Atrial fibrillation (AF) is the commonest cardiac rhythm disorder worldwide, affecting 1% of the general population. It is estimated that up to 16 million people in the US will suffer from the arrhythmia by 2050. AF is an independent stroke risk factor and associated with more severe strokes. For six decades, warfarin has been the only truly effective therapy to protect against stroke for patients with atrial fibrillation. Despite the proven worth of warfarin, its limitations have seen reluctance amongst physicians and patients to utilise this efficacious agent. This has meant that substantial numbers of patients are either unprotected against stroke or suboptimally protected with antiplatelet therapy.2DS2-VASc) now permit rapid but comprehensive evaluation of a patient’s risk for thromboembolism, allowing better identification of low-risk patients who do not require antithrombotic therapy, and whilst for those with ≥1 stroke risk factors require formal oral anticoagulation. Aspirin has been proven to be inferior to anticoagulation, and is not free of bleeding risk. We also have simple scores to easily evaluate a patient’s risk of haemorrhage (e.g. HAS-BLED).Contemporary well-validated stroke risk factor schemes (CHAThe emergence of new oral anticoagulants should further improve stroke prevention in AF, and they successfully negotiate many of the hurdles to oral anticoagulation generated by warfarin’s limitations. Monitoring, reversal, and perioperative management are areas which require further investigation to enhance our ability to safely and effectively utilise the new agents. Atrial fibrillation (AF) is the commonest cardiac rhythm disorder worldwide, affecting 1% of the general population ,3, and 1AF leads to a prothrombotic state  which prFor six decades, vitamin K antagonists (i.e. warfarin) have been the only truly effective therapy to protect against stroke for patients with atrial fibrillation. Despite the proven worth of warfarin as thromboprophylaxis in AF, its limitations and inconveniences have seen reluctance amongst physicians and patients to utilise this efficacious agent . This haThe last decade has been an intensive period of research in AF which has finally seen the emergence of novel oral anticoagulants to complement and compete with warfarin. There has also been evidence to highlight the limitations of aspirin as thromboprophylaxis in AF, and improved risk stratification schemata to allow for the identification of patients truly at low-risk for thromboembolism and simple assessment of the bleeding risk. These advancements should combine to reduce the variability of management in AF stroke prevention and lead to more patients receiving anticoagulation to protect them against thromboembolism. The new agents, although overcoming many of warfarin’s limitations, will present their own set of challenges and considerations as they are incorporated into clinical practice.2 score [1). Its original validation categorised patients as “low-risk” if they scored 0, “intermediate-risk” if they scored 1-2 and “high-risk” if they scored 3 or higher. Although AF is an independent risk factor for stroke, this risk is not homogenous and depends on the presence or absence of specific risk factors for stroke in AF , 20. The2 score , which i2 had a rate of thromboembolism of 1.67 per 100 person years, and those deemed at intermediate risk had a rate of 4.75 per 100 person years. The CHADS2 score characterises 65% of patients into the “intermediate” risk grouping [2 score alone would result in the undertreatment of a cohort of patients at significant risk of thromboembolism.There are several drawbacks associated with this scoring schema. Older guidelines used this grouping to recommend oral anticoagulation to high-risk patients, aspirin for low-risk patients, and a choice of either anticoagulation or aspirin for the intermediate grouping. Thus a patient with a previous stroke could be classified as “intermediate risk” and be given aspirin in preference to warfarin. An analysis of hospital inpatients with AF  found thgrouping . This hagrouping  and thatgrouping . Thus re2DS2VASc score [2DS2VASc score has now been well-validated in a number of cohorts [2 [2DS2VASc score (see Table 2) has been incorporated into major international guidelines [A new risk scoring system would have to be more reliable in identifying truly low-risk patients and minimising patients being denied anticoagulation while they remained at risk of stroke. The CHASc score  has beenSc score . The CHA cohorts  and has horts [2 . The CHAidelines .3). Bleeding is the most feared adverse effect associated with anticoagulation. Many of the risk factors for bleeding are also risk factors for thromboembolism, so the limiting effect of bleeding risk on the prescription of antithrombotics means a number of patients are untreated despite clear indications for anticoagulation . The HASThe HAS-BLED score should prompt clinicians to consider the correctable risk factors for bleeding, such as labile INR or concomitant drugs. It allows for periodic reassessment of the bleeding risk and considers the individual quality of INR control in each patient . The HASWarfarin exerts its anticoagulant effect by interfering with the cyclic interconversion of vitamin K and its epoxide. Carboxylation of clotting factors by vitamin K is required for them to be biologically active , therefoWarfarin’s efficacy in the prevention of stroke in AF was irrefutably proved in a clutch of randomised control trials in the eighties and nineties -39. DataA study in 2006 was undertaken with the intention of proving aspirin and clopidogrel in combination as non-inferior to warfarin for the prevention of thromboembolism in AF . The triThe optimal INR for patients with AF on warfarin for protection against thromboembolism has been established as 2.0-3.0 . INR bel4), warfarin requires frequent laboratory monitoring of its coagulation effect and subsequent dose alterations. This necessitates frequent clinic attendance with a consequential increase in healthcare costs and patient inconvenience.While the efficacy of warfarin is unequivocal , the lim5).The limitations associated with warfarin inform many of the characteristics that are sought by a successful novel oral anticoagulant . To rep6). The new drugs may eliminate the need for an initial two-drug regimen as they reach maximal effect within a few hours. The does responses are predictable and they have not been shown to have numerous significant drug or dietary interactions. None of these drugs require routine monitoring of coagulation.The last few years have seen the emergence of several new oral anticoagulants which are poised to entire routine clinical practice and have the potential to offer effective thromboprophylaxis against stroke for a subset of patients who are not receiving anticoagulation despite their high-risk of thromboembolism. These new agents have more predictable pharmacokinetic and pharmacodynamics properties than warfarin, with their key attributes summarised in (Table 1) [While several potential targets for new anticoagulants have been identified  , the two1) .via the kidneys [Dabigatran etexilate is an oral prodrug which is converted to the active compound (dabigatran) in the liver . Dabigat kidneys . 2 score of 1 or higher were included or who were older than 65 years with coronary artery disease (see Table 7) [RE-LY  was a laMajor bleeding was the primary safety outcome . Dabigatran 110mg was superior to warfarin with a 20% reduction in major bleeding (p=0.003), whereas dabigatran 150mg led to similar rates of major bleeding as warfarin (p=0.031). Both doses of dabigatran caused significantly less intracranial bleeding than did warfarin.Warfarin was better tolerated than dabigatran: discontinuation rates were 21% for dabigatran 110 mg, 21% for dabigatran 150 mg, and 17% for warfarin at the end of the second year of the trial (p<0.001 for dabigatran vs warfarin). The main driver for drug discontinuation in the dabigatran arm was dyspepsia, felt likely to be due to the tartaric acid core of the compound.Dabigatran 150 mg was found to have an increased rate of myocardial infarction (0.74%) when compared with warfarin (0.53%/year), although this effect did not reach statistical significance . Warfarin has been proven in the past to protect against myocardial infarction , and it Rivaroxaban is an oral, reversible, direct factor Xa inhibitor . It has 2 score of 3 or greater. In contrast to RE-LY, this was a double-blind double-dummy trial with sham INRs. Patients were randomised to rivaroxaban 20 mg once daily , or dose-adjusted warfarin. The primary end point was stroke or systemic embolism. Rivaroxaban was non-inferior to warfarin for the prevention of stroke: HR 0.79, 95% CI 0.66–0.96, p<0.001 for noninferiority. The intention-to-treat analysis could not demonstrate the superiority of rivaroxaban: HR 0.88, 95% CI 0.74–1.03, p=0.117 for superiority. Superiority was, however, demonstrated in the per-protocol analysis of patients who continued to receive treatment for the 40-month trial period with a 21% reduction in stroke or systemic embolism .ROCKET-AF  was a phBleeding was similar overall in the two groups  although rivaroxaban led to significantly less fatal bleeding and intracranial haemorrhage. Paradoxically, more patients in the rivaroxaban arm suffered a haemoglobin decrease of 2 g/dl or more or required a blood transfusion. The two drugs were equally well-tolerated.Similarly to rivaroxaban, apixaban is an oral, selective, reversible direct factor Xa inhibitor  with higThe AVERROES trial  evaluateThe ARISTOTLE study  was a laThere are several similarities that can be drawn from the three major phase III trials published which compare warfarin to novel oral anticoagulants. All three agents significantly reduced the rates of haemorrhagic stroke, and this was the primary driver in the reductions for the primary endpoints in all trials. Only dabigatran 150mg BD was shown to significantly reduce the risk of ischaemic stroke. All three drugs also demonstrate positive bleeding profiles  when compared to warfarin. Apixaban is the only new agent which demonstrated a statistically significant reduction in all-cause mortality compared to warfarin, although dabigatran 150mg BD and rivaroxaban also showed a trend towards reduction in the risk of death. 