Genomic variations are associated with the metabolism and the occurrence of adverse reactions of many therapeutic agents. The polymorphisms on over 2000 locations of cytochrome P450 enzymes (CYP) due to many factors such as ethnicity, mutations, and inheritance attribute to the diversity of response and side effects of various drugs. The associations of the single nucleotide polymorphisms (SNPs), the internal pharmacokinetic patterns and the vulnerability of specific adverse reactions become one of the research interests of pharmacogenomics. The conventional genomewide association studies (GWAS) mainly focuses on the relation of single or multiple SNPs to a specific risk factors which are a one-to-many relation. However, there are no robust methods to establish a many-to-many network which can combine the direct and indirect associations between multiple SNPs and a serial of events . In this paper, we present a novel deep learning model based on generative stochastic networks and hidden Markov chain to classify the observed samples with SNPs on five loci of two genes (CYP2D6 and CYP1A2) respectively to the vulnerable population of 14 types of adverse reactions.A supervised deep learning model is proposed in this study. The revised generative stochastic networks (GSN) model with transited by the hidden Markov chain is used. The data of the training set are collected from clinical observation. The training set is composed of 83 observations of blood samples with the genotypes respectively on CYP2D6*2, *10, *14 and CYP1A2*1C, *1 F. The samples are genotyped by the polymerase chain reaction (PCR) method. A hidden Markov chain is used as the transition operator to simulate the probabilistic distribution. The model can perform learning at lower cost compared to the conventional maximal likelihood method because the transition distribution is conditional on the previous state of the hidden Markov chain. A least square loss (LASSO) algorithm and a k-Nearest Neighbors (kNN) algorithm are used as the baselines for comparison and to evaluate the performance of our proposed deep learning model.There are 53 adverse reactions reported during the observation. They are assigned to 14 categories. In the comparison of classification accuracy, the deep learning model shows superiority over the LASSO and kNN model with a rate over 80 %. In the comparison of reliability, the deep learning model shows the best stability among the three models.Machine learning provides a new method to explore the complex associations among genomic variations and multiple events in pharmacogenomics studies. The new deep learning algorithm is capable of classifying various SNPs to the corresponding adverse reactions. We expect that as more genomic variations are added as features and more observations are made, the deep learning model can improve its performance and can act as a black-box but reliable verifier for other GWAS studies. Genomewide association study (GWAS) is to explore the correlations among genomic variations and a series of genetic risk factors. It aims to reveal the complexity of the changes of DNA sequence and their corresponding effects on gene expression, proteins and finally leading to the macro factors such as disease susceptibility, prognostic factor and pattern of metabolism etc . With thThe objective of pharmacogenomics is to study how the comprehensive genomewide variations (i.e. groups of relevant SNPs) systematically affect the patterns of pharmacokinetics and pharmacodynamics of individuals and the variation of biological patterns to the same or similar substances in different subjects . It is wThe literature review shows that with the disseminations of new genome sequencing technologies especially the deployment of NGS, the cost-effective of genome sequencing has reached a good ratio that makes large-scale individual studies feasible in both laboratory and clinical context. However, as a large sum of sequencing and SNPs data is generated at low cost, the conventional GWAS analytic methods have become the bottleneck for many study purposes that stress the complex association network connected to numerous SNPs and events with direct, indirect, unilateral and bilateral linkages. As indicated in Fig. In order to solve this difficulty, we propose the machine learning method which can effectively seal the complexity of the SNPs and adverse drug associations into a computational model trained by empirical data. This study will analyze the complex associations among SNPs on two loci (CYP2D6 & CYP1A2) of cytochrome P450 enzymes and the occurrences of adverse drug reactions (ADRs) which are observed in a clinical observation. The goal is to demonstrate the proposed deep learning model can accurately classify the human participants with the different combinations of SNPs to the susceptibility of ADRs. The overall procedure is shown as Fig. The blood samples collected from the 83 human participants were kept in ultra-low temperature freezer before processing. The DNA was extracted and preserved at −80 °C. The alleles on the CYP1A2 and CYP2D6 gene were sequenced and genotyped by Polymerase chain reactions (PCR) with Pfu enzyme (using TianGEN Pfu PCR Mastermix kit). The sizes of the amplified five alleles of CYP2D6*2, *10, *14 and CYP1A2*1C, *1 F are respectively 312 bp, 443 bp, 235 bp, 597 bp, and 847 bp. The electrophoresis for the CYP2D6 locus was performed with 100 ng DNA in 2.5 % of agarose gel for 40 min. The gel with ladders for the CYP2D6 locus is illustrated in Fig. The sequencing and genotyping of the two genes was done by Applied Biosystem 3130xl with the Invitrogen Bigdye® terminator v3.1 cycle sequencing kit (Life Technology). The frequency of different SNPs is presented in Table There are 53 ADRs reported from clinical observation. The ADRs are categorized into 14 groups as listed in Table In order to explore the association of ADRs and SNPs on the two target, two data sets are set up respectively for data conversion of the trial group and the blank group. The data include: group ID, doses, the genotypes of the alleles and all reported ADRs. The five alleles  of the two loci are coded by 15 dummy variables to indicate specific allele combinations of the of the diploid , where we use “1” to represent a positive result to the corresponding allele and use “0” to represent a negative result to the corresponding allele. Accordingly, we use ordinal variables to represent the ADRs, where a “2” means an ADR with extremely increased level, a “1” means ADR occurrence with increased level, a “0” means no ADR occurrence, a “−1” means an ADR with decreased level, and a “−2” means ADR with extremely decreased level. All missing data are filled with “0” too. This preprocessing strategy will not add extra information to the model and thus it minimizes the influence to the outcome of data analysis.P(X), we assume X = , then P(X) can be modeled by a given training of observed samples. Since the training data set D is acquired from different individuals, we can assume them as independent from P(X). In order to model P(X|D), we use a Markov chain formed by the data points. The transition matrix between the points is considered reflecting the ground truth distribution of P(X). A two-dimension Gaussian distribution that contains 2000 states points is illustrated in Fig. In the generative stochastic networks (GSN) X− is a sample set independently from P(X). The probability of P(X|X−) is calculated by the Bayes' theorem. It is noted that both P(X|X−) and P(X) can be re-evaluated and be used to produce another distribution of sample space if the prior distribution P0(X|X−) is known. Their relation is presented by β is a constant independent of X.Then we assume xi ~ P0(X−|xi) and xi + 1 ~ P, where xi is the ith outcome regarding the probabilities of ADRs of the study, and θ stands for the parameters (mean and covariance) of the Gaussian distribution. A hidden variable Hi is assumed to govern the result xi via a serial of unknown associations. Thus, the hidden Markov chain with X and H as its state variables can be expressed by Eq.  with the parameters d by Eq. :1\documeH0 and which can determine x0 and H1. Unfortunately, the value of H0 is not directly acquired because we do not have direct information regarding H0. An alternative method to get the value of H0 is to set it as a constant  where d stands for the dimension of training data sample. Finally, the time complexity of the algorithm is a polynomial function of D set at the beginning.As shown above, the training subsets are generated by a Gibbs sampling procedure in order to measure ined DAE . ThroughIn order to evaluate the effectiveness of the new algorithm, we provide two conventional algorithms from the previous studies , 16 for n test samples can be indicated be the average of accuracy loss defined in Eq.  in the predictions to all categories compared to M1  and M2 . In the evaluation of the impacts of sample size and noise, we continuously change the volume of the training set and noise, and then we compute the average accuracy losses (la) given a specific ratio of the training set and test data set. The whole data set is respectively partitioned into the training set and test data set with the ratios from 1:9, then 2:8, and to 9:1, where the data points are randomly selected. The test results are shown in Fig. la decreases) when more data are allocated to the training set. The GNS generative algorithm (M3) has the lowest average loss (la) when the training/testing set ratio is over 0.2. This indicates that M3 starts and keeps having the best performance over the three algorithms when the training set is 20 % in size of the testing set.The results from Table a be the average value of a given feature. The algorithm changes the mean from 0 to 0.5a with the step of 0.1a. And the variance is defined as half of the corresponding mean. By the above settings, the impact of noise on the prediction can be evaluated by observing the average loss of accuracy (la) at different noise levels. The evaluation results are illustrated in Fig. To evaluate the influence of noise level to model performance, we divide the whole data set evenly in half for training and testing (i.e. the training/testing set ratio is 5:5). The Gaussian noise is assumed to affect all the features of the sample points in the training sets. Since the stratification effects brought by the factors such as ethnicity, geographic region, and social environment can be adjusted by expanding the whole data set, and no systematic bias is identified, we assume all the features are independent and thus the noise can be effectively controlled by the mean and variance estimated based on Gaussian distribution. Let The data from Fig. Genomewide association study or GWAS is one of the main trends in genomics research. GWAS aims to explore the variations across the human genome in order to identify the genetic risk factors associating with specific events in health  and to generalize the research results in the population. GWAS provides a valuable solution for pharmacogenetics whose goal is to identify the DNA sequence variations or SNPs and their association with drug metabolism, efficacy and adverse effects . The conOne solution to reveal