ‘benefits arguments’. Almost all were not convinced of the moral permissibility of AR by ‘characteristics of non-human-animals arguments’, including that non-human-animals may not be sentient, or are simply property. Most were not convinced of the moral permissibility of AR by ‘human exceptionalism’ arguments, including that humans have more advanced mental abilities, are of a special ‘kind’, can enter into social contracts, or face a ‘lifeboat situation’. Counterarguments explained much of this, including that not all humans have these more advanced abilities [the argument from species overlap], and that the notion of ‘kind’ is arbitrary . Pediatrician and nurse/RT responses were similar.Most respondents were not convinced of the moral permissibility of AR when given common arguments and counterarguments from the literature. HCW should seriously consider arguments on both sides of the AR debate. How we justify animal research (AR) is a controversial issue. Nevertheless, this is an important issue in pediatrics for several reasons. Pediatric health care workers (HCW) often perform (and are expected to perform) AR, promote AR directly with trainees and indirectly as role models, and advocate for use of public funds (from granting agencies and charitable foundations) toward pediatric related AR. Therefore, it might be expected that the arguments for and against AR are well settled. However, standard ethical arguments and counterarguments for AR are rarely discussed in the medical literature, and it is likely that many are not aware of the debate.prior injury/disease], and non-consensual [conducted with subjects who have not voluntarily agreed to participate] , non-therapeutic [does not aim at restoring the health of a research subject with icipate] . In otheicipate] . Second,icipate] ,3. Thirdicipate] ,5. The qThere are several general types of arguments used to justify AR -7. FirstAll pediatricians and pediatric intensive care unit nurses and respiratory therapists (RTs) who are affiliated with the Canadian University were e-mailed the survey using an electronic, secure, survey distribution and collection system . A cover letter stated that “we very much value your opinion on this important issue” and that the survey was anonymous and voluntary. We also offered the incentive that if the response rate was at least 70% we would donate $1000 to the Against Malaria Foundation or the PICU Social Committee. Non-responders were sent the survey by e-mail at 3-week intervals for up to 3 additional mailings.The development of the questionnaire followed published recommendations . To geneThe background section described the situation: “In this survey, ‘animals’ means: mammals, such as mice, rats, dogs, and cats. It has been estimated that over 100 million animals are used in the world for research each year. There are many good reasons to justify AR, which is the topic of this survey. Nevertheless, some people argue that these animals are harmed in experimentation, because their welfare is worsened. In this survey, ‘harmful’ means such things as: pain, suffering , and early death. We value your opinion on the very important issue of the ethical dimension of AR”.We presented demographic questions, 3 questions about support for AR, and 12 arguments with their counterarguments to consider. The arguments and counterarguments were introduced as follows: “a) First, we give you an argument to justify harmful AR, and we ask you if you agree with that argument; b) then we give some responses to the argument, and we ask if you think each response would make it harder for someone to justify harmful AR using the initial argument . All the arguments and responses in this survey are those commonly made in the literature on AR”. When each argument was presented it was followed with the question: “Is this a good enough reason to justify using animals in medical research?” When the responses to an argument were presented, they were introduced with the question: “Do any of the following responses make it harder for someone to justify AR using Argument X [i.e. make Argument X much less convincing]?” Responses were in a ‘yes’ or ‘no’ format.The study was approved by the health research ethics board of our university, and return of a survey was considered consent to participate.P < .05 after Bonferroni correction for multiple comparisons considered significant.We used REDCap as our survey management tool . This weThe response rate was 53/115 (46%). Demographics included: males 24/51 (47%); age <35 yr 5/52 (10%), 35-44 yr 20/52 (39%), and >44 yr 27/52 (52%); those who have done AR in the past 19/52 (37%), currently do AR 3/52 (6%), and never done AR 31/52 (58%).Responses are shown in Table Responses are shown in Table Responses are shown in Table We asked at the beginning and again at the end of the survey if “in order to achieve human benefits, research that results in harm to animals should be supported”. At the