2 score of 3 or higher. RE-LY and ARISTOTLE had significantly more low-risk patients . ROCKET-AF, however, had poorer quality of INR control with mean TTR of 55% whereas the mean TTR was 62% in ARISTOTLE and 64% in RE-LY. Meaningful comparisons between the agents will only be achieved in head-to-head trials. There are also differences between the three trials in design and study population. The RE-LY trial was an open trial, with a PROBE design. It is difficult to offer conclusive deductions on the new agents based on the differences in the results from the three distinct trials. ROCKET-AF had a generally higher risk patient group, with 86% of the total population possessing a CHADSThus far only dabigatran and rivaroxaban has been approved by the FDA, and dabigatran has been incorporated into guidelines in Europe , the US As well as appreciating the great potential these new agents have in improving our ability to effectively treat AF patients with thromboprophylaxis, we must also appreciate their potential pitfalls. Dabigatran and apixaban require twice-daily dosing, which favours forgetfulness and raises the possibility of more missed doses. Due to the short half-lives of these drugs, omitted doses may place the patient in a prothrombotic state and increase the risk of thromboembolism. This effect may be compounded by the fact that routine coagulation monitoring is unnecessary: regular INR checks permit clinicians to assess the compliance of a patient, as well as dispense health education and affirm how crucial it is for patients to adhere to their anticoagulation. There is a danger that adoption of the novel oral anticoagulants may lead to the situation seen with other cardiovascular drugs, where poor adherence is a widely-encountered problem -85. Dabigatran is contraindicated in patients with severe renal insufficiency and must be used in caution in patients with moderate renal dysfunction (as must rivaroxaban). The dose of dabigatran approved by the FDA in renal impairment was never actually studied in the RE-LY trial . The ratAs well as the appropriate dose reductions, a position paper from the Italian Federation of Thrombosis Centres  advises The RE-LY trial also highlighted dabigatran’s propensity to cause dyspepsia and other gastrointestinal upset, likely caused by the tartaric acid core of the dabigatran core which leads to an acidic microenvironment. The Italian position paper suggests patients with recurrent dyspepsia should remain on warfarin, although rivaroxaban and apixaban may be potentially effective therapeutic strategies for patients with GI dysfunction who are truly unable to take warfarin. The Italian consensus paper also recommends patients with a previous myocardial infarction remain on warfarin and do not receive dabigatran. This stems from the RE-LY trials reporting of an increase in the number of myocardial infarctions in the dabigatran group – although this trend did not reach statistical significance and was less pronounced in a report of supplementary findings from the RE-LY trial . NonetheAlthough dabigatran and the other novel oral anticoagulants do not require routine monitoring of coagulation effect, there are certain situations when a laboratory measure of anticoagulant effect would be needed . The aPTT may offer a qualitative measure of the anticoagulant effect of direct thrombin inhibitors , it is nThere are situations when anticoagulation needs reversal, generally either in the emergency setting due to bleeding or for planned elective surgery. Warfarin has an established antidote in the form of vitamin K, but it should be remembered that even after intravenous administration of vitamin K then INR takes several hours to normalise . The novIn patients with normal renal function dabigatran can be discontinued 24 hours prior to surgery, increasing to 2-4 days if the patient has impaired renal function or is deemed at high risk for bleeding . A laborWhen bleeding is encountered in patients taking a novel antithrombotic agent, first stop the drug as the relatively short half-lives ensure the blood levels of the drug will fall rapidly. For predominantly renally-excreted drugs such as dabigatran, it is crucial to ensure an adequate level of diuresis. Supportive measures should be given as with all cases of bleeding, including transfusion of packed red cells or fresh frozen plasma as the clinical scenario dictates. If life-threatening bleeding is encountered and supportive measures are insufficient, recombinant factor VII or prothrombin complex concentrates  may have8) and recognise the factors and nuances pertinent to each clinical encounter which will favour differing antithrombotic strategies.Clinicians are well aware of the limitations of warfarin and the need for alternative therapies. We must also now be aware of the shortcomings of the new agents  first stroke, systemic embolism, myocardial infarction, cardiovascular death; 2) first unplanned cardiovascular admission, death from any cause. After over 3000 patients had been enrolled, the trial was prematurely halted due to a two-fold increase in cardiovascular events in the dronedarone group  . Thus th2DS2-VASc) now permit rapid but comprehensive evaluation of a patient’s risk for thromboembolism. We are better equipped to accurately identify low-risk patients who do not need to be burdened with antithrombotic therapy, and are clearer that the majority of AF patients (those with at least one other stroke risk factor) require formal oral anticoagulation. Aspirin has been proven to be an inferior option to anticoagulation, and is not free of bleeding risk. We also have the simple validated tools to evaluate a patient’s risk of haemorrhage (HAS-BLED). The interplay of these three factors  should mean fewer patients are undertreated with an ineffective therapy and more receive appropriate protection from thromboembolism.A decade of intensive research into AF has resulted in major progress in the management of this disorder. Contemporary well-validated stroke risk factor schemes (CHAThe emergence of a clutch of new oral anticoagulants should further shift the landscape towards improved stroke prevention in atrial fibrillation . These a


Cfs). To examine the validity of the proposed algorithm, we carried out a clinical test in which 12 oriental medical doctors made pairwise diagnoses on the pulses of volunteering subjects. 169 subjects were simultaneously diagnosed by paired doctors, and the diagnoses in 121 subjects were concordant, yielding an accuracy of 72% and a Matthews correlation coefficient of 0.42, which indicates reasonable agreement between doctors. Two sample T-tests showed that subjects in the sunken pulse group had significantly higher BMI and Cfs (P < .05) than those in the floating pulse group. The pulse classification by the algorithm converged with the diagnoses of paired doctors with an accuracy up to 69%. With these results, we confirm the validity of the novel classification algorithm for the floating and sunken pulses.We propose a novel classification algorithm for the floating pulse and the sunken pulse using a newly defined coefficient ( Pulse diagnosis is one of the four important diagnostic methods in oriental medicine, which are inspection, listening and smelling, inquiring, and palpation –4. The pPulse diagnosis requires sophisticated palpation techniques and interpretation on the pulse qualities demands enduring efforts with many clinical trials and case studies. For this reason, experiential bias and subjective judgments remain difficult to avoid. To overcome technical difficulties, to answer doubts and skepticism on its clinical validity, and to go beyond its current state of the art, it is necessary to develop objective techniques for pulse-taking using standardized protocols and quantitative definitions of the pulse qualities in terms of a few measureable physical parameters –7.To quantify and standardize pulse diagnosis, it is essential to develop palpation devices that provide objective pulse signals as a function of the hold-down pressure, and to develop classification algorithms of the pulse qualities by analyzing the measured pulses. At the same time, the validity and efficiency of pulse diagnosis need to be verified clinically by objective methods. Thus far, some progress has been made on the quantification and standardization of pulse diagnosis. For example, pulse devices equipped with arrays of piezoresistive pressure sensors have been developed. In addition, clinical data have been collected by pulse devices and it has been analyzed by statistical methods. Studies on the fundamental aspects such as the blood flow dynamics along the radial artery and classifications of pulse waveforms are currently under investigation –18. Pulse-taking devices should be able to measure pulse waveforms for several different steps of hold-down pressure, and it should be able to record the spatial distribution of the pulse strength along the axis of the radial artery and in its cross-section . With a For developing a reliable classification algorithm for the pulse qualities, the algorithm needs to be validated by a clinical test with a relevant protocol. The pulse-taking device used should have guaranteed repeatability and reproducibility, and the pulse qualities determined by the algorithm should be tested for consistency with the OMDs' diagnoses. As a first step towards developing reliable classification algorithms, in this work, we introduce a novel classification algorithm for the floating and sunken pulses. To study the validity of the suggested algorithm, we study clinically the correlation between algorithmic outcomes and the OMDs' diagnoses. The floating and sunken pulses belong to four principal pulses together with the rapid and slow pulses . TherefoAccording to “Mai Jing”, the floating pulse is a pulse potent when felt with no pressure applied but impotent when felt with pressure applied, and the sunken pulse is a pulse impotent when felt with no pressure applied but potent when felt with pressure applied, while the hidden pulse is a pulse imperceptible until the fingers touch the bone with extremely heavy pressure . SimilarP-H curve (the curve of the pulse strength (H) as a function of the hold-down pressure (P)) as shown in P-H curve alone is insufficient to distinguish the floating or sunken pulses from more complex pulses. However, for the sake of simplicity, in this work, we do not distinguish the floating and sunken pulses from floating-type and sunken-type pulses and suppose them as the floating and sunken pulses. As shown in There have been suggestions on how to quantify the pulse qualities defined only by the level of depth using pulse signals obtained by pulse-taking devices. As expounded by Fei , the floCfs) defined by . WeH1 to H5) for each pressure-step (P1 to P5) as shown in the bottom panel in Hshallow and Hdeep, we proceeded to calculate Cfsnew(1) and Cfsnew(2).As shown in Paired OMDs diagnosed 169 subjects simultaneously either as floating or sunken pulses. Among them, the diagnoses on 121 subjects (71.6%) were concordant, while the diagnoses of the remaining 48 subjects (28.4%) were divergent. Among the concordant cases, 49 subjects (40.5%) were diagnosed with floating pulses, and 72 subjects (59.5%) with sunken pulses. We assumed diagnoses by paired OMDs returned reliable concordance. For the 121 subjects who were concordantly diagnosed with floating or sunken pulses by paired OMDs, we tabulate some physiological quantities stratified by pulse quality and gender in Cfsnew(1) and Cfsnew(2) were significantly different between the two pulse groups and in both genders at the significance level of P = .05, while blood pressures, heart rate, or Hmax remained not different significantly. Here Hmax is the maximum