the associations inside the network of SNPs and ADRs is to use machine learning methods in which the complex internal relations can be concealed into a black box. After trained by the labeled data set, the classifier will develop a capacity to differentiate the latent patterns and label the new data. In this study, we propose a deep learning model based on Generative Stochastic Network (GSN) as the implementation to solve the associations between the SNPs on two loci of cytochrome P450 enzyme and the ADRs in clinical observation. The generative model is considered more cost-effective compared to the conventional Bayesian models because it does not need to compute the joint likelihood and posterior distribution at a high computational cost. The GSN will learn the transition operator of a hidden Markov chain via the labeled training set, and the probability distribution of the training set can be estimated by the stationary distribution of the transition operator learned from the training set. This generative model is more efficient compared to the Bayesian algorithms because in a certain state of the Markov chain the transition operator is conditional on its previous state thus it only needs to compute a small step between the current and former states with a significantly lower cost at computation compared to the Bayesian models.On the other hand, GWAS requires a large sample of observations covering numerous SNPs (i.e. thousands of genetic loci for example) in order to acquire a result of associations between SNPs and the due events with an acceptable degree of power. This usually causes the potential problem of a false positive result because except for the high cost of organizing a large-scale clinical study, the SNPs truly related to the risk factor are likely to be confounded by the false relations of the SNPs in the same or the adjacent high DL region. This risk is unable to be effectively prevented by the current mainstream GWAS analytic methods so far. The advantage of using machine learning model to classify the associated and unassociated SNPs is that the model performance is expected to be enhanced by increasing the size of the training set which can be acquired from empirical data. The GSN based deep learning model shows its robustness in that it is insensitive to system noise compared with other non-generative models. And this feature is important to classify a skewed sample.The current studies imply that the cytochrome P450 enzymes play an important role in the metabolism of most drugs  and the The uncertainty of ADRs is one of the major threats to healthcare. The economic loss caused by various ADRs relevant to medications is over 100 billion dollars annually in the US, and the expense of treatments for ADRs are actually comparable to the cost of the normal healthcare , 18. A sThe study results indicate the GSN based generative algorithm is able to provide reliable and accurate predictions of risk levels to different ADRs after the deep learning model is trained by a relatively small data set . As impl


We aimed to evaluate and define the general clinical applicability and impact of pulmonary radioaerosol mucociliary clearance (PRMC) on the work up of patients suspected of having primary ciliary dyskinesia (PCD). In addition, we wanted to evaluate the accuracy of the reference values used in the PRMC test.99mTc-albumin colloid aerosol was carried out on 239 patients (4–75 years of age) during a 9-year period. All were referred to the nuclear medicine department because of clinical suspicion of PCD. The results were compared primarily to results from nasal ciliary function testing, to electron microscopic (EM) examination of the ultrastructure of the cilia, and to the final clinical diagnosis.Measurement of PRMC after inhalation of Of the 239 patients, 27 ended up with a final clinical diagnosis of definitive PCD. No patients with a PRMC test that was normal or otherwise not consistent with PCD ended up with PCD as final clinical diagnosis (though a minority of patients in this group ended up unresolved in regard to PCD). Forty percent of patients with an abnormal PRMC test ended up with PCD as final clinical diagnosis. Furthermore, the PRMC test had a high rate of conclusive results (90 %). Children <14 years of age with normal PRMC measurements showed significantly faster lung clearance than adults with similarly normal PRMC measurements.To this date, PRMC is the only test providing evaluation of the mucociliary clearance of the entire lung. Its greatest strength is its ability to reject a suspected PCD diagnosis with great certainty. In our material, this accounted for 2/3 of referred patients. In addition, the test has a high rate of conclusive results. According to our analyses, reference equations on children would benefit from updated data. Mucociliary clearance is known to be an important innate defence mechanism against inhaled microbes and irritants. Potentially harmful substances get trapped in the mucus layer lining the airways, and subsequently, the synchronised movement of the cilia propels the mucus to the pharynx where it will be swallowed. This mechanism can be hampered either by conditions affecting the constitution of the mucus  or by conditions directly affecting the movement of the cilia. This last group of conditions has traditionally been divided into primary and secondary ciliary dyskinesia (PCD and SCD). PCD is a genetic disorder caused by mutations in genes in relation to the axoneme. Clinically, the patients present with cough, dyspnea, and recurrent airway infections starting from early childhood. However, it is both genetically and phenotypically a heterogeneous condition presumably reflecting the molecular complexity of the axoneme, and for this same reason, diagnosing these patients can be challenging. SCD on the other hand includes a variety of temporary, acquired defects of ciliary movement caused by viral or bacterial infection or by certain air pollutants. In some instances, it can be difficult to differentiate SCD from PCD because of overlapping findings in the tests meant for diagnosing PCD .Although there is no definitive evidence that early diagnosis of PCD is beneficial, there is emphasis on finding and treating these patients as early as possible, since early and aggressive treatment of lung infections is thought to be crucial in the prevention of future lung damage –4.The diagnosis relies on the clinical assessment of the patients combined with an array of different tests. These typically include measurement of nasal nitric oxide  where a low NO-value points towards PCD and vice versa, electron microscopic (EM) examination of the ultrastructure of the cilia, and microscopy of ciliary beat pattern and frequency. Genetic testing is developing rapidly, but to this date, only some of the disease-causing genes are known and currently around 65 % of patients could be detected this way . Highly While EM of the cilia and ciliary function testing are both time consuming and somewhat invasive, nasal NO measurement is a fast, non-invasive tool that can generally be used from 5 years of age (modified techniques have been shown to be applicable from 2.5 years)  when it The aim of this study is to evaluate and define the general clinical applicability and impact of PRMC on the work up of patients suspected of having PCD. Moreover, the reference values used today will be scrutinised—especially their accuracy when used in the work up of children.The study was carried out as a register-based follow-up study. The patients were included continuously over 9 years, and their final clinical diagnosis was evaluated at the end of the study period.In total, 239 patients were included in the study, and all were referred to the nuclear medicine department for PRMC test because of clinical suspicion of PCD. The vast majority of these had PRMC done as part of their work up for PCD in the National PCD Centre in Copenhagen at Rigshospitalet, whereas 64 patients were referred directly from departments of pulmonary medicine in other hospitals, and they were only referred to the National PCD Centre in Copenhagen if the clinical suspicion of PCD sustained after completion of the PRMC test.Preceding the PRMC test, patients were informed that they had to be without symptoms of acute airway infections 4–6 weeks prior to the test. Some patients had their PRMC test repeated during the study period; but in this paper, when nothing else is stated, only results from the first PRMC test are used.99mTc-albumin colloid with a mass median aerodynamic particle diameter of 3.4 μm. They did 20 tidal breathings with slow inspiration followed without breath hold by forced expiration, and the radioaerosol was administered during the whole inspiration phase. Inhalation volume was not measured. Immediately after the inhalation procedure, the patients rinsed their mouth by gargling three times. Thereafter, lung radioactivity was detected by placing the patients in a supine position against a posteriorly positioned gamma camera, and it was ensured that a target rate of 1000 counts per second was obtained. If not, extra inspirations were made.The PRMC technique has been described in detail earlier . In brieMeasurement of lung radioactivity was performed for the next 2 h with static acquisitions at 0, 60, and 120 min together with two 20-min dynamic acquisitions (films) in the first hour. This was supplemented with a 15-min static acquisition after 24 h.81mKr-gas scintigram. The initial 99mTc aerosol distribution was compared to this 81mKr ventilation distribution by calculating a penetration index (PI), which reflects how far the radioaerosol had penetrated into the lungs, thereby indicating how far the radioaerosol had to move before being cleared from the airways. Using our reference equations which are based on a cross-sectional study of 53 healthy never-smoking adults (18 to 84 years) [1 and pLR2). These predicted values were then compared to the actual measured lung retention at 1 and 2 h (LR1 and LR2) corrected for background and physical decay.Regional ventilation distribution was assessed by an 4 years) , the PI,Z-score for LR1 and LR2, respectively. The upper limit for a normal test was defined as a Z-score of >1.645 , since an abnormal mucociliary clearance can only be too slow, resulting in too high retention.The difference between predicted and actual lung retention divided by the standard deviation was calculated as a The two 20-min dynamic acquisitions were used to assess mucus transport in the larger airways. When bolus transport occurred, distinctive radioactive boluses could be seen to move through the main stem bronchi and the trachea Fig. . The mov1 and LR2 outside the predicted values? (2) Was the bolus transport in the trachea abnormal? And (3) was focal retention seen in the airways after 24 h? A “yes” to all questions indicated abnormal clearance, while “no” to all indicated normal clearance. A regional impaired clearance was the case, if there were one or a few focal retentions at 24 h, while the other two parameters were normal. In other cases of inconsistencies, the test was read as inconclusive (see below).The interpretation of the PRMC test was based on the following three questions: (1) Were LRGenerally, the interpretation of the PRMC tests can be divided into three main groupings: (1) where the test is clearly abnormal rendering PCD or SCD possible explanations, 2) where the test is inconclusive whereby PCD cannot be ruled out, or (3) where the test is normal or otherwise not consistent with PCD. If for example one or a few focal retentions were seen at 24 h, this would imply overall normal clearance with only regional impairment. Such a test would therefore be placed in group (3). As another example, some patients show signs of SCD but not PCD , and such a test would as well be placed in group (3). This means that PRMC has the ability to discern between PCD and SCD in some but not all cases. Examples of static acquisitions from an abnormal, a normal, and a regional abnormal test, respectively, are seen in Fig.  where th1 and LR2 values, bolus transport, and 24-h retention, or (3) very peripheral radioaerosol deposition, which can cause the predicted normal range for lung retention  to include a retention of 100 %. This problem is minimised by means of the inhalation technique described earlier, which serves to promote deposition of the radioaerosol in the central airways.An inconclusive test is usually a result of one of the following causes: (1) cough during examination, (2) inconsistency between LRAs noted, if the patient coughs during the first 2 h of the PRMC test, an apparently normal test will be interpreted as inconclusive. This is due to the fact that the clearance measured may be the result of cough clearance instead of clearance due to ciliary movement. Hence, an effective cough clearance would be able to disguise a true abnormal clearance leading the test to appear as being normal . In contrast, an abnormal test is still considered reliable despite coughing . Consequently, a staff member monitored the patients during the first 2 h of the test and registered time and number of coughs and throat clearing. Subsequently, all patients who had coughed in this timeframe had to perform a 1-min voluntary cough clearance test immediately after the 2-h acquisition, which served as a measure for the effectiveness of their coughing with regard to airway clearing. If this test showed minimal cough clearance, an otherwise normal PRMC test could be regarded as being normal provided that the cough during the first 2 h of the test had been very limited . The voluntary cough clearance test is not thought to affect the interpretation of the 24-h scan, since this scan is predominantly determined by retention in the smaller bronchi, bronchioles, and possibly bronchiectasis which is only marginally affected by cough clearance, since coughing is thought mainly to affect the first six airway generations . In addiThe final clinical diagnoses were primarily obtained via national electronic hospital records, in which we were able to search for each patient by means of their unique social security number. In a few cases, this information was complemented with non-electronic records from the National PCD Centre in Copenhagen. Results from EM and ciliary function testing were found in a database in the National PCD Centre in Copenhagen where these tests are being performed. A description of these two methods can be found in the ERS recommendations for PCD . All patOptimally, all patients in the study should have had their tentative PCD diagnosis confirmed or refuted after the completion of both thorough clinical assessment, nasal NO measurement, EM, ciliary function testing, PRMC, and in some cases gene testing. Because of the set-up of the study, the different tests were done only when considered pertinent by the treating doctor, meaning that only some of the patients were examined this comprehensively.For a minority of the patients (32 out of 239), a conclusion could not be reached with regard to their final clinical diagnosis. This was most often due to lack of information in the national electronic hospital records or due to on-going work up of a patient with contradicting test results.t test for means was used for comparisons unless both groups were larger than 30, in which cases a z test was performed instead. Comparisons were done between PI in patients with a normal and abnormal PRMC test and between PI and Z-scores for LR1 and LR2 for children vs. adults with a normal PRMC.Two-sample The Danish Data Protection Agency has approved the study.In total, 239 patients with an age range of 4 to 75 years were included in this study Table . 23.8 % No patient with a PRMC test that ruled out PCD ended up with PCD as final clinical diagnosis Table —that is,The final clinical diagnoses of patients with an abnormal PRMC test were somewhat more variable. 40 % ended up with verified PCD while 39 % had the PCD diagnosis rejected—that is, 39 % of the positive PRMC tests were false positives. At the end of the study, 21 % had an uncertain diagnosis. A similar variability in the final diagnoses was seen amongst patients with an inconclusive PRMC test. PCD was rejected in 64 % of cases while it was verified in 16 %. At the end of the study, 20 % had an uncertain diagnosis.1 and LR2 values within the normal range while bolus transport and 24-h static acquisition were abnormal.The highest percentage of patients ending up with verified PCD as final clinical diagnosis were seen amongst children 4–14 years old  while no patients ≥55 years ended up with the final clinical diagnosis of PCD ).Z-scores and PI to age was compared amongst patients who had a perfectly normal PRMC test , a normal EM was found while the rest of the tests were abnormal together with a final clinical diagnosis confirming PCD. This is in line with a growing number of publications showing normal EM tests in PCD patients , 25, 26.1 and LR2. But if PI had been significantly higher in patients with an abnormal PRMC test, this would have implied that we did not to a satisfactory degree take into account the magnitude of PI when calculating predicted values. Fortunately this does not seem to be the case, which means that abnormal test results are most likely caused solely by slow clearance of the radioaerosol.We found no significant difference between the initial aerosol distribution in the lungs (indicated by the mean PI) in patients with a normal and abnormal PRMC test. The magnitude of PI is accounted for when calculating predicted values for LRZ-scores for LR1 and LR2) in children than in adults. This might be an expression of the fact that children under 14 years of age have smaller lungs and thereby generally shorter airways than adults [When comparing children and adults presenting perfectly normal PRMC tests, our results show markedly faster clearance  than the mean PI seen in the corresponding adults. According to the discussion above, this should not affect the results of the test seeing that the magnitude of PI is accounted for in the calculation of predicted LRWhatever the explanation, according to our results, it seems fair to state that the reference material used for the PRMC test today does not in a satisfactory degree take into account the effect of age (or height) on lung clearance, which is most evident for children under 14 years of age. This is not surprising since our reference material is based upon a cross-sectional study of 53 healthy never-smoking adults (18 to 84 years) meaning that predictive norms have been extrapolated for ages <18. In the reference material, the dependency of age on PRMC was weak . But accBased on our experience with work up of patients suspected of having PCD, we propose the following algorithm for PCD investigation Fig. .Fig. 4SuWhen patients have been referred to a specialist centre with expertise in PCD work up, the ensuing work up should take into account the strength of suspicion of PCD based on history and clinical presentation of the patient . HoweverPatients with initially high suspicion of PCD and all patients with abnormal or inconclusive nasal NO values should have PRMC performed. If the PRMC test is normal, PCD can be ruled out in accordance with the fact that we found no false negatives in the present study. In our setting, PCD could be ruled out in roughly two out of three patients. An inconclusive test would require a re-test (in the present study 80 % acquired a conclusive result after only one re-test) while an abnormal test should lead to more intensive work up with EM, ciliary function testing, and possibly gene testing.One of the main strengths of the present study is that it shows what to expect of the PRMC test if implemented in the everyday clinical work up of PCD. Another strength is the large number of patients that are included. By far, it is the largest study to date concerning clinical use of the PRMC test. Furthermore, even when taking into account the weaknesses mentioned in the following, the fact that no patient with a normal PRMC test ended up with PCD as final clinical diagnosis is quite convincing. It is also worth noticing the very high rate of conclusive results achieved with the PRMC test.The main weakness of this study is that all of the PRMC results have been used in the subsequent work up of the patients, which means that these results have inevitably affected the final clinical diagnoses. In addition, not all patients underwent the same thorough work up for PCD. This was due to the fact that in some patients, the assumed PCD diagnosis was easily rejected leaving some tests unnecessary in the everyday clinical work up for PCD. It would of cause be advantageous to make a similar study with blinded results and with strict protocols for the work up of all patients suspected of having PCD. On a smaller scale, this was done by Marthin et al.  in our sIn this study, we have evaluated the use of the PRMC test in the work up of patients suspected of having PCD. To this date, it is the only method for testing the mucociliary function of the lower airways, and when compared to other existing tests, one of its great strengths is that it is not affected by secondary mucociliary defects affecting nasal cilia. In addition, the test is non-invasive and is applicable to children as young as 5 years of age. The method is relatively easy to introduce and perform and could therefore potentially be of interest for general nuclear medicine departments if used for research purposes such as to investigate possible links between mucociliary dysfunction and pathophysiology of lung diseases or pharmacological challenges to the mucociliary apparatus . But if From the above, we can conclude the following: (1) in the present study, we found no false negatives. We therefore believe that PRMC has the ability to reject a suspected PCD diagnosis with great certainty, when used in daily clinical work up (in the present study in 2/3 of referred patients). (2) PRMC has the ability to find candidates for further work up. In the present study, 40 % of patients with an abnormal PRMC test ended up having PCD as final clinical diagnosis. This number will of course depend greatly upon the group of patients being investigated. (3) PRMC has a high rate of conclusive results (90 % in the present study). (4) In addition, our results indicate that there might be a need for reference material for children.


