Background. There is a current debate in the medical literature about plasma calcitonin screening in patients with nodular goiter (NG). We decided on analyzing our 20-year experience with patients in an iodine-deficient region (ID). Patients and Methods. 22,857 consecutive patients with NG underwent ultrasonography and aspiration cytology (FNAC). If FNAC raised suspicion of medullary cancer (MTC), the serum calcitonin was measured. Results. 4,601 patients underwent surgery; there were 23 patients among them who had MTC . Significantly more MTC cases were diagnosed cytologically in the second decade than in the first: 11/12 and 6/11, respectively. The frozen section was of help in 2 cases out of 3. Two patients suffered from a 3-year delay in proper therapy, and reoperation was necessary in 1 case. FNAC raised the suspicion of MTC in 20 cases that were later histologically verified and did not present MTC. The diagnostic accuracy of FNAC in diagnosing MTC was 99.2%. Two false-positive serum calcitonin tests  and one false-negative serum calcitonin test occurred in 40 cases. Conclusion. Regarding the low prevalence of MTC in ID regions, calcitonin screening of all NG patients does not only appear superfluously but may have more disadvantages than advantages. Medullary thyroid carcinoma (MTC) is accounted for 3.5–10% of all thyroid malignancies . As for On the one hand, the serum calcitonin level is considered to be the most sensitive and specific marker of MTC; on the other hand, it must be taken into consideration that not all MTCs produce calcitonin , and hypThere is an ongoing debate in the medical literature about benefits from routine measurement of plasma calcitonin levels in patients who have thyroid nodules . The newDuring the 20-year period of 1991–2011, all patients, who were present at our thyroid outpatient department for the first time, underwent ultrasonography and TSH determination. 22,857 patients with nontoxic thyroid nodular goiters underwent FNAC. Patients with a solitary nodule larger than 1 cm in the maximal diameter underwent US-