value among the measured pulse strengths ranging from H1 to H5, where Hi = (Hi1 + Hi2 + Hi3)/3 is the pulse strength at the ith pressure-step averaged over the three palpation locations. Other parameters such as diastolic blood pressure, difference between systolic and diastolic blood pressure were not significantly different between the floating pulse group and sunken pulse group.Cfsnew(1) and Cfsnew(2) for the 121 concordant subjects is 0.96. Despite strong correlation between Cfsnew(1) and Cfsnew(2), Cfsnew(2) is more meaningfully different than Cfsnew(1) between the floating and sunken pulse groups in both genders. It implies that Cfsnew(2) may give improved performance over Cfsnew(1) in predicting the floating and sunken pulses. Based on this observation, we examined the diagnostic performance of pulse qualities using Cfsnew(1) and Cfsnew(2), with reference to the OMDs' diagnoses. The performance of the algorithm with reference to the OMDs' diagnoses is slightly better in male volunteers than in female volunteers. Since the difference between two gender groups is minor, hereafter we do not distinguish genders and present results only for the entire subject group.The Pearson correlation coefficient (PCC) between Cfsnew(1) and Cfsnew(2). A simple decision rule can be made by We attempt to classify the measured pulses into the floating or sunken pulses using Cfs* is the discriminant for the floating and sunken pulses. Applying this rule, we obtain Cfs*. Results by using Cfsnew(1) is shown in (a) and Cfsnew(2) in (b). As plotted by the asterisked curve, the number of concordant decisions for the floating pulse increase with increasing Cfs*, while it was the opposite for the sunken pulse (squared curve). The overall maximum in the concordant decisions for the floating and sunken pulses lies in the central regime of Cfs*. Specifically, the accuracy (ACC), which is the ratio between the concordant decisions and total decisions, is maximally given by ACC = 82/121 = 67.8% at Cfs* = 0.45 with Cfsnew(1) (boxed region in (a)), and ACC = 84/121 = 69.4% at Cfs* = 0.53 with Cfsnew(2) (solid box in (b)). On the other hand, the Matthews correlation coefficient (MCC) is maximally given by MCC = 0.31 at Cfs* = 0.45 with Cfsnew(1) (boxed region in (a)), and MCC = 0.40 at Cfs* = 0.68 with Cfsnew(1) (dotted box in (b)).Cfsnew(1) and Cfsnew(2) show similar patterns in accuracy with maximum values given similarly, that is, ACC = 67.8% with Cfsnew(1) and ACC = 69.4% with Cfsnew(2). However, compared to Cfsnew(1), Cfsnew(2) shows improved correlations with decisions by OMDs. Noting that the MCC between pairs of OMDs is 0.42, the MCC between diagnoses by Cfsnew(2) and OMDs is reasonably good. Both the Matthews correlation coefficient and the two-sample T-test in Cfsnew(2) yields better concordance with the pulse determinations by OMDs than Cfsnew(1). It implies that an adequate pair of the low and high applied pressures to discriminate the floating pulse from the sunken pulse is given by  = . We will discuss this result further in the section of Discussion and Conclusions.Decisions by both Cf* and that for the sunken pulse by Cs*. Then, the classification rule  (we skip the result for Cfsnew(1)). Since the rule in  to count the determined pulses either as floating or sunken pulses with respect to the total number of subjects to be determined, that is,duces to  in the c[If  Cfs≤Cfsnew(2). Both the total number of decisions (asterisked curve) and the concordant decisions with OMDs' (squared curve) increase with increasing Cf* for the floating pulse, while they increase with decreasing Cs* for the sunken pulse. On the other hand, the accuracy (plain curve) tends to increase with decreasing number of decisions made. The sudden drop in accuracy for the sunken pulse close to 1 (Cs* ≥ 0.85) is unimportant anomaly that may occur with small sample size. An optimal pair of two discriminants turns out to be  =  (boxed areas) with respective accuracy given by ACC = 29/47 = 61.7% for the floating pulse, and ACC = 35/40 = 87.5% for the sunken pulse. The overall accuracy for the 87 determined cases is ACC = 64/87 = 73.6%, which is improvement of about 4.2% compared to the result by (Cfsnew(1). For instance, for an optimal pair of  = , ACC = 24/38 = 63.3% for the floating pulse and ACC = 43/57 = 75.4% for the sunken pulse. The overall accuracy for the 95 determined cases is ACC = 67/95 = 70.5%, which is improvement of about 2.7% in accuracy with 26 subjects (21.5%) left undetermined.Cf*, Cs*). For instance, choosing Cs* much larger than Cf* is expected to increase the accuracy but decrease the selection rate, while choosing an equal value for Cs* and Cf* will maximize the selection rate (SR = 1) but at the expense of the accuracy. To examine gain and loss between the selection rate and accuracy as a function of the discriminants , we calculated the number of concordant decisions and the number of decisions made on two-dimensional space spanned by Cf* and Cs*.Depending on one's preference for accuracy versus selection rate, one can have different combinations for an optimal pair of discriminants (Cfsnew(1) and the squared curve presents results by Cfsnew(2). There exists an optimal pair of discriminants  with which the concordant decisions become maximal for a given number of decisions made as the floating or sunken pulses (refer Cfsnew(2) than with Cfsnew(1) in the entire range of the selection rate. Especially, for Cfsnew(2), a meaningful improvement in accuracy is observed at the expense of selection rate.As a result, in es refer . InformaCfs satisfying Cf* < Cfs ≤ Cs* can be classified into the group of middle-depth pulse. Then . It shows a clear tendency for Cfsnew(2) (squared curve) that increasing the number of the decisions into the middle-depth pulse group increases the accuracy in the decisions into the floating and the sunken pulse groups.[If  Cfs≤Cfs) which is bounded between 0 and 1. When Cfs is close to 0, it is classified as a floating pulse, When Cfs is close to 1, it is classified as a sunken pulse. Using the classification algorithm based on the new Cfs method, we performed a clinical study to determine correlation between diagnoses made by the OMDs and by the algorithm. We proposed a novel classification algorithm for the floating pulse and sunken pulse and carried out a clinical test to examine its validity. First, we introduced the coefficient of the floating and sunken pulses , and the rest (28%) showed divergent diagnoses, yielding a Matthews correlation coefficient of 0.42. It lies within a reasonable range of agreement between the OMDs. Out of the 121 concordant diagnoses, 72 were diagnosed as the sunken pulses (sunken pulse group), and 49 were the floating pulses (floating pulse group). T-tests between the floating pulse group and the sunken pulse group showed that BMI and Cfs were significantly different (P < .05) in both genders. This result is in accordance with an experiential knowledge that obese individuals are likely to have sunken pulses [P = .1, while it did not show differences in heart rate, maximum pulse strength, and so forth It indicates that average blood pressure is another factor which determines the pulse depth. By these observations, we suggest that two major factors determining the pulse depth are the thickness of skin tissue above the radial artery and the average blood pressure. Two-sample n pulses . We can Cfsnew(1) or Cfsnew(2), while the Matthews correlation coefficient reached was up to 0.40 for Cfsnew(1) and 0.31 for Cfsnew(1). It suggests that the classification algorithm using either coefficient yields an acceptable concordance with OMDs' diagnoses, and Cfsnew(2) yields an optimal performance in its concordance. It suggests that an adequate pair of low and high applied pressures for our algorithm is given by  = . Since our algorithm simulates diagnosis by OMDs who compare pulse strengths at low and high hold-down pressures to determine the pulse depth, the above result implies that OMDs are likely to determine the pulse depth by comparing pulse strengths at a weak applied pressure of Pshallow ≈ 37 mmHg and at a strong pressure of Pdeep ≈ 143 mmHg, one of which lies much below the normal range of the blood pressure and the other one lies above. For the 121 subjects diagnosed concordantly by paired OMDs, we tested for consistency with the novel classification algorithm. For the entire study cohort, the maximum accuracy reached was about 68% ~ 69% using coefficient Pshallow than at Pdeep, the maximal pulse strength will be positioned well below the average blood pressure (Pavg ≈ 85 mmHg) and it shows a typical floating characteristic, and when the pulse strength at Pdeep is larger, it shows a sunken characteristic. On the other hand, if pulse strengths at Pshallow and Pdeep are not so different, the maximum pulse will be observed around Pavg, yielding a middle-depth pulse. A more accurate choice for the low and high pressures to be used in the algorithm can be made with a pulse-taking device that offers more narrowly spaced hold-down pressures. A pulse-taking device with 9 steps of the hold-down pressure that are spaced equally up to 180 mmHg is an appropriate candidate for such purpose.When the pulse strength is larger at Cf* < Cfs ≤ Cs*, which is more useful since OMDs in clinics diagnose a pulse either as floating, sunken, or neither of them corresponding to the middle-depth pulse. The implication of the middle-depth pulse is a sign of balance between Yang/function (Qi) and Yin/form (blood) [Finally, we suggested an updated version of the above-tested algorithm by allowing a regime of the middle-depth pulse group for the pulses satisfying  (blood) .Cfs. By a clinical test, primarily we confirmed the validity of the novel classification algorithm and secondly we found appropriate numerical values of the pressure steps which are apt for determining the level of pulse depth. To our knowledge, this study is the first report on developing classification algorithm for the floating and sunken pulses based on a massive clinical data with human subjects of the order of hundred. This work may motivate further research activities towards developing objective classification algorithms for other pulse qualities.In conclusion, we proposed a novel algorithm to classify pulse into floating, sunken, or middle-depth categories using a coefficient called