Pharyngeal segmentation is a defining feature of vertebrate embryos and is apparent as a series of bulges found on the lateral surface of the embryonic head, the pharyngeal arches. The ancestral condition for gnathostomes is to have seven pharyngeal segments: jaw, hyoid, and five posterior branchial arches. However, within the sarcopterygians, the pharyngeal region has undergone extensive remodelling that resulted in a reduction in the number of pharyngeal segments, such that amniotes have only five pharyngeal arches. The aim of this study is to probe the developmental basis of this loss of pharyngeal segments.Hoxa2 aligns with the second pouch and arch in both chick and catshark, while Hoxa3 is transiently associated with the third arch and pouch. Surprisingly, we have found that Hoxb1 expression is spatially and temporally dynamic and that it is always associated with the last most recently formed pouch and that this domains moves caudally as additional pouches are generated.We have therefore compared the development of the pharyngeal arches in an amniote, the chick, which has five segments, with those of a chondrichthyan, the catshark, which has seven segments. We have analysed the early phase of pharyngeal segmentation and we find that in both the most anterior segments form first with the posterior segments being added sequentially. We also documented the patterns of innervation of the pharynx in several vertebrates and note that the three most anterior segments receive distinct innervation: the first arch being innervated by the Vth nerve, the second by the VIIth and the third by the IXth. Finally, we have analysed Hox gene expression, and show that the anterior limit of We propose that the first three pharyngeal segments are homologous, as is the posterior limit of the pharynx, and that the loss of segments occurred between these two points. We suggest that this loss results from a curtailment of the posterior expansion of the pharyngeal endoderm in amniotes at relatively earlier time point, and thus the generation of fewer segments. Pharyngeal segmentation is a characteristic of all vertebrates. During development it is first seen in the appearance of a series of bulges on the lateral surface of the head, the pharyngeal arches . These pLatimeria and the lungfishes, the sister group to the tetrapods [A general feature of gnathostomes is to have seven pharyngeal segments; jaw, hyoid, and five posterior branchial (gill-bearing) arches . This aretrapods –6. HowevThese alterations to the pharyngeal region that occurred with the evolution of the tetrapods are obviously underpinned by changes to the developmental programme. The loss of overt pharyngeal segmentation is due to the overgrowth of the second arch to cover the more posterior arches, followed by the fusion of the caudal edge of the second arch to the subjacent tissue which results in the internalisation of the posterior arches . HoweverThe segmentation of the endoderm, which results in the formation of the pharyngeal pouches, is central to the development of the pharyngeal arches . The phaGallus gallus), an amniote, which has five segments, to those in the catshark (Scyliorhinus canicula), a chondrichthyan, which has seven segments. We have analysed the formation of pharyngeal pouches, patterns of innervation of the pharyngeal arches, and the expression of Hox genes, which are markers of axial identity in the pharynx, and in particular how these relate to the endodermal pouches. Our data support the view that the three most anterior pharyngeal segments are homologous between different vertebrate classes, and that the reduction in the number of segments must have been achieved by a loss of those lying more caudally. Notably, we find that in both chick and catshark embryos Hoxb1 expression marks the posterior limit of the pharynx. However, this expression pattern is spatially and temporally dynamic, with Hoxb1 labelling the posterior limit of the pharynx at early and late stages irrespective of the number of segments that have been generated. Thus we conclude, that the posterior limit of the pharynx is likely to be homologous across the gnathostomes and that the reduction in the number of pharyngeal segments that accompanied the evolution of the tetrapods will have involved a heterochronic shift such that the posterior expansion of the pharynx was curtailed at a relatively earlier time point and thus less segments were generated.To begin to address the route through which the number of pharyngeal segments has been reduced, we have compared the development of the pharyngeal arches in the chick , staged [Fertile hen’s eggs were incubated at 38 °C to the required stages (HH st)  and the , staged  and fixePreviously fixed embryos were washed three times 30 min in PBS/1 % TritonX-100 (PBSTx) before being washed in a blocking solution of 10 % goat serum in PBSTx twice for one hour at room temperature. The relevant primary antibody was diluted in the blocking solution with 0.02 % sodium azide and the embryos incubated at 4 °C for 1–2 weeks. Embryos were then rinsed in blocking solution and washed three times for one hour in blocking solution before adding the secondary antibody diluted in blocking solution with 0.02 % sodium azide. This was incubated at 4 °C for 1–2 weeks. The primary antibodies used were rabbit anti-laminin at 1:100 (Sigma); mouse anti- NFM 1:10000 (Zymed). Secondary antibody was Alexa 488-conjugated goat anti-mouse IgG, and Alexa 568 goat anti-rabbit IgG, both used at 1:1000 (Molecular Probes). For sectioning, embryos were washed into PBS, embedded in gelatin, fixed, and vibratomed at 50 μm slices.2O2 in methanol for 1 h and then rehydrated. Embryos were then treated with 10 μg/ml Proteinase K in PBST for 20 min, postfixed with 4 % PFA/0.1 % gluteraldehyde and then washed twice in PBST. The embryos were incubated at 70 °C in hybridisation buffer  for 1 h, followed by overnight at 70 °C in digoxigenin-labelled riboprobes. Embryos were washed four times with hybridisation buffer at 70 °C, then three times 30 min with MABT , and blocked with 2 % BBR (Roche Diagnostics)/20 % goat serum/MABT and incubated overnight in anti-DIG-AP antibody (Roche) diluted 1:2000 in the same block. Embryos were washed extensively with MABT and the alkaline phosphatase activity detected using NBT and BCIP in NTMT. The reaction was stopped by washing in MABT and fixing in 4 % PFA.Previously fixed embryos were washed twice in PBST (PBS + 0.1 % Tween-20), dehydrated through a methanol series in PBST, bleached with 6 % HWe used anti-laminin immunostaining to conduct a general analysis of the formation of the pharyngeal pouches in both chick and catshark embryos. The pharyngeal segments form first anteriorly and then subsequently posteriorly, in both species Fig. . The firAn examination of the patterns of cranial nerve innervation of the pharynx in different vertebrate embryos can prove useful in focussing on which specific arches are likely to have been lost. We have therefore documented the patterns of innervation of the pharyngeal segments in two amniotes, chick and mouse, which have five segments, in a chondrichthyan, the catshark, which has seven segments, and in the lamprey which has nine. Wholemount immunofluorescence with an anti-neurofilament antibody is shown in Fig. Hox genes are useful as they can be used as markers of axial identity. Although the expression patterns of Hox genes in the pharyngeal arches have been well documented in amniotes, these studies have largely reported expression within the mesenchyme of the arches and they have often not explicitly analysed the limits of expression of these genes within the pharyngeal endoderm [To ascertain which pharyngeal segments/pouches have been lost from extant tetrapods, we need to be able to align the reduced number of posterior pouches of an amniote, in this case the chick, with those of the catshark, which represents the more basal condition. With regard to this issue, endoderm , 18.Hox1-5 gene paralogues, as these are known to be expressed in the pharyngeal region [Hox genes. However, we found that Hoxa2 was expressed in the second arch mesenchyme and the endoderm posteriorly from the second pouch  has been described previously and it is broadly similar to the situation observed in amniotes, with Hox genes of paralogous groups (PG) 1–4 being expressed in the pharyngeal region, and Hox gene of paralogous groups (PG) 5–8 not being expressed [Hoxd3 expression had shifted posteriorly and was associated with the fourth arch, and that this segment additionally expressed Hoxd1, d2 and d4. The precise relationship between the expression of Hox genes and the pharyngeal pouches has not been scrutinised in any chondrichthyan, but such analysis is vital if we wish to align the pharyngeal pouches across the gnathostomes. The key questions are: Does Hoxa2 expression align with the second pouch in both amniotes and a chondrichthyan, and does Hoxb1 expression which highlights the fourth and last pouch of amniotes align with the fourth pouch in a chondrichthyan, or is this gene expressed by multiple posterior pouches, or indeed only in the sixth and last formed pouch?The overall relationship between the pharyngeal arches and xpressed . There wHoxa2, Hoxa3 and Hoxb1 and we therefore sectioned wholemount in situ hybridisations of catshark embryos processed for these genes. We found that Hoxa2 expression was present in the mesenchyme of the second and more posterior arches and the endoderm posterior of the second pouch between stages 20 to 24 (Fig. Hoxa3 expression was found in the mesenchyme of arch 3 at stages 21 to 24, but that expression in the endoderm of the third pouch was weak at stage 21 and absent at later stages (Fig. Hoxb1 was found in the third pouch at stage 20, and at stage 24 in the fifth pouch (Fig. Hoxb1 expression is associated with different pharyngeal pouches at different developmental stages.Our analysis of the chick highlighted the fact that the different pouches were primarily marked by the expression of  24 Fig. , d. Contges Fig. , e. Finauch Fig. , f. ThusHoxb1 with different pouches at different stages of development in Scyliorhinus was