beginning, pediatricians responded ‘yes’ in 32/52 (62%); at the end in 24/41 (59%) [p = 0.77]. Finally, we asked “what is it about vulnerable humans  that makes it wrong to use them in experiments?” Pediatricians responded as follows: 5/42 (12%) “these vulnerable humans are able to experience things like pleasure, joy, happiness, sadness, pain, and suffering”, 11/42 (26%) “these humans are vulnerable to physical and psychological harm; using them in experiments is harmful for them”; 5/42 (12%) “we care about them”; and 21/42 (50%) “they are still human”.The response rate was 73/120 (61%). Demographics included: males in 12/72 (17%); nurses in 56/68 (82%) and RTs in 12/68 (18%); age <35 yr in 45/73 (61%), 35-44 yr in 11/73 (15%), and >44 yr in 17/73 (23%); have done AR in the past in 8/73 (11%), currently do AR in 0, and never done AR in 65/73 (89%).Responses are shown in Table Responses are shown in Table Responses are shown in Table We asked at the beginning and again at the end of the survey if “in order to achieve human benefits, research that results in harm to animals should be supported”. At the beginning, nurses/RTs answered ‘yes’ in 31/72 (43%); at the end in 19/59 (32%) [p = 0.20]. Finally, when asked about what makes it wrong to use vulnerable humans in experiments, nurses/RTs responded as follows: 12/59 (20%) “these vulnerable humans are able to experience things like pleasure, joy, happiness, sadness, pain, and suffering”, 20/59 (34%) “these humans are vulnerable to physical and psychological harm; using them in experiments is harmful for them”; 5/59 (9%) “we care about them”; and 22/59 (37%) “they are still human”.No statistically significant differences were found between the subgroups in responses to any of the three categories of arguments/counterarguments. At the beginning (p = 0.036) and end (p = 0.009) of the survey, nurses/RTs were less likely to support AR. There were no statistically significant differences in the response to the final question about what makes it wrong to use vulnerable humans in experiments.There are four main findings from this survey. First, pediatricians (62%) and nurses/RTs 43%) are supportive of AR. Second, ‘benefits arguments’ were usually thought sufficient to justify AR; however, when confronted with counterarguments pointing out that alternative research methods may be available, or suggesting that it is unclear why the same ‘benefits arguments’ do not apply to using humans in research, most were not as convinced. Third, almost all respondents were not convinced by ‘characteristics of NHAs arguments’ , including that NHA may not be sentient, or are simply property. Fourth, a large majority of respondents were not convinced by the main arguments that have been offered in favor of ‘human exceptionalism’ , those arguments that justify AR due to the benefits, but that claim the same benefits do not justify human research. These include arguments that humans have more advanced mental abilities than NHAs, that humans are of a special ‘kind’ , that humans can enter into social contracts, or that we face a lifeboat situation where human interests trump NHA interests -15. Fina3% are suPrevious public surveys have generally asked only whether people support AR for human benefit, and not asked people to evaluate their reasons for supporting (or not) AR . For exaThis study has several limitations. Response rates for pediatricians and nurses/RTs were 46% and 61%; thus we cannot rule out biased participation in the survey. Arguments/counterarguments presented needed to be short and concise, and this may have left out important details that would have influenced the understanding of and response to the text. The moderate sample size from one University limits the generalizability of our results. Nevertheless, this is the first survey we are aware of that asks any group not just to consider whether they support AR; rather, to consider the most common arguments in the literature in favor of and against AR. Strengths of this study include the rigorous survey development process, and the inclusion of the most common and accepted arguments in the literature. Future study should determine the generalizability of our results.In conclusion, when presented common arguments to justify AR, most respondents accepted ‘benefits’ arguments, and only a minority found the ‘characteristics of NHAs’ and ‘human exceptionalism’ arguments convincing. Most found the arguments to justify AR significantly weakened by common counterarguments, including those who initially found the ‘benefits’ arguments convincing. Engagement with and serious discussion of the arguments on both sides of the AR debate, deliberate extensive investigation of alternative research methods, and examination of the return on investment from AR and alternative research methods are potential ways forward in the debate about the moral permissibility of AR. We consider this survey an initial step toward this goal.