Salmonella in alternative cages systems have been raised. The extent to which cage systems may affect the intestinal microbiota of laying hens is not known, and different microbiota may demonstrate different resistance towards colonization with Salmonella. To investigate this, ileal and caecal samples from two experimental studies where laying hens were inoculated with Salmonella Enteritidis and housed in different systems , were compared using Terminal Restriction Fragment Length Polymorphism (T-RFLP). The distribution of genera in the microbiota in caecum was furthermore described by next generation sequencing of 16S rDNA libraries.In the EU conventional cages for laying hens are forbidden beginning in January 2012, however concerns about a higher transmission rate of Salmonella positive and negative samples from same cage. By deep sequencing of 16S rDNA libraries from caecum, 197 different Operational Taxonomic Units (OTU) were identified, and 195 and 196 OTU respectively, were found in hens in aviary and furnished cages, but only 178 OTU of these were recovered from conventional cages. The ratio between the dominating phyla or families and genera in the microbiota remained fairly constant throughout the study. Faecalibacterium and Butyricimonas were the most prevalent genera found in the caecal microbiota of layers irrespective of the cage type.Hens in the same cage type developed similar T-RFLP fingerprints of the ileal and caecal microbiota, and these could be separated from layers in the other cages types. No significant difference in the fingerprint profiles could be observed between Salmonella from these groups. We suggest, that differences in group size and exposure to a more faecally contaminated environment in the alternative systems may explain the observed differences in diversity of the caecal microbiota.Hens confined in the same cage group tend to develop similar microbiota in their ileum and caecum possibly due to isolation, while differences in the microbiota between cages may be caused by environmental or individual bird factors. Although the cages type had influence on composition of the microbiota in the layers by promoting higher diversity in furnished and aviary systems, we did not observe differences in colonization and excretion pattern of  Salmonella to humans due to a general higher level of microbial contamination of the shell of eggs derived from hens housed in alternative housing systems [Salmonella. Stressing laying hens by feed withdrawal is a traditional method to induce molting, and in several studies this have resulted in an increase in the susceptibility towards colonization by Salmonella [Due to animal welfare considerations the EU has banned the use of conventional cages (CC) for laying hens from 2012, and alternative systems such as furnished cage systems (FC), floor systems or aviaries (AV) have been proposed to replace these [ systems . It is nlmonella ,5. The mlmonella , as a reIn the investigations of the dynamics in the intestinal microbiota, molecular methods have superseded conventional culture methods due to an increased sensitivity, and have become powerful tools. Terminal Restriction Fragment Length Polymorphism (T-RFLP) and Denaturant Gradient Gel Electrophoresis (DGGE) have been used to describe variations and diversity of the microbiota in the intestinal tract in broilers -10. HoweSalmonella Enteritidis.The microbiota of laying hens experiencing nutritional stress has been investigated by 454 pyrosequencing [D), however to compensate for the variation between individual comparisons, the mean of the SD values was calculated and used to compare cage groups. The Dice coefficients from the first experimental study are shown in Table D 54.3 ± 9.6) with FC being in between, sharing profiles with both the other cages (CC SD 67.4 ± 9.9 and AV 66.8 ± 11.4). When sampling was done 4 weeks later, higher SD values were calculated within cage, while values between cages were in the range 65.5-67.5. This shows that layers sharing the same environment also had comparable ileal microbiota, and this similarity increased over time. The height of the T-RF peaks reflected the prevalence of individual species in the microbiota. Ileum was characterized by having the same 3-4 dominating T-RFs in all cage groups, but other individual T-RFs were also present. Before inoculation 10.5 ± 1.7 different T-RFs were detected in CC, while FC had 6.5 ± 2.7 and AV 7.3 ± 3.5. These were maintained throughout the study, although an increase was found in AV (10.7 ± 2.7). The four most dominating T-RFs in all samples were 393 bp, 406 bp, 597 bp, and 550 bp. These T-RFLP fragments could be equated with by different Lactobacillus species by in silico digest of 16S rDNA. Although the total number of detectable T-RFs remained constant in the ileum, an inverted relationship was found between one group of T-RFs: 406 bp, 606 bp and 550 bp which decreased in height, whereas as a new and unidentified T-RF 813 bp emerged. This shift was primarily found in layers from FC and a few layers from other cages, and this may explain some of the differences observed in SD between cages.The microbiota in ileal and caecal samples from the first experiment were characterised by creating individual T-RFLP fingerprint profiles for each sample. Profiles were generated on the basis of the number of Terminal Restriction Fragments (T-RFs) in the range of 60 - 850 bp. The relationship between two profiles could then be calculated by pair wise comparisons as a Dice similarity coefficient (SD 45.4 ± 14), however the profiles were more uniform on the second sampling 4 weeks later (AV 74.2 ± 4.9). The SD values were higher within the same group than between cage groups, and an increase in SD over time was observed, in accordance with the findings from the ileum.The T-RFLP profiles from the caecum contained a higher number of T-RFs reflecting a much more complex microbiota than in the ileum, and an increase in the amount of T-RFs was observed in all caecal microbiota over time Table . The majLactobacillus spp., while no specific genera could be identified for the other fragment, as several different genera  may be represented by this T-RF.To test whether the differences in profiles between cages were caused by a specific cage factor or merely a reflection of isolation between cages, we included samples from the second experimental study . Apart fS. Enteritidis on intestinal microbiota was also evaluated. After inoculation, no clinical signs of infection were detected in the layers. However, colonisation of the intestinal microbiota was established, and S. Enteritidis could be detected in samples from internal organs as well as in cloacal swabs [Salmonella was found in a few layers by culture and PCR. In the ileal samples, Salmonella was detected in 2/8 from AV by PCR, while other samples were negative. In the caecum, S. Enteritidis could be cultured in 2/8 samples from AV, 3/8 from both FC and CC. The concentration of S. Enteritidis in the positive samples was generally low, as culture positive samples not always were positive by real-time PCR. T-RFLP profiles of intestinal microbiota positive for S. Enteritidis were compared with profiles where it had been eliminated. On the basis of the mean SD values calculated between Salmonella negative and positive samples from the same cage, no differences could be detected between positive and negative samples within same cage (data not shown). When profiles were analysed by PCA, no discrimination was found between positive or negative samples within the same cages .The consensus sequence from each OTU was compared against the Ribosomal Database (RDP server) to find the most related species or genus. Though many of the bacterial species in the caecal microbiota still remain to be characterized, it was possible to classify 92% of all OTUs to phylum level, and out of these were 86% classified to class level and 55% to genus level. Although variation was observed in the relative presence that colonized the caecum, it was the same group of genera that were dominating in all cages before and after inoculation, accounting for more than 74% of the total amount of reads Table . The caea Figure , and witTo investigate the impact of different housing conditions on the intestinal microbiota in ileum and caecum in laying hens, samples obtained from two experimental inoculation studies previously reported by De Vylder et al. ,19 have The differences in the evolution of the microbiota were further analysed by deep sequencing of 16S rDNA libraries from pooled caecal samples. When 16 week old laying hens were moved from a floor system and into conventional cages, their caecal microbiota changed towards a less diverse microbiota compared to hens from the same flock that were allocated to aviary and furnished cages. Sequencing of rDNA libraries revealed that hens housed in conventional cages showed a progressive decrease in the number of different OTUs in their caecal microbiota, compared to hens housed in aviary or furnished cages. The decline was already observed after 2 weeks in the cage, and it was even more pronounced after 4 weeks. The same reduction was not observed in the other cage systems. The OTUs that were not recovered in conventional cages were all represented in the other cages, however in low numbers reflecting that they belong to the group of less abundant species. As each OTU represents unique genera or even species, this reflects an overall decrease in diversity of their caecal microbiota towards fewer and more dominating species. Alternative cage systems are characterized by having larger cages due to flock sizes and facilities for enhancing natural behaviour. These facilities may, however, hinder the removal of manure compared to conventional cages, and an overall higher bacterial level has been noted in these systems [The vast amount of sequence reads generated from each sample by 454 pyrosequencing allowed quantification of the individual OTU in relation to the total flora. Only minor differences were observed in the relative distribution of phyla and classes of bacteria in the caecal microbiota between cages, but quantitative variations that were not cage specific were observed between different genera. However, when OTUs were grouped according to phyla and classes, comparable groups were found in all samples. This indicates that the cage system itself did not influence the balance between the large classes, but pinpoints the caecal microbiota as a dynamic, highly competitive organ where a decrease in one genus may be compensated by an increase in a closely related species, or other species belonging to the same functional guild that shares the same requirement for substrates.Bacteroidetes, with Firmicutes being the second most prevalent. The ratios between these two phyla (F/B) remained fairly equal between the CC and AC, but a decrease was observed for CC. A major reason for this difference was promoted by a shift from Faecalibacterium to Butyricimonas. Whether this change was mediated by the cage system of a coincidence remains to be established, but we did not find that it changed the susceptibility for Salmonella, probably because both species produces butyric acid. There are indications that the feed may have large influence the F/B ratio. In domestic and wild turkeys, Scupham et al. [Firmicutes, with Bacteroidetes only present at much lower level. An explanation for this may be the different feeding strategies that are used. Broilers are normally fed a high energy diet that sustains fast growth, which possibly leaves more digestible nutrients for the intestinal microbiota. In contrast, laying hens are fed a much more restricted diet containing less energy and higher amounts of digestive