surprising, and we therefore set out to determine if this was a general feature of pharyngeal development in both chick and catshark. The first two pharyngeal pouches form in the chick at stage 13 and we found that even at this early time point Hoxb1 expression was already established in the most caudal pouch, pouch 2 (Fig. Hoxb1 expression was now associated with that newly formed pouch (Fig. Hoxb1 expression had shifted to this last pouch (Fig. Hoxb1 expression was associated with the newly formed fourth pouch (Fig. Hoxb1 expression had shifted to this pouch (Fig. Hoxb1 expression shifted to the lastly formed sixth pouch from stage 23 onwards Fig. The association between the expression of h 2 Fig. . By staguch Fig. . Finallyuch Fig. , d. Intruch Fig.  while atuch Fig. . FinallyHox genes in the forming arches, and in particular how these relate to the pharyngeal pouches, which dictate the number of segments formed. Our results show support for the first three anterior segments being homologous across the gnathostomes. Each of these had a distinct innervation, and both the mesenchyme of these arches and the corresponding pouches expressed the same repertoire of Hox genes in both chick and catshark. To gain insights into how the posterior pouches align between chick and catshark, we analysed the expression of Hoxb1, which is expressed in the most posterior pouch in amniotes. Somewhat unexpectedly, we found that Hoxb1 exhibits a dynamic expression profile during the formation of the pharyngeal segments, with the expression domain of this gene progressively moving caudally such that at any given stages it is expressed in the last formed pouch. This leads us to suggest that the posterior limit of the pharynx is homologous between chick and catshark, and that a reduction in the number of pharyngeal segments has been achieved by the earlier termination of the caudal expansion of the pharyngeal endoderm. Thus the caudal limit of the pharynx is established at a relatively more anterior position and therefore fewer pharyngeal pouches form and correspondingly fewer pharyngeal segments.In the present study, we have attempted to address the route through which the reduction in the number of pharyngeal segments that occurred within the sarcopterygians, and which is evident in extant tetrapod embryos. To do this, we compared the development of the pharyngeal segments in an amniote, the chick, with that in a chondrichthyan, the catshark. We show that in both the overall trend in the formation of the pharyngeal pouches is similar, with the most anterior forming first and then the posterior segments forming sequentially. We further analysed the patterns of innervation and the expression profiles of Hoxa3, in chick and catshark in both the mesenchyme and the corresponding pouch endoderm is also a conserved feature of the third segment; although, the expression of this gene is lost as development progresses. However, it is less clear which of the more posterior arches have been retained and which have been lost. In part this is due to the fact that the expression patterns of Hox PG 3 and 4 do not simply align with distinct arches or pouches in either chick or catshark embryos. Finally, our study suggests that the posterior limit of the pharynx may be homologous, in that it is defined by the expression of Hoxb1 in both species (Fig. From our analysis we can begin to understand which of the posterior, or branchial, segments have been lost. Our results clearly indicate that the most anterior of the branchial series, the third pharyngeal segment is homologous across the gnathostomes and is retained in amniotes. In lamprey, catshark, chick and mouse, this segment is distinctly innervated by the IXth nerve. Furthermore, we show that the expression of ies Fig. . Indeed,Gogonasus and Eusthenopteron, which are tetrapodomorph fish, have lost the fifth arch. Thus it has been suggested that the absence of the fifth branchial arch is a derived feature of advanced sarcopterygians.While our work gives an insight into the possible mechanism that underpinning the reduction in arch number that occurred within the sarcopterygians, the morphological data form fossils also provides a very useful complement that give us insights into when the reduction in the number of pharyngeal segments started . Thus whHox1-defined posterior limit of the pharynx and more anterior segments, is likely to extend beyond events within the sarcopterygians. An analysis of Hox gene expression in the lamprey, Lethenteron japonicum, found that the Hox1 gene, LjHox1w, in this this species also marks the caudal limit of the pharynx, although it is unclear whether LfHox1w displays a similar temporally and spatially dynamic pattern of expression [Saccoglossus kowalevskii, which concludes that this process is homologous to the formation of the pharyngeal segments of vertebrates, also reported spatially and temporally dynamic expression of Hox1 [Hox1 expression was restricted to the posterior boundary of the pharyngeal endoderm at the three gill-slit stage and that it was still restricted to the posterior boundary of the pharyngeal endoderm at the four gill-slit stage. Collectively, our work and those of others therefore suggest that a possible role for Hox1 genes in defining the caudal limit of the pharynx extends beyond the vertebrates, and is likely to have evolved much earlier with the emergence of the deuterostomes.Importantly, other studies suggest that the mode of arch reduction that we have identified, in which deletion occurs between a pression . Perhaps of Hox1 . In thatHox1 expression domain and the last formed anterior segment. Correspondingly, an increase in the number of pharyngeal segments, Thus the number of pharyngeal segments could be increased, such as is seen in the extinct jawless vertebrate Endeiolepis which had up to thirty pairs of gill slits [Hox1 expression limit and the anterior.This is also significant in that it could help us understand how the number of pharyngeal segments has been modified across the deuterostomes. Thus while enteropneust hemichordates and cephalochordates have numerous pharyngeal gill slits , 25, whill slits , by alloOur results support the view that the three most anterior pharyngeal segments are conserved across the vertebrates and that the caudal limit of the pharynx is also conserved. Thus, within the sarcopterygians, the segments that were lost were those form the “branchial”/posterior region which lie between the third arch and the caudal limit. Furthermore, our results would suggest the reduction in the number of pharyngeal segments was achieved as a result of the premature termination of the posterior extension of the pharyngeal endoderm which in turn would result in the generation of fewer segments. Finally, we suggest that such a mechanism may also account for the variability in the number of pharyngeal segments seen across the vertebrates and other deuterostomes.


Stomoxys calcitrans (stable flies).A laboratory study was conducted to assess the repellent and insecticidal efficacy of a new combination of fipronil and permethrin  against 2 and crushed to determine feeding status (fed or unfed). Any dead flies remaining on the dog or in the cage were crushed to determine feeding status and counted as fed or unfed. Repellency was defined as the percent reduction in the number of fed flies in the treated group as compared to the untreated control group, and insecticidal efficacy was defined as the reduction in the number of live flies at the end of each exposure period in the treated group as compared to the control group.Sixteen dogs were allocated to two treatment groups. Eight dogs were treated with a new topical spot-on formulation containing 6.76% w/v fipronil + 50.48% w/v permethrin on Day 0 and eight dogs served as untreated controls. Each dog was exposed to approximately 100 stable flies on Days 1, 7, 14, 21, 28 and 35. After a one-hour exposure period, live flies were carefully aspirated into a vial, anesthetized with COPercent repellency was ≥96.6% through Day 28, and 88.7% on Day 35. Percent insecticidal efficacy was ≥ 98.3% through Day 35.S. calcitrans on dogs for at least 5 weeks.A single topical administration of a new combination of fipronil and permethrin provides protection  from  Stomoxys calcitrans) and the fruit fly (Phortica variegata), a vector of Thelazia callipaeda in domestic and wild carnivores  as well as humans, have an important veterinary impact , where C is the GM of the control group and T is the GM of the treated group.Percent insecticidal efficacy was defined as the reduction in the number of live flies at the end of each exposure period in the treated group as compared to the control group. The number of live flies after each exposure was transformed to the natural logarithm of (count + 1) for calculation of GM by treatment group as previously described . ParasitFor both repellency and insecticidal efficacy, the treated group was compared to the untreated control group using the Friedman rank test with blocks defined as the allocation blocks . The tesS. calcitrans exposures. The affected dogs recovered after treatment with diphenhydramine . One dog in the untreated control group and two dogs in the treated group developed hypersalivation following administration of the anesthesia reversal agent, which resolved without treatment.No adverse reactions to the treatment were observed in any dog during the study, including the 4-hour period after treatment. Four dogs in the untreated control group developed suspected allergic reactions  following one or more of the The stable fly challenge was robust on all exposure days as evaluated by the number of live flies and the number of fed flies in the untreated control dogs at the end of each exposure period. The GM of live flies in the untreated control group ranged from 92.9 to 101.3 and the number of fed flies ranged from 75.1 to 91.6 during the study (Tables p = 0.005).The GM number of fed flies in each group and the percent repellency on each exposure day is shown in Table p = 0.005).The GM number of live flies in each group and the percent insecticidal efficacy on each exposure day is