Arenimonas donghaensis is the type species of genus Arenimonas which belongs to family Xanthomonadaceae within Gammaproteobacteria. In this study, a total of five type strains of Arenimonas were sequenced. The draft genomic information of A. donghaensis DSM 18148T is described and compared with other four genomes of Arenimonas. The genome size of A. donghaensis DSM 18148T is 2,977,056 bp distributed in 51 contigs, containing 2685 protein-coding genes and 49 RNA genes. Arenimonas donghaensisTDSM 18148 (= HO3-R19T = TKACC 11381) was isolated from seashore sand [Xanthomonadaceae. So far, the genus Arenimonas contained seven species, Arenimonas donghaensis (type species) [Arenimonas malthae [Arenimonas oryziterrae [Arenimonas composti [Arenimonas metalli [Arenimonas daejeonensis [Arenimonas daechungensis [A. composti [Aspromonas composti [ malthae , Arenimoeonensis  and Arenungensis . These bungensis , rice rhungensis  and sediungensis , respectcomposti .Arenimonas strains are Gram-staining-negative, aerobic, rod-shaped, non-spore-forming, oxidase-positive, non-indole-producing, non-nitrate-reducing, containing iso-C16:0 and iso-C15:0 as the major fatty acids, phosphatidylglycerol and phosphatidylethanolamine as the major polar lipids, Q-8 as the major respiratory quinone, and possessing relatively high DNA G + C content (63.9–70.8 mol %) [The common characteristics of the ent 63.9–0.8 mol %Arenimonas species, we performed genome sequencing of strains A. donghaensisTDSM 18148, A. compostiTKCTC 12666, A. malthaeTCCUG 53596, A. metalli CF5-1T and A. oryziterraeTKCTC 22247. In this study, we report the genomic features of A. donghaensisTDSM 18148 and compare it to the close relatives.In order to provide genome information and determine genomic differences of A. donghaensisTDSM 18148 shares 93.1–95.7 % 16S rRNA gene identities with the other six type strains of Arenimonas species, A. malthae CC-JY-1T (DQ239766) (95.7 %), A. daejeonensis T7-07T (AM229325) (95.7 %), A. metalli CF5-1T (HQ698842) (94.6 %), A. oryziterrae YC6267T (EU376961) (94.3 %), A. composti TR7-09T (AM229324) (94.3 %) and A. daechungensis CH15-1T (JN033774) (93.1 %). A 16S rRNA gene based neighbor-joining phylogenetic tree of the related strains was obtained using MEGA 5.05 software [Strain A. donghaensisTDSM 18148 are Gram-negative, aerobic, non-spore-forming, straight or slightly curved rods, motile by means of a single polar flagellum. Colonies are yellowish white, translucent and convex on R2A agar after 3 d cultivation .A. donghaensisTDSM 18148 was determined using the Illumina Hiseq2000 [The whole-genome sequence of iseq2000  with theiseq2000  , amino acid transport and metabolism (8.7 %), coenzyme transport and metabolism (5.8 %), lipid transport and metabolism (5.1 %), translation, ribosomal structure and biogenesis (12.4 %), replication, recombination and repair (5.2 %), cell wall/membrane/envelope biogenesis (5.9 %), posttranslational modification, protein turnover, chaperones (6.3 %), general function prediction only (8.4 %), function unknown (7.3 %) and signal transduction mechanisms (5.3 %) , general function prediction only (8.5 %), function unknown (7.3 %) and signal transduction mechanisms (9.0 %). The remaining 242 unique genes (40.3 %) are not classified into any COG categories  value of 0.18, which means an approximate genus-wide nucleotide sequence homology of 82 %.There are 601 strain-specific genes for  unknown .3 % and n-genome  size of n-genome  and DnaSArenimonas genomes using CRISPRfinder program online [A. compostiTKCTC 12666. This CRISPR length is 5331 bp, with 29 bp direct repeat (DR) sequences be separated by 87 spacers.The clustered regularly interspaced short palindromic repeats (CRISPRs) mediate resistance to foreign genetic material and thus inhibit horizontal gene transfer . Screenim online  found thXanthomonadaceae were chosen for genomic based phylogenetic analysis, including the five Arenimonas genomes that were sequenced by us. In total, 1014 core protein sequences were extracted using the cluster algorithm tool OrthoMCL with default parameters [Arenimonas species clustered into the same branch (Fig. Fifteen available genome sequences of the family rameters . The neinch Fig. , which iA. donghaensisTDSM 18148, the TCA cycle is complete and hexokinase is absent in all the five Arenimonas strains. The proteins responsible for the oxidative phase of pentose phosphate pathway are also incomplete in five Arenimonas strains, this may be part of the reasons that the five Arenimonas strains can only use several single carbon sources.Similar to Arenimonas. The genomic based phylogeny is in agreement with the 16S rRNA gene based one indicating the usefulness of genomic information for bacterial taxonomic classification. Analysis of the genome shows certain correlation between the genotypes and the phenotypes especially on utilization of sole carbon sources.To the best of our knowledge, this report provides the first genomic information of the genus