fibers, which instead may favour genera from Bacteroidetes. The same phenomena has been described for the microbiota in obese humans, where Ley et al. [Bacteroidetes during long term restricted diet.When the consensus sequences from 197 OTUs were aligned with the RDP database, more than 91% were identifiable at least to phylum level, and more than 55% could be identified to genus level. The most prevalent phyla in the caecal microbiota were m et al.  found sim et al. ,13,21,22y et al.  observedFaecalibacterium and Butyricimonas constituting more than one third of the total microbiota in all sequenced caecal samples. The first species is a well known colonizer of the caecal microbiota of poultry; however Butyricimonas has just recently been described in rats [Salmonella in the caecal microbiota [Butyricimonas was the most dominant genus in caecum samples from conventional cages, but this difference was not reflected in any variations found in the colonization level of S. Enteritidis as reported by De Vylder et al. [The two most dominating genera found in this study were  in rats , and has in rats , and colcrobiota . Butyricr et al. , who fouS. Enteritidis to the intestinal microbiota were able to change the species diversity in ileum or caecum. When individual T-RFLP profiles from Salmonella positive layers were compared with cage mates that had cleared the infection no differences were observed. When comparing the distribution of OTU in each group before and after inoculation, the balance between different classes and genera were also maintained throughout the study. The low impact on the intestinal microbiota may be explained by the fact that inoculation only induced a subclinical infection, in contrast to experimental studies where a more profound disturbance of the microbiota has been observed in cases where diarrhoea has followed infection [We did not find evidence that the introduction of nfection ,28.Salmonella (competitive exclusion). However, we did not find that decreased diversity in the layers had a significant impact on the colonization and elimination of Salmonella. It is likely that this colonisation resistance is highly important in broilers where a mature flora has not been established yet, but in layers this may not be as important. Furthermore, in the second inoculation study where seeder birds were housed together with non-infected birds, De Vylder et al. [S. Enteritidis was higher among hens housed in aviary or floor system than in conventional and furnished cages. A likely explanation for our observation is that direct contact to faecal material from infected hens is very important for the transmission of S. Enteritidis in a flock, and that the higher species diversity found in layers with more contact with faecal material does not prevent colonization, but keeps it at a relatively low level.In the early studies of Nurmi and Rantala , it was r et al.  found thSalmonella, and moreover the higher diversity found in aviary systems did not protect laying hens from being infected. We did not find evidence, that the cage systems itself was able to change the intestinal microbiota in a way which made it more sensible towards colonization with Salmonella, but it highlights that hygiene in alternative systems is a particularly critical factor for preventing the spread of Salmonella within a flock.In the present study, we have compared the intestinal microbiota in layers from different housing systems under experimental conditions. When laying hens were housed in conventional cages, a change was observed in their caecal microbiota towards a less diverse flora, with the most prevalent genera being more dominating compared to aviary and furnished cage. This decrease in diversity did not have an impact on the elimination of 8 CFU of a nalidixic acid resistant S. Enteritidis PT 4 strain (76Sa88), which previously had been isolated from an outbreak of salmonellosis in laying hens [Intestinal content samples from ileum and caecum were received from two experimental infection studies previously described by De Vylder et al. ,19. Brieing hens  chain fa9 CFU of the same nalidixic acid resistant Salmonella Enteritidis strain. At day 22 post-infection, the seeder hens were randomly divided into four groups and housed together with the non-infected contact hens in the different housing systems such that in each housing system fifty percent seeders and fifty percent contact animals were present. Samples of ileal and caecal content were collected for analysis of the microbiota at the end of the experiment 4 weeks later. Al experiments were approved by the Ethical Committee of the Faculty of Veterinary Medicine, Ghent University.In the second experiment 16 week old laying hens raised in a floor systems, were accommodated for two weeks in one isolation unit (floor system) to adjust to their new environment. Then the flock was randomly divided in two groups, and one hundred and twenty-six non-inoculated contact animals were housed in 3 different housing systems; (1) 36 hens in battery cages, (2) 30 hens in a furnished cage, (3) 30 hens in an aviary. The remaining one hundred and twenty-six hens, called seeder-hens, stayed on the floor and were individually inoculated orally with 10During necropsy of layers, samples were collected from the ileum and caecum. The gut samples were stored by diluting 1 g with 3 ml of 98% ethanol and kept at 4°C until purification, where the ethanol was removed by washing twice with 1 ml of Buffered Peptone Water . Oviduct samples were stored at -20°C until preparation, where surface samples from these organs were collected by scraping the mucosal lining after gentle thawing. Two hundred milligrams of gut contents (ileum and caecum) or oviduct were used for total DNA extraction using the QIAamp DNA Stool Mini Kit  system. The extraction was carried out in accordance with the instructions of the manufacturer, with an additional step of lysozyme treatment, which was added to the procedure before the use of InhibitEX tablets provided in the QIAamp DNA Stool Mini Kit. After incubation of the sample in ASL buffer at 95°C for 5 min, 140 μL of a 10 mg/ml solution of lysozyme  in Tris-EDTA buffer (10:1 mM), pH 8, was added to each extraction tube and samples were incubated at 37°C for 30 min. The purified DNA was eluted in 200 ml buffer AE (Qiagen) and DNA was stabilized by adding 4 μL of a 50 mg/ml BSA solution  and 2 μL of Ribonuclease-A . The purity and concentration of DNA was measured using NanoDrop . All samples were stored as concentrated samples at -20°C until use. Samples were diluted to a concentration of 5 mg DNA per ml before use.Salmonella by a LNA real-time PCR method described by Josefsen et al. [Extracted total DNA samples from the ileum and caecum were tested for n et al.  with minGeneration of a PCR fragment of the 16S ribosomal gene was done as described previously . BrieflyHha1  in a 20 μl reaction mixture.The PCR products from the four replicates were pooled into two samples, purified with QIAquick PCR purification kit , and finally eluted in a volume of 30 μl EB buffer . Then 15 μl purified PCR product was digested overnight (or 3 hours) at 37°C with 0.02 U of Each sample was analysed as two replicate fragments (T-RFs) by electrophoresis on an automatic sequence analyzer . Aliquots (2 μl) of T-RFs were mixed with 2 μl of deionized formamide, 0.4 μl of loading buffer (PE Biosystems), and 0.6 μl of DNA fragment length standard . The T-RF mixture was denatured at 94°C for 2 min and chilled on ice prior to electrophoresis. Five microliter aliquots of the mixture were loaded on a 36-cm, 6% denaturing polyacrylamide gel. Electrophoresis settings were 2,500 V and 40 mA for 10 h, using the B filter set. Due to sequence species specific variations in the ribosomal gene, a restriction digest will give rise to T-RF of different size, and when many species are mixed as in the intestinal microbiota this can be visualized as a pattern of peaks in an electropherogram, a fingerprint profile. These profiles were collected by the software and analysed by the use of BioNumerics software . The length of each band was determined by comparing it towards the internal standard ladder. From each sample two replicates were compared, and weak bands that were only represented in one of the two were rejected to exclude false T-RFs from the fingerprint. After normalization of all profiles towards the internal standard, they were compared using BioNumerics. The comparisons between cages were based on calculating the Dice similarity coefficient and the unweighted pair group method using arithmetic averages for clustering. Principal Component Analysis (PCA) was performed to reflect the grouping and relatedness of samples.Bacteria. To increase the number of reads, all samples were run as multiplex on the same ¼ picoplate using nucleotide barcodes tags on primers, allowing sample identification to each sequence read.Samples (n = 10) from the same cage types , and sampling date (before inoculation and 4 weeks PI.), were pooled by mixing 250 ng of purified DNA from each sample in one tube, in total making up 6 samples. After adding twice the volume of 70% ethanol to each sample for conservation, they were submitted for pyrosequencing at LGC Genomics, Berlin, Germany, using the Genome Sequencer FLX Titanium system's standard amplicon sequencing protocols . There, a 410-420 bp fragment spanning two variable regions (V4 and V5) in 16s rDNA genes was amplified using the primers 519F 5'-CAGCAGCCGCGGTAATAC-3 and 926R 5'-CCGTCAATTCCTTTGAGTTT-3, targeting http://rdp.cme.msu.edu/ was used to assign the phylogenetic position of each OTU. The search criteria were for both type and non-type strains, both environmental (uncultured) sequences and isolates, near-full-length sequences (> 1200 bases) of good quality. If there was a consensus at the genus level, the tag was assigned this taxonomic classification. If no such consensus was found, the classification proceeded up one level to family, and again if no taxonomic affiliation could be assigned the tag continued to be proceeded up the tree, as described by Huse et al. [All sequences in the output file from the FLX sequencer was sorted into sample groups based on the barcode tag. After trimming all sequences for barcodes and fusion primers using the FLS software, sequences were imported into the CLC bio software , where they were checked, aligned and filtered for high quality sequences. OTU's were generated by CLC based on 99% similarity on the data set that had a sequence longer than 400 bp. The Sequence match analysis tool in the Ribosomal database project 10 e et al. . In someSN carried out the molecular work, did the the statistical analysis of T-RFLP and wrote the manuscript. LM preformed sequence alignment and analysis of the pyrosequencing data. LB participated in the design of the molecular study. JDV and FVI designed and conducted the experimental studies, and KP conceived of the study. All authors read and approved the final manuscript. The authors declare that they have no competing interests.


Haptocorrin (HC) carries cobalamin analogues (CorA), but whether CorA are produced in the body is unknown. All cobalamins (Cbl) to the foetus are delivered by the Cbl-specific protein transcobalamin (TC), and therefore analysis of cord serum for CorA may help to clarify the origin of CorA.HC-CorA were quantified in paired samples of cord serum from newborns and serum from mothers (n = 69).The CorA-concentration was higher in cord serum  than in serum from the mothers , (p<0.0001). HPLC-analysis showed CorA-peaks with retention times of 13.5, 14,5 and 16.5 min in samples from both the mother and cord serum. The peak with retention time 16.5 min constituted 24% (mother) and 45% (cord serum) of the total amount CorA, and eluted as does dicyanocobinamide.Our results support that CorA in the human body are derived from Cbl. Vitamin B12  is a cofactor for two enzymes in humans; methionine synthase and methyl malonyl coenzyme A mutase The presence of circulatory CorA was demonstrated in the late 1970's by Allen and co-workers using measurements of plasma corrinoids with radioimmunoassay involving the two Cbl-binding proteins intrinsic factor (IF) and haptocorrin (HC) We have confirmed the presence of CorA on circulatory HC, while no CorA was observed on transcobalamin (TC) The origin of CorA in the human body is unknown. One possibility is that CorA are derived from passive absorption of analogues synthesized by the microbiota of the intestine, and not endogenously created in the body from circulating Cbl in the human organism. A recent finding of large amounts of analogues in human stools In the present study, we report CorA to be present in cord serum from newborns in concentrations far exceeding those in the corresponding samples from the mother and we identify the same three molecular forms of analogues in serum from mother and child. Our results strongly supports that CorA are produced within the body from circulating Cbl.Blood samples were collected from 69 at term mothers between 1 and 12 hours before they gave birth, and cord blood from the umbilical vein of their newborn babies collected immediately after expulsion of placenta. This study cohort was recruited among deliveries at the Department of Obstetrics and Gynaecology at the University of Saarland, Germany in 2004 and has previously been characterized Samples of amniotic fluid were collected from 21 healthy pregnant women, 14–23 gestational weeks. These samples were leftover material from routine analysis, performed at the Department of Clinical Biochemistry, Aarhus University Hospital. According to Danish ethical rules there was no request for ethical approval.Deglycosylation, immunoprecipitation and proteolytic degradation of serum HC or HC in amniotic fluid in order to release the protein bound corrinoids from 100 µL serum or amniotic fluid, was performed as previously described 2Cbi, Sigma-Aldrich, Denmark) were also exposed to KCN prior to HPLC. The KCN exposed corrinoids present in the samples or standards were separated with HPLC essentially as described by Jacobsen et al A pool of serum from five mothers and a pool of cord serum from their babies were prepared. Corrinoids were released from HC present in 1000 µL of each of the two serum pools as described above. Before separation with reversed phase high performance liquid chromatography (HPLC), and the released corrinoids were converted into their cyano forms by exposure to potassium cyanide . Standard solutions containing 200 pmol/L CNCbl or 100 pmol/L dicyanocobinamide (CNt-tests and paired t-tests) were performed with the software program GraphPad Prism version 4.00 . Original data, as part of this study, can be requested from the corresponding author.All statistical analysis . In addition, the fraction of corrinoids on HC accounted for by CorA, was higher in the newborn babies, than in the mothers . While the concentration of HC-Cbl showed a significant direct correlation between values in cord serum and that in maternal serum  the contents of HC-CorA showed only a weak correlation  (In this study we examined Cbl and CorA attached to HC in serum from term mothers and cord serum from their newborn babies.  = 0.01) . Serum f2 = 0.19, p = 0.0004). Because gestational age was unknown on 6 of the newborns in the cohort, only 63 datasets was included in this calculation  14–23 (20) weeks (n = 21) showed a range (median) of 43–740 (170) pmol/L. The corresponding data for HC-CorA was 2–218 (147) pmol/L and for the fraction HC-CorA/(HC-Cbl+HC-CorA) the results was 0.01–0.59 (0.36). We found no significant correlation between gestational week and the concentration of HC-Cbl or HC-CorA in amniotic fluid.2Cbi.Enzymatically released corrinoids from HC in pooled serum from mothers and in pooled serum from their babies were separated with HPLC after converting of all the corrinoids forms into cyano forms, and post column fractions were analyzed. As expected, cobalamin eluted as cyanocobalamin upon treatment with KCN, giving rise to a peak with a retention time of 15 min in both chromatograms . This peThis study demonstrates the presence of relatively high amounts of CorA in cord serum from newborn babies. We also detected both Cbl and CorA in amniotic fluid. Because of that it may be questioned whether foetal CorA is derived from the amniotic fluid, through foetal swallowing and intestinal uptake. We find this very unlikely since the intestinal IF-Cubam mediated pathway is specific for Cbl, and do not promote the uptake of CorA Foetal Cbl stems from placental uptake of TC-bound Cbl, while no transport of HC-bound corrinoids across the placenta seems to occur The fact that Cbl in the foetus is derived from the mother, explains the correlation between maternal and cord sera HC-Cbl concentrations. The correlation is driven by two other correlations. The Cbl-status of the newborn mirrors that of the mother, and the HC-Cbl status is related to the Cbl status HPLC studies of corrinoids from cord serum, revealed that a substantial amount (45%) eluted with the same retention time as did KCN exposed Cbi. Even though more CorA was released from cord serum than from the maternal serum, the molecular forms of CorA appears to be the same, and comparable to peaks observed in KCN exposed corrinoids present on serum HC from healthy donors Our results have several implications. First, the relatively high occurrence of a compound eluting as does Cbi, suggests an ordered cleavage of the phosphate group linking the corrin ring to its lower ribose-benzimidazole ligand. Second, the observation that the same types of CorA are present in the foetus and in the mother suggest that also in the mothers the CorA is derived from a degradation of Cbl rather than from intestinal absorption of CorA. A high amount of CorA has recently been reported to occur in stools In conclusion our data suggests that CorA is produced in the human body from Cbl. The exact nature of the corrinoids and also the mechanism by which they are formed remains to be clarified.The technical assistance of Anna Lisa Christensen and Jette Fisker Petersen is highly appreciated.


Current surgical approaches to the distal radius include dorsal and palmar plate fixation. While palmar plates have gained widespread popularity, few reports have provided data on long term clinical outcomes in comparison. This paper reports the result of a randomised clinical study comparing dorsal Pi plates and palmar, angle-stable plates for treatment of comminuted, intraarticular fractures of the distal radius over the course of twelve months.42 patients with unilateral, intraarticular fractures of the distal radius were included and randomised to 2 groups, 22 were treated with a palmar plate, 20 received a dorsal Pi-plate. Results were evaluated after 6 weeks, 3, 6 and 12 months postoperatively focussing on functional recovery as well as radiological results.The palmar plate group demonstrated significantly better results regarding range of motion and grip strength over the course of 12 months. While a comparable increase in function was observed in both groups, the better results from the early postoperative period in the palmar plate group prevailed over the whole course. Radiological results showed a significantly increased palmar tilt and carpal sag in dorsal plates, with other radiological parameters being comparable. Pain levels were decreased in dorsal plates after hardware removal and failed to show significant differences after 12 months. However, complications such as tendon ruptures were more frequent in the dorsal plate group.Functional advantage of palmar plates gained within the first 6 weeks prevails over the course of a year. Both groups demonstrate further gradual increase of function after 6 months, although dorsal plates did not catch up completely. Improved early postoperative function seems to be the cornerstone for the best possible results. Patients with dorsal plates benefit from hardware removal more than palmar plates in terms of reduction of pain levels. The advantage of palmar plates is a faster functional recovery with lower complication rates. This is especially important in the elderly population. Radiological results did not show a superiority of palmar plates over dorsal plates. Fractures of the distal radius are the most common fractures in the upper extremity and treatment options have been controversially discussed throughout the literature over the last decades -5. Espec®, Basel, Switzerland) or open reduction and internal fixation with the dorsal Pi-plate . Randomisation was carried out preoperatively after inclusion criteria were met. Closed, identical envelopes were placed in a box and were drawn by an uninvolved nurse. Only patients over the age of 50 with unilateral AO-type C fractures without any other injuries of the upper extremity were included. Patients with intercarpal injuries such as SL-ligament dissociation, fractures older than 8 days, open fractures and patients with premorbid conditions precluding surgical intervention were excluded. Inclusion criteria were deliberately strict to limit this study to fracture patterns not amenable to other means of fixation.50 patients, operated on in a period of 8 months were initially registered in the study and 42 patients with unilateral AO-type C1, C2 and C3 fractures of the distal radius were included as they completed the complete follow up including hardware removal after 6 months. Patients were randomised to two groups: open reduction and internal fixation with a palmar, angle-stable plate  to detect a difference of one standard deviation (assumed to be 10°) in motion after 12 months between two balanced groups, 17 cases would be needed. With an assumed follow up rate of 85% 20 patients per group were required.All patients with radius fractures were followed up after 6 weeks, 3, 6 and 12 month postoperatively. Hardware was removed in all patients after 6 months. Only 42 patients with unilateral AO-type C1, C2 and C3 fractures of the distal radius did the complete follow up of 12 months and thus were included. 8 Patients were lost for follow up, one died due to unrelated cause and 7 could not be reached or were unwilling to return for the final follow up.Group 1  were treated by fracture fixation with a palmar, plate allowing multidirectional, angle stable screw placement. This group included 22 right-hand dominant patients, 19 women and 3 men, with a mean age of 67.7 (range 52-92) years. The left extremity was injured in 12 patients  and the right in ten . All injuries occurred as a result of a fall onto the hand. Ten fractures were classified as AO-type C1, 7 as AO-type C2 and 5 AO-type C3.®, Basel, Switzerland) (Volar Plate Group) or a dorsal Pi-plate  . Surgical techniques and postoperative treatment and assessment have been described in the previous article [®  software. After explorative analysis, the Student-T test was used except in 2 occasions were the Mann-Whitney test was applied when the Kolmogorov-Smirnov test showed that non-parametric variables were not distributed normally. The level of significance was set at p < 0.05.In Group 2  fracture fixation was performed with the dorsal Pi plate. This group included 20 right-hand dominant patients, 17 women and 3 men, with a mean age of 67.6 range 52 - 85) years. The right extremity was injured in 8 patients  and the left in 12 patients . Except for one patient who sustained the injury in a motor vehicle accident, all other injuries were caused by falls onto the hand. Nine fractures were classified as AO-type C 1, 5 as AO-type C 2 and 6 as AO-type C 3. Patients underwent open reduction and internal fixation with either a palmar, angle-stable plate  and the dorsal plate group (22 degrees), with no statistically significant difference between the two groups (p = 0.29)  The Gartland-Werley score identified statistically significant differences between both groups. Patients in the palmar plate group showed a score of 2.1 compared to 9.2 in the dorsal group (p = 0.001).No cases of posttraumatic non-union were observed. No case of plate or screw loosening was encountered. Nine complications occurred in six patients in the palmar group. The most common complication was transient paraesthesia in the median nerve in 5 cases which resolved the latest after 6 weeks, not requiring carpal tunnel release. Complex regional pain syndrome Type 1 was diagnosed in two patients. Both improved with physiotherapy and nasally applied calcitonin. Furthermore one tendon irritation, an adhesion of the FPL tendon treated with hardware removal after 4 months occurred. In one patient SL- dissociation was missed intraoperatively and only noticed 6 weeks later. In the dorsal group 11 complications occurred in 7 patients. One patient complained of transient radial nerve dysaesthesia which resolved at the 3 months follow up appointment. Median nerve irritation occurred in one patient, also resolving spontaneously, while CRPS developed in 3 patients and was treated in the same way as in the palmar group. Three secondary fragment dislocations were seen in the dorsal plate group, all in AO-type C3 fractures. Two patients underwent revision, with conversion to a palmar plate, the third refused a second operative intervention. In this patient an intraarticular step of more than 2 mm remained, while another patient of this group developed an ulnar impingement syndrome, requiring ulnar shortening osteotomy. Also two ruptures of the EDC II tendon occurred and were treated with a tendon transfer to EDC III. No complications were seen after hardware removal in both groups.While AO type A and B fractures have been treated with palmar plates for an extended period of time, highly comminuted fractures have either been treated with a dorsal or combined approach, but not regularly through a palmar approach only. Only the invention of angle stable implants has opened the door to consider this approach in severely comminuted fractures. While palmar fixed angle implants could be the future for treatment of most Colles' fractures, the dorsal approach remains a good choice in highly comminuted fractures with a metaphyseal defect, when a bone graft is also required . A previRadiological results did not show statistically significant difference in regard to radial inclination. Palmar tilt did show a slightly increased result in the dorsal group compared to the palmar group. This proved to be statistically significant. It seems that if increased palmar tilt is desired, some degree of "overcorrection" has to be obtained. This may prove difficult since palmar plates would need additional bending. Nevertheless the palmar tilt of 9 degrees in palmar plates is sufficient in terms of function. A steeper palmar tilt in dorsal plates may be the result of the surgical technique, when the whole distal part of the fracture is reduced to the palmar side with the dorsal plate rigourosly preventing dorsal displacement. This aspect also can be noticed in the carpal sag. The carpal sag was increased in the dorsal plate group, which shows that a complete palmar shift of the fragment and the carpus occurs when the fracture is stabilized from dorsally. This may be due to a straight plate being applied to a curved dorsal aspect of the radius, especially as features like the Lister's tubercle would require a dorsal bend of the plate. A palmar overcorrection will, in some cases, lead to palmar dislocation of the fractures which has occurred in three patients. It has to be stated clearly that, in terms of anatomical restoration, a dorsal implant is by no means inferior to a palmar implant, and in certain aspects such as palmar tilt proved more effective in our group. Nevertheless, radiographs are not the only decisive aspect to judge a plating system as function is the most important aspect for the patient.The complications encountered in the patients are similar to those described in the current literature and our earlier articles ,5. For dThere are limitations to this report. The cohort of patients was collected at a tertiary care center with expertise in both plating systems. It is not known whether these results can be generalised, as there is a referral-bias in our patient population. Also our inclusion criteria were deliberately strict to limit our patient population to elderly patients in which plate fixation may be problematic and produce less than ideal results. These patients require stable fixation for poor bone quality to allow quick rehabilitation . This agOverall we have found an advantage of palmar over dorsal plating in comminuted fractures of the distal radius, which supports our earlier findings . While iThis study was in part financially supported by IBRA and by the University of Wuerzburg.None of the authors has any conflict of interest in terms of commercial or financial involvement. No agreement with IBRA was made regarding the prohibition of publishing positive or negative results.MJ drafted the manuscript, was involved in the design of the study, did the statistical interpretation and analysisJG carried out the examinations, was involved in the development of the studyRJ developed the design of the study, carried out the examinationsAll authors performed the surgeries.All authors read and approved the final manuscript.


GPX1-4 and SEPP1 were genotyped. Serum selenium was not associated with the risk of neoplastic progression to EA, aneuploidy or tetraploidy (P for trend = 0.25 to 0.85). SEPP1 concentrations were positively associated with the risk of EA  and with aneuploidy , but not selenoenzyme activity or oxidative stress markers. No genetic variants, overall, were associated with the risk of neoplastic progression to EA . Our results do not support a protective effect of selenium on risk of neoplastic progression to EA. Our study is the first to report positive associations of plasma SEPP1 concentrations with the risk of EA and aneuploidy, which warrants further investigation.Clinical trials have suggested a protective effect of selenium supplementation on the risk of esophageal cancer, which may be mediated through the antioxidant activity of selenoenzymes. We investigated whether serum selenium concentrations, selenoenzyme activity, oxidative stress and genetic variation in selenoenzymes were associated with the risk of neoplastic progression to esophageal adenocarcinoma (EA) and two intermediate endpoints, aneuploidy and tetraploidy. In this prospective cohort study, during an average follow-up of 7.3 years, 47 EA cases, 41 aneuploidy cases and 51 tetraploidy cases accrued among 361 participants from the Seattle Barrett's Esophagus Research Study who were free of EA at the time of blood draw and had at least one follow-up visit. Development to EA was assessed histologically and aneuploidy and tetraploidy by DNA content flow cytometry. Serum selenium concentrations were measured using atomic absorption spectrometry, activity of glutathione peroxidase (GPX) 1 and GPX3 by substrate-specific coupled test procedures, selenoprotein P (SEPP1) concentrations and protein carbonyl content by ELISA method and malondialdehyde concentrations by HPLC. Genetic variants in  Results from a few clinical trials and observational studies support a protective effect of selenium on the risk of esophageal cancer Here we investigated the association of serum selenium with the risk of neoplastic progression to EA. Persons with Barrett's esophagus (BE), a premalignant metaplasia of the lower esophageal epithelium, develop EA at a rate of 6–7 per 1,000 person-years, which is substantially higher than in the general population This prospective cohort study was conducted through the Seattle Barrett's Esophagus Research Study, a dynamic cohort of persons diagnosed with BE Endoscopic biopsy protocols and evaluations used in the Seattle Barrett's Esophagus Research Study have been published previously A total of 361 participants free of EA at the time of blood draw and with at least one follow-up visit were included in analyses based on serum selenium. For the analysis for aneuploidy and tetraploidy, 37 and 36 participants, respectively, were excluded from the analysis due to the diagnosis of the respective endpoint at the time of blood draw. As a result, analyses for EA, aneuploidy and tetraploidy had 2647.2, 2378.2 and 2290.0 person-years, respectively. For the subset of 198 participants who had given sufficient blood at the first or second follow-up visit for the laboratory assays, selenoenzyme activity, or selenoprotein concentration and two measures of oxidative stress were made. In addition, single nucleotide polymorphisms (SNPs) in selenoenzyme/selenoproteins were genotyped in this subset.EA was assessed histologically, and aneuploidy and tetraploidy were assessed as previously described Serum selenium concentrations were measured by atomic absorption spectrometry  according to the standard protocol The activity of glutathione peroxidase (GPX) 1 in white blood cells and of GPX3 in plasma were measured by applying our standardized protocol using OXItek commercial kit  based on Paglia and Valentine's method To assess lipid peroxidation, malondialdehyde (MDA) was assayed in EDTA-treated plasma using HPLC as previously described GPX1, GPX2, GPX3, GPX4 and SEPP1) was selected because these genes are associated with oxidative stress and expressed in the gastrointestinal tract r2≥0.8 and minor allele frequency ≥5% A set of tagging SNPs (tagSNPs) in each of five selenoenzyme/selenoprotein genes  All analyses were controlled for gender, baseline values of age (5 categories), waist to hip ratio (quartiles), smoking status  and the use of non-steroid anti-inflammatory drugs . Genetic analysis was also controlled for Caucasian ethnicity (European American or other). All covariates each affected the hazard ratio (HR) of at least one of the outcomes by a minimum of 10%.For genetic analysis of five selenoenzyme/selenoprotein genes, the overall variation within a gene was first evaluated by global gene test, which compared the log likelihood ratio statistics between the model with and without all SNPs in a given gene To assess if prevalence of high-grade dysplasia, aneuploidy or tetraploidy influenced the results, a sensitivity analysis was conducted in two ways: 1) excluding participants with high-grade dysplasia and 2) excluding those with aneuploidy and/or tetraploidy at baseline and considering two outcomes (EA and aneuploidy and/or tetraploidy). The results from the sensitivity analysis were compared with those from the overall analyses. All statistical analyses were conducted by SAS 9.2  and STATA 11 .The majority of study participants were Caucasian and male Table 1,Serum selenium concentrations were not associated with the risk of neoplastic progression to EA Table 2.SEPP1 concentrations were positively associated with the risk of EA and aneuploidy, but not tetraploidy Table 3.GPX3 gene were statistically significantly associated with the risk of EA (P for trend = 0.03 for rs4958872 and P for trend = 0.04 for rs3792797); however, the overall