shown in Table The results of this study demonstrate that a single topical treatment of the new combination of fipronil and permethrin (Frontline Tri-Act®/Frontect®) provides excellent repellency and insecticidal efficacy for at least 5 weeks. The product provided immediate effects after administration as demonstrated by 100% repellency and insecticidal efficacy one day after treatment. Both effects persisted for over 1 month with 96.6% repellency 28 days after treatment and 88.7% repellency 35 days after treatment. Insecticidal efficacy remained over 98% for 35 days after treatment.The repellent effects of the combination of fipronil and permethrin will help prevent painful bites as well as more severe sequelae such as bleeding, infection and necrosis for at least 5 weeks after treatment. In addition to the direct benefits from the repellent effect, the insecticidal effects of the product may also help reduce overall stable fly populations in kennels or other situations where the flies rely on dogs as their sole source of blood. This reduction in fly population may further reduce the challenge to dogs in these areas.et al. [, in which an average of 12.6 of 50 flies took a blood meal during a 30-minute exposure period and 22.5 of 50 flies survived the exposure period. The reason for the higher feeding and survival rate in the current study is unknown but may be related to the source, age, rearing or holding conditions of the stable flies, longer exposure time or other reasons. Flies used in this study were younger and fasted from blood for a shorter time prior to exposure, which may explain some of the difference. Regardless of the reason for the high feeding rate in the current study, it demonstrates that the challenge was robust and a strong test of the product.The stable flies in this study provided a strong challenge to the product on each exposure day. A high percentage of the flies fed on the untreated control dogs on each day of the study, with 75.1 to 91.6 of approximately 100 flies having fed by the end of the exposure period. The viability of the flies was high with 92.9 to 101.3 of the flies remaining alive at the end of the exposure period. The percentage of flies that fed and survived the exposure period in this study was higher than previously reported by Fourie et al. [The repellency and insecticidal efficacy of the combination of fipronil and permethrin was higher than previously reported for a commercial permethrin-containing product. Fourie et al.  reportedS. calcitrans on dogs for 5 weeks. The product also provided a high insecticidal efficacy against flies exposed to treated dogs for 5 weeks following treatment. The product has been shown to be safe for dogs and will provide reliable protection against a variety of ectoparasites, including stable flies, for at least 1 month.A single topical administration of a combination of fipronil and permethrin provided repellent (anti-feeding) protection from


Hip hemiarthroplasty is a common treatment for femoral neck fracture. However, the acetabular cartilage may degenerate after hemiarthroplasty leading to postoperative failure and the need for revision surgery. The clearance between the acetabular cartilage and head of the prosthesis is one of the potential reasons for this failure. In this study, the influence of joint clearance on the biomechanical function of a generic hip model in hemiarthroplasty was investigated using biphasic numerical simulation. Both a prolonged loading period of 4000 s and dynamic gait load of 10 cycles were considered. It was found that a larger clearance led to a higher stress level, a faster reduction in load supported by the fluid and a faster cartilage consolidation process. Additionally, the mechanical performance of the acetabular cartilage in the natural model was similar to that in the hemiarthroplasty model with no clearance but different from the hemiarthroplasty models with clearances of 0.5 mm and larger. The results demonstrated that a larger clearance in hip hemiarthroplasty is more harmful to the acetabular cartilage and prosthesis heads with more available dimensions  could be manufactured for surgeons to achieve a lower clearance, and reduced contact stress in hemiarthroplasty surgeries. Although it is less destructive, less costly and requires shorter surgical time than a total hip replacement procedure, the acetabular cartilage, when articulating with a metallic head component, may degenerate, resulting in pain, immobility and the need of a revision surgery Selection of the femoral component size is crucial for hip hemiarthroplasty, as it is directly linked with acetabular function and degeneration Mechanical factors have long been recognised as the primary contributor to cartilage damage. The function and degeneration of cartilage is closely linked with its biphasic (i.e. fluid–solid) nature, because the fluid phase is able to support most of the compressive load applied to the tissue and it also provides an excellent lubrication environment The time-dependent tribological performance of the hip following hemiarthroplasty has been measured experimentally by Lizhang et al. 2E = 1.2 MPa, Poisson's ratio ν = 0.045) with a constant permeability (K = 0.0009 mm4/N s) The hip hemiarthroplasty FE model used in this study was composed of a pelvis with the acetabular cartilage in articulation with a metallic prosthetic head component . The labThe metallic head component was represented by a rigid and impermeable sphere. To evaluate the influence of head size on the model predictions, four different radial clearances  were evaluated by varying the size of the head . The conmrl.sci.utah.edu/software/febio) The modelling procedure has been previously validated by comparing the model predictions to experimental tests, and good agreement in contact mechanics was achieved 3Contours of contact stress for all the models under the static load at 0.6 s and 4000 s are presented in Results of the models under the static load over 4000 s period are summarised in The fluid flux for the hemiarthroplasty models with 0 mm and 2 mm clearances is illustrated in A summary of results for the gait loading case is presented in 4In this study, the influence of joint clearance on the biphasic performance of the hip following hemiarthroplasty was investigated using a FE model. Both a static load during a prolonged loading period and a dynamic gait load over several cycles were considered in order to simulate the circumstances commonly experienced by the hip. A similar investigation was conducted by Pawaskar During the early loading period, a larger clearance leads to a smaller contact area, substantially increased peak contact stress, but a slightly lower fluid support ratio, which would contribute to a higher stress level in the solid matrix of the acetabular cartilage, a greater level of friction and a greater potential to degenerate. Over a prolonged loading period, the cartilage consolidated faster in the model with larger clearance, because of the faster fluid exudation, also suggesting a more harmful mechanism with a large clearance.Over 10 cycles of gait, an increased peak contact stress and faster cartilage consolidation process were also observed for the model with larger clearance. In particular at mid-swing, the fluid support ratio was lower and decreased substantially faster in the model with larger clearance when contact occurred near the interior edge of the acetabular cartilage. This is because the tissue around the interior region of the acetabular cartilage was less confined and thus had a lower capability to support fluid than the tissue in the central acetabular cartilage In most of the previous numerical studies on the hip, the cartilage was assumed to be incompressible and monophasic (e.g. hyperelastic) to simulate the biphasic response of the joint during the early period of loading. As shown in the static loading case, it takes more than 1000 s to observe an obvious cartilage consolidation when the contact occurs around the central acetabulum region. However, for the time at which the contact slides toward the edge region, cartilage consolidation becomes obvious only over 10 cycles of gait (∼10 s). This suggests that monophasic joint simulations are appropriate for very specific circumstances. On the other hand, in the dynamic loading case, the pattern of fluid exudation is subject to the variation in the loading magnitude and loading direction over time, suggesting that dynamic loads should be applied in a time-dependent way for biphasic simulations.The main limitations of this study are the adoption of a generic joint geometry and the assumption of a linear elastic solid phase for the cartilage. The linear elastic solid phase assumed in this study is not able to represent the inhomogeneous fibre-reinforced structure where the tensile stiffness is higher than the aggregate stiffness In both the dynamic loading and prolonged static loading cases, a larger clearance of the hip in hemiarthroplasty was found to be more harmful to the acetabular cartilage, as evidenced by a higher stress level and faster cartilage consolidation process. The biomechanical function of the acetabular cartilage in the natural model was similar to that in the hemiarthroplasty model with no clearance but different from the hemiarthroplasty models with clearances of 0.5 mm and larger, suggesting that clearance needs to be avoided or minimised to ensure the joint following hemiarthroplasty is as close to the normal healthy mechanical environment. It is also recommended that prosthesis heads with more available dimensions  should be manufactured for surgeons to achieve a minimal clearance during hemiarthroplasty surgery. Further studies will focus on subject-specific evaluations to provide more stratified intervention guidelines.


This correction does not affect the manuscript’s results: the correct region-level test statistics were used in the analysis; only the written definition of the region-level test statistics was incorrect.2.An incorrect version of Figure 3.We note that there is also a published correction to the supplement that was originally published with this manuscript.


To implement a biometric patient identification system in the field of radiation oncology.A biometric system using palm vein scanning technology has been implemented to ensure the delivery of treatment to the correct patient each and every time. By interfacing a palm vein biometrics system (PVBS)  with the radiation oncology patient management system (ROPMS)  one can integrate patient check-in at the front desk and identify and open the correct treatment record of the patient at the point of care prior to the initiation of the radiation therapy treatment.The learning time for the use of the software and palm scanner was extremely short. The staff at the front desk and treatment machines learned the procedures to use, clean, and care for the device in one hour’s time. The first key to the success of the system is to have a policy and procedure in place; such a procedure was created and put in place in the department from the first day. The second key to the success is the actual hand placement on the scanner. Learning the proper placement and gently reminding patients from time to time was found to be efficient and to work well. The use of a biometric patient identification system employing palm vein technology allows one to ensure that the right care is delivered to the right patient each and every time. Documentation through the PVBS database now exists to show that this has taken place. The overall goal of patient safety measures in radiation therapy is to ensure that the right treatment is delivered to the right patient and the correct anatomical site at every treatment visit. As part of our continuous quality management program , we sougAfter mapping out our clinical workflow, two areas were identified for integration—the front desk and the treatment machine. To map out the workflow, we followed the patient through the department to see the points of contact with individuals and technology to ascertain where and how a misidentification can take place. We were also interested to determine where the process could be automated or where technology could be added to help mitigate the risk of patient misidentification. The crucial area identified was the point of care at the treatment machine. At the treatment machine, prior to care initiation, there is a manual process of selecting the patient from a list of patients on treatment for the day. The list, or queue for the day, can contain patients with the same first and last names. Our database has over 20,000 records of patients with many instances of patients with the same or very similar first and last names. More often than not, two such patients are under treatment at the same treatment machine increasing the risk of patient misidentification using a manual patient identification methodology. There may be up to 180 patients on treatment on any given day. This is where a palm vein biometrics system (PVBS)  can add an extra layer of safety. At the front desk, the staff or registrar has the patient registration module of the ROPMS  along with other electronic medical records (EMR) applications open at their workstations. Patient tracker views in the ROPMS are customizable and can be integrated with the PVBS. The PVBS utilizes its own server for patient record and palm vein image storage and utilizes the ADT  interface for communication and patient identification in our ROPMS. The patient’s name, DOB, and address are fed into the PVBS application prior to patient identification through the palm vein scanning. Upon a patient’s first arrival to the department, a face photo is taken and entered into the ROPMS. The patient's demographic information is also checked for accuracy and missing information is entered. Then, in the PVBS application, the DOB is entered and a palm vein scan is registered or recorded for the first time. The registration requires two scans of the palm. Instructions are given to the patient for correct palm placement and hand position. The correct technique is to make a “five”—extend all fingers with the fingers spread apart and then to slide the hand onto the reader until the guides stop the hand. The majority of patients have no problem learning or applying this technique within one or two tries. However, some patients have difficulty either understanding or opening their fingers due to arthritis and require multiple tries before mastering the technique to allow reliable and reproducible identification. To help with the technique, an illustrated hand guide placement picture has been placed next to the reader Figure . For subsequent visits by the patient, the registrar clicks on the patient tracker screen in ROPMS and then onto the PVBS screen. The registrar then verifies the DOB and scans the patient’s palm Figure . After aThe following hardware—a PVBS server, the existing ROPMS servers, an ADT interface, USB Fujitsu Palm Readers , and PalAs with any new technological initiative, tracking its use and accuracy helps determine the value obtained. As a result, automated monthly SQL (Structure Query Language) reports were built and analyzed. These monthly SQL reports included data on additions to the department, positive matches found for patients in the PVBS database, and unsuccessful scans or no matches found for patients in the PVBS database.The learning time for the use of the software and palm scanner was extremely short. The staff at the front desk and treatment machines were able to learn all necessary procedures of how to effectively and safely employ the system within one hour. It was found that it is instrumental to have a policy and procedure in place; this was created and put in place from the first day for the successful deployment of this technology throughout the department. Moreover, it was found that proper hand placement on the scanner and having an illustrated hand guide placement picture next to the palm scanner to remind patients of the proper hand placement are key ingredients to reliable and reproducible identification using this technology. Supporting patients in learning the proper placement by using gentle reminders was found to be an extremely effective technique, allowing the majority of patients to master the palm scanning technique within the first few treatment sessions. The success of these efforts is reflected in the low number of unsuccessful matches found in the reports, which is on average three per patient Figure . While tIt has been found that a PVBS can add an additional layer of safety to patient identification and that such a technology can be integrated into the radiation oncology care workflow requiring only minimal modifications of the existing patient care workflow. This integration has been achieved by utilizing the ADT for communication and patient identification in our radiation oncology patient management system (ROPMS). In the hierarchy of safety effectiveness, by the Institute for Safe Medication Practices, -7 technoThe question one might ask is what if the hand is fractured during the course of treatment? If the right hand is fractured, then of course the left hand can be employed. The right hand record can be deleted in the PVBS system and the left hand palm print can be stored instead. The PVBS has an administration tool that select users can have access to for performing such tasks. In the event that a patient is a double amputee having lost both hands or both hands are fractured during the course of treatment, one has to revert to the traditional manual way of patient identification. Moreover, since both of these events are low probability events, the existence of such a patient in the department does not present an undue safety risk for patient misidentification since any other patient with two intact or one broken hand can still utilize the technology. While the unique palm vein pattern of a patient does not change from birth to death, special workflow procedures are recommended for the use of the PVBS technology with pediatric patients. For patients 16 years of age or younger, it is recommended that the patient be re-enrolled following successful authentication if it has been more than a year since their last visit. While the vein pattern does not change, the size of the hand is growing and that can affect consistent hand placement. The system automatically prompts for the re-enrollment based on patient age, current date of service, and last date of service. One way to further enhance the patient experience with PVBS is to add a self check-in kiosk at the front desk. This will allow patients during their daily visits to quickly and accurately self check-in for their daily appointment.The use of palm vein technology allows one to ensure that the right care is delivered to the right patient at the point of care prior to care initiation. With the PVBS database, clear documentation exists that the correct treatment was given to the correct patient for each treatment. This documentation could not be done without biometrics. The use of PVBS technology can help in significantly reducing the risk of patient misidentification at the point of care. There are 1225 misadministrations each year in radiation therapy across the United States . The use


Assessment of causes of death and changes in pattern of causes of death over time are needed for programmatic purposes. Limited national level data exist on the adult female causes of death in Bangladesh.Using data from two nationally representation surveys, the 2001 and 2010 Bangladesh Maternal Mortality Surveys (BMMS), the paper examines the causes of adult female death, aged 15–49 years, and changes in the patterns of these deaths. In both surveys, all household deaths three years prior to the survey were identified. Adult female deaths were then followed by a verbal autopsy (VA) using the WHO structured questionnaire. Two physicians independently reviewed the VA forms to assign a cause of death using the ICD-10; in case of disagreement, a third physician made an independent review and assigned a cause of death.The overall mortality rates for women aged 15–49 in 2001 and 2010 were 182 per 100,000 and 120 per 100,000 respectively. There is a shift in the pattern of causes of death during the period covered by the two surveys. In the 2001 survey, the main causes of death were maternal (20 %), followed by diseases of the circulatory system (15 %), malignancy (14 %) and infectious diseases (13 %). However, in the 2010 survey, malignancies were the leading cause (21 %), followed by diseases of the circulatory system (16 %), maternal causes (14 %) and infectious diseases (8 %). While maternal deaths remained the number one cause of death among 20–34 years old in both surveys, unnatural deaths were the main cause for teenage deaths, and malignancies were the main cause of death for older women. Although there is an increasing trend in the proportion of women who died in hospitals, in both surveys most women died at home (74 % in 2001 and 62 % in 2010).The shift in the pattern of causes of adult female deaths is in agreement with the overall change in the disease pattern from communicable to non-communicable diseases in Bangladesh. Suicide and other violent deaths as the primary cause of deaths among teenage girls demands specific interventions to prevent such premature deaths. Prevention of deaths due to non-communicable diseases should also be a priority. Knowledge about causes of death is crucial for public health planning and resource allocation . HoweverInformation on causes of death is especially important in view of the epidemiological transition that developing countries are experiencing. Although limited evidence exists, Bangladesh, a developing country in South Asia, is going through an epidemiological transition -- a shift in the mortality pattern from communicable diseases (CDs) to chronic diseases , 5. AnalIn Bangladesh, there is a paucity of data on the causes of death at the population level. Although the Bangladesh Bureau of Statistics (BBS) maintains a nationally representative Sample Vital Registration System (SVRS) which records causes of death based on the information collected by a lay reporting system, there are reservations about the accuracy of the causes of death information from this system . StudiesVA has increasingly been used to determine causes of death, especially in settings where deaths occur outside a hospital. Recent studies have suggested that VA can provide causes of death information that, at the population level, is similar to death certification in high-quality hospitals . In thisThe present paper uses VA data from two nationally representative surveys, the 2001 and 2010 