genetic variation was not significant  (Two SNPs in the  = 0.33) Table 4.GPX1-4 and SEPP1 genes was significantly associated with the risk of neoplastic progression to EA.Our study found no evidence of an association of serum selenium concentration with the risk of neoplastic progression to EA, aneuploidy or tetraploidy. However, SEPP1 concentration was strongly and significantly positively associated with risk of EA and aneuploidy, though not tetraploidy. None of the overall variation in Our null finding on serum selenium is consistent with the case-cohort study in The Netherlands that observed no association between toenail selenium and the risk of progression from BE to EA or high-grade dysplasia Within the general population, clinical trials of selenium supplementation with or without other micronutrients in China and the U.S. reported non-significant 6% to 70% decreased risk of esophageal cancer with supplementation, although their 95% CI were large, likely due to their small sample sizes Few studies have investigated associations of selenoenzyme activity or selenoprotein concentrations with cancer −) 2− •) and nitric oxide (NO) To our knowledge, our study is the first to report a positive association of SEPP1 concentrations with the risk of progression from BE to EA and aneuploidy. Aside from being a carrier of selenocysteines, SEPP1 itself has antioxidant properties GPX2 gene  and the risk of EA in a case-control study; however, no association was found GPX2 with the risk of neoplastic progression to EA, nor did we find such an association with GPX1, GPX 4 and SEPP1 genes. Although two GPX3 variants were individually significantly associated with the risk of EA, the overall variation was not significant. Hence, this finding may be due to chance. Our study was limited in sample size; in our post-hoc power calculation while adjusting for alpha level to 0.0114 to account for multiple comparisons of SNPs per gene, the powers of detecting the risk estimate of 1.20 to 1.50 ranged from 1.4% to 3.2% for the observed minor allele frequency of 5% and from 2.4% to 11.9% for the observed minor allele frequency of 49%, respectively for all three measures of the risk of neoplastic progression. Hence, our study was almost certainly underpowered to detect the type of weak associations found in genome-wide scans for other cancers.Only a single previous study has investigated the association between two potentially functional candidate variants in the Strengths of our study include the prospective design, the long follow-up (on average 7.3 years) and the high frequency of follow-up visits (on average 5.7 visits) for biospecimen collection. We were also able to evaluate flow cytometric abnormalities  that reflect neoplastic progression, which extended our ability to measure progression. Detailed exposure assessments allowed us to adjust for important potential confounding in our analysis. Finally, we extended the evaluation of common variants in selenoenzyme/selenoprotein genes by including more genes than in a previous study of EA One very important limitation of our study is the relatively small number of endpoints in our cohort. To some extent, this is mitigated by the involvement of high-risk participants and the use of valuable intermediate markers of neoplastic progression. Our ability to detect an association between selenium and the risk of neoplastic progression to EA also may have been limited by the relatively high selenium concentrations in our cohort. We used a single serum selenium measurement, which may not capture participants' selenium intake during the entire follow-up period. Finally, serum selenium concentrations may not reflect tissue concentrations, which may be the exposure of most importance.In summary, we found no evidence of association of selenium concentrations with the risk of neoplastic progression to EA. This finding is inconsistent with our previous cross-sectional analysis and suggests that findings from cross-sectional studies of selenium and neoplastic progression need to be interpreted with caution. Our study is the first to observe positive associations of plasma SEPP1 concentrations with the risk of neoplastic progression to EA, a finding that warrants further investigation.


The combination therapy of interferon beta-1b and tacrolimus over the 10-month period of the study was shown to be safe and relatively well tolerated. There were no unexpected adverse events occurring as the result of the combination therapy. Further study of this combination therapy in patients with multiple sclerosis unresponsive to conventional therapy is warranted.Tacrolimus is a calcineurin inhibitor which works to induce immune suppression by preventing cytokine transcription and lymphocyte activation. Combining the immunomodulator interferon beta-1b (Betaseron) with the immunosuppressant tacrolimus (Prograf) may have the potential of additive therapeutic benefit through the complementary mechanisms of action of these two therapeutics. In this randomized, open-label, multicenter, two-arm pilot study, the authors examined the safety and tolerability of the combination of interferon beta-1b and tacrolimus in relapsing remitting (RRMS) and secondary progressive (SPMS) multiple sclerosis patients who have failed one or more immunomodulatory therapies. Patients ( The autoimmune pathophysiological process by which the immune system carries out its attack of the central nervous system in multiple sclerosis (MS) is complex and multipronged and can vary between patients and even within the same patient, over time . As in aStreptomyces tsukubaensis and is currently used as an immunosuppressant to prevent allograft rejection in transplant patients  every other day for 2 weeks, prior to escalating to the fixed 250 μg dose every other day. All patients received injection training [Patients started therapy with interferon beta-1b at half the standard dose (125 training .Tacrolimus therapy was initiated at week 6 (or when full-dose interferon beta-1b was tolerated) at a starting dose of 0.1 mg/kg/day administered twice daily. The dose was adjusted in order to reach the desired low or high blood concentration range (dose adjustments took from 2 to 4 weeks) and was evaluated monthly to ensure that the level remained in the assigned range. The use of corticosteroids  was allowed for the treatment of acute relapses.Safety and tolerability was assessed with physical examinations, laboratory evaluations, and recording of adverse events. The latter were collected at each clinic visit as shown in P values of <0.05 were considered to be statistically significant.All statistical analyses were performed according to the intention-to-treat principle. Descriptive statistics of baseline data for both groups are summarized in This randomized, open-label, multicenter, two-arm dosage trial took place between February 2003 and October 2006 at three MS clinics in Canada. The flow of participants through the different stages of the trial is shown in Baseline patient characteristics of both groups are shown in All adverse events (AEs) were recorded for all patients. Five total patients dropped out of the pilot study. One patient was removed due to a revised diagnosis of MS. Combination therapy-related AEs resulted in three patients discontinuing combination therapy but continued on monotherapy . One patient completely discontinued all therapy. Two patients were lost to followup.n = 3) included hyperglycemia, headache, injection site reactions, tremor, and diarrhea.Twenty patients completed the study in an intention to treat manner. There were no unexpected AEs occurring as the result of the combination. Eighteen different AEs were noted in more than 10% of patients see . Of thosNo significant increase in baseline values of mean creatinine or glucose was seen in either group. A mild decrease was noted in mean magnesium and platelets levels in both groups. No clinically significant differences in lab values were noted between groups over the 10-month period. The mean tacrolimus level during the study was 4.68 ng/mL (sd 0.90) in the LBL group and 7.63 ng/mL (sd 1.44) in the HBL group.P = 0.013).Only the HBL group showed an increase (9.1 mmHg) in mean end-of-study systolic blood pressure when compared to baseline and was considered to be significant when compared to the LBL group (No therapy-related serious adverse event (SAE) occurred. There were no deaths, and no permanent morbidity was noted for any patient.P = 0.089).Only the LBL group showed an improvement in quality of life as measured by the median change from baseline in the visual analogue scale (VAS)  for the HBL group. However, this difference between groups was not found to be statistically significant (This pilot safety study is the first to describe the safety and tolerability of the combination of interferon beta-1b and tacrolimus in MS patients.Combination therapy can result in an additive or even synergistic effect in benefit and/or increased incidence of adverse events (AEs). The combination therapy did not generate any untoward drug interactions or unexpected side effects. There was no evidence of opportunistic infection or impaired immune surveillance. The lack of tacrolimus-related pancreatic or renal dysfunction is reassuring and likely explained by the lower blood levels of tacrolimus attained as compared to transplant trials; however, a longer duration of study is needed to confirm this.n = 2) or tacrolimus (n = 1) monotherapy, and there was no patient withdrawal as a result of perceived lack of efficacy or due to combination therapy-associated adverse events. Discounting the patient withdrawn due to a revised diagnosis of MS from would adjust the withdrawal rate to 16%, thus brining it closer in line with previous findings [The study withdrawal rate of 20%  was higher than the 13% obtained in the 8 MIU arm of the interferon beta-1b pivotal trial . 1/5 patfindings . OverallSome side effects were more frequently found in the HBL group , but interestingly, injection site reactions were significantly lower in the HBL group possibly as a result of the higher dose of tacrolimus. Otherwise, no other difference was found in the incidence or type of AE between groups. The lack of difference between groups, however, may be explained by the small size of the cohort in this study.Several patients continued on the combination therapy initially through a controlled one-year extension (unpublished data) and then in an uncontrolled fashion for several additional years (some up to 8 years). Within that small group's uncontrolled long-term experience, no additional side effects other than what has been previously described in this study occurred, and specifically no significant increase in blood pressure or occurrence of opportunistic infections or cancer was observed (unpublished observations).Lastly, it was observed that both tacrolimus treatment groups experienced a nonsignificant decrease in EDSS over the course of the study, and MRI scan results indicated a median number of T2 and T1 lesions per scan remaining unchanged over the duration of the study (data not shown). While this pilot study was not designed to evaluate efficacy, we believe these findings lend support to the safety profile of this particular combination therapy. Future studies with larger patient cohorts will be designed to expand on these observations and to answer questions regarding the clinical efficacy of combination therapy.Overall, results of this pilot safety study suggest that further studies with larger numbers and longer duration may be warranted. An add-on approach rather than a complete switch may prove to be more beneficial for some patients. The combination of interferon beta-1b and tacrolimus may potentially be considered as a therapeutic option for patients with aggressive disease unresponsive to conventional DMD agents, provided, as for all combination therapies in general, close monitoring and predefined efficacy goals and safety stopping rules are in place.