Bangladesh Maternal Mortality Surveys (BMMS), to examine the causes of adult female death aged 15–49 years, and the changes in the patterns of causes of death during the 9 year period between the two surveys. Both surveys were large and covered approximately 100,000 and 174,000 households respectively using nationally representative samples, sufficient to detect a 20 % decline in reduction of maternal mortality with 95 % confidence and 80 % power. In both surveys samples were selected through a three-stage sampling design . Both suth Revision (ICD-10) classification. The underlying cause of death is grouped into eight categories: maternal; infectious diseases; malignancies; diseases of the circulatory system; suicide; other violent deaths; miscellaneous, and deaths for which cause of death was impossible to assign, or for which the reviewing physicians could not agree on a single cause of death. In this analysis, deaths that occurred in the three years preceding the two surveys among women aged 15–49 years are included.Cause of death was determined by independent review of the VA forms by two physicians. If they could not agree on the final cause of death, the case was then reviewed by a third physician. For a small number of cases where the three physicians agreed that the death was maternal but could not agree on a single cause of death, an expert committee of obstetricians was consulted to assign a cause of death. For each case, an underlying cause of deathp < 0.05. Data were analyzed using SPSS version 17.0 and Microsoft Excel 2007. As the sample was not self-weighted household weights were used to get unbiased estimates at the national level.Analysis included mortality rates and proportional mortality showing age specific patterns of causes of death, place of death and health seeking behavior before death. The mortality rate is calculated as the ratio of deaths in the three years preceding the survey to the person years lived by women aged 15–49 years  in the same period expressed per 100,000 women aged 15–49 years. Proportional mortality (%) was calculated by dividing the number of deaths attributed to a specific cause by the total number of deaths for which a VA was carried out. The difference between the two death rates in 2001 and 2010 was compared by calculating the 95 % confidence intervals (CI), and the difference between two proportions was compared by z-test for statistical significance at 80 % power and This paper is a secondary analysis of existing de-identified data sets. Ethical approval for the BMMS 2001 and 2010 were obtained from the Ethical Review Committee (ERC) of the Bangladesh Medical Research Council (BMRC).In the 2001 BMMS, a total of 696 deaths were reported among women 15–49 years in the three years prior to the survey. The number of such deaths was 749 in the 2010 survey. The overall mortality rates for this group of women in 2001 and 2010 were 182/100,000  and 120/100,000  respectively  whereas the proportion of deaths due to maternal causes and infectious diseases decreased significantly (p < 0.05). The proportion of deaths due to suicide and other unnatural causes remained the same in both surveys.When the proportion of deaths by different causes were compared between the two surveys Table , in the As expected, cause specific death rates varied by different age groups Table . The deap > 0.05). Unnatural deaths (suicide and other violent deaths) were the second leading cause of death for women aged 20–24 years after maternal deaths in both surveys; in 2001, 1 in every 5 women had suicidal deaths and an additional 7 % had other violent deaths. This figure decreased to 10 % for suicide and 9 % for other violent causes in 2010. A similar pattern of change in the causes of death is observed for 25–29 years age group for maternal and unnatural deaths. Diseases of the circulatory system and malignancies were the two leading causes of death among older women aged 35–49 years. These two diseases together accounted for 45 % of total deaths in 2001 and 50 % of total deaths in 2010 in this age group. In 2001, diseases of the circulatory system were the primary cause of death in this age group, followed by malignancies whereas malignancies ranked as the main cause in 2010 followed by diseases of the circulatory system. Women died from different types of malignancies and there was little variation in the type of malignancies between the two surveys. In 2001, among women who died from malignancies, the distribution by type malignancy was: cancers of the gastrointestinal tract, including liver (37 %), blood and lymphatic system (19 %), reproductive cancer including cancers of the reproductive organs including breast and cervical cancer (28 %) and other cancers (18 %). In 2010, women died from cancers of the gastrointestinal tract, including liver (30 %), cancers of blood and lymphatic system (28 %), reproductive cancers including cancer breast and cervical cancers (29 %) and other cancers (13 %). Under the miscellaneous category, diabetes, hepatitis and chronic respiratory diseases were the main causes of death in both surveys.Distribution of deaths by different cause categories also varies by age groups Table . While mp < 0.05) as shown in Table Although there is an increasing trend in the proportion of women who die in health facilities, the majority of women still die at home. In the 2010 survey, 38 % women died in a health facility compared to 26 % in 2001 . While this increase was observed among all death categories except unnatural deaths, the increase was highest for deaths due to circulatory diseases , followed by miscellaneous causes  and maternal causes . In both 2001 and 2010, public facilities were the most prominent source of care. However, in the 9 years between the two surveys, there has been a substantial increase in seeking care from private health facilities (increased from 12 to 46 %).Care seeking before death also improved Table . In the This paper presents, for the first time ever, provides national estimates of causes of adult female deaths and provides a unique opportunity to to examine changes in the patterns of causes of death over time. Overall, there has been a major shift in the pattern of causes of death over the 9 year period. While deaths due to maternal and infectious origins are significant contributors of adult female deaths, deaths due to non-communicable diseases (NCDs) such as circulatory diseases, malignancies, diabetes and other chronic conditions are on the rise. Unnatural deaths including deaths due to suicides are also a major killer.While this study provides national level estimates of cause specific female mortality and the causes of death, the findings should be interpreted with caution, given the difficulties in assigning an exact cause of death using the VA method. Verbal autopsy, as a method of ascertaining causes of death has increasingly been recommended, especially when death occurs outside of health facilities and without the presence of a medically trained person to assign a cause of death , 23–25. We used an adapted version of the WHO questionnaire for adult deaths , have emA methodological challenge in assigning an exact cause of death using the VA method is also reflected in the proportion of deaths that are being categorized as “not-classified”. In both surveys, about 10 % of deaths were categorized as “not-classified” for which it was either impossible to assign a cause of death on the basis of information collected through VA or the reviewing physicians could not agree to a single cause of death. If an appropriate diagnosis could be made for these cases, the cause specific mortality reported here might be affected to some extent. However, previous studies using similar verbal autopsy method as a means to assess the causes of death have reported either higher or similar figures for “unknown” or “unclassified” categories of death , 8.Our findings are in agreement with other studies documenting NCDs as a major cause of adult death including female deaths in Bangladeshi , 30, 31.Similar to many other developing countries, Bangladesh is going through a shift in the disease and mortality patterns from acute infectious and parasitic diseases to non-communicable, chronic diseases. The 2010 update of Global Burden of Disease Study estimated that between 1990 and 2010, proportion of deaths due to NCDs in Bangladesh increased from 34 to 60 %, whereas contribution of communicable diseases  nearly halved (56 % to 30 %) and the proportion of injury remained around 10 % (NCDs) . Given tOur results indicate that a substantial proportion of female deaths occurred due to intentional and unintentional harm, with suicide being the leading cause of death among female teenagers in both surveys. While limited information exists on the extent and the causes of suicide among the Bangladeshi population, a recent study conducted to understand the epidemiology of suicide in Bangladesh found that suicide is the fourth injury related deaths in Bangladesh and the leading cause of injury related deaths among adolescents aged 10–19 years, with slightly higher risk of suicide among females compared to males . In thisBecause of methodological differences we cannot compare previous study findings with our study findings. However, suicide is an issue of growing concern in Bangladesh with some areas reporting 37 % of all deaths of women aged 15–44 years attributable to suicide . LongituWe are also unable to explain the underlying causes of this high level of unnatural deaths. However, other studies have identified a number of circumstances leading to suicides among young girls and women including emotional and verbal abuse by husbands and other family members, financial hardship in the family, childlessness and rejection of marriage offer or unacceptable partner . PreventUsing BMMS data, this paper presents the cause and age specific adult female deaths at the national level in two surveys, almost a decade apart, and comparing them between the two surveys. The sample size for the two surveys was calculated to have sufficient statistical confidence and power to estimate the national MMR and calculate the changes in MMR over the 9 year period between these surveys but not to estimate cause specific mortality or age and cause-specific mortality or to compare them between the two surveys. However, the results are strongly indicative of the decreasing trend for death rates for some diseases .There is an increasing trend in seeking care before death and in the proportion of deaths that happened in health facilities. The increase in seeking care is more marked for conditions like circulatory diseases and diseases like diabetes, chronic respiratory conditions etc. The majority of women visited public facilities for seeking care. Given that more than 70 % of the country’s population live in rural areas where the first point of care usually is public facilities that are primarily designed to provide maternal and child health care services, whether these facilities are equipped to manage the increasing number of NCD cases needs to be explored.Bangladesh has made remarkable progress in the health and development sector in the last three decades. Bangladesh is one of the nine countries that are on track to achieve the Millennium Development Goal (MDG) 5 of reducing maternal mortality. This was made possible through improvements in the health sector as well as in non-health sectors. However, much still needs to be done to prevent untimely deaths of women. Our study provides a solid basis in support of the argument that Bangladesh is going through an epidemiological transition and NCDs are significant contributors to adult female deaths. Given the prominence of NCDs in the mortality statistics and their potential impact on the socioeconomic development of the country, the government of Bangladesh has identified NCDs as a priority area and has taken steps to strengthen activities for the prevention and treatment of these conditions. However, only 4.5 % of the national health budget in the Health Population and Nutrition Sector Development Program (HPNDSP 2011–16) has been allocated for these diseases. The Government efforts should be strengthened further to take this trend into account and to increase the proportion of the health budget for NCDs, and to use evidence-based information on how to tackle these conditions. While prevention efforts should be focused on identifying and modifying risk factors through practicing healthy life-style, early detection and treatment are also important to decrease the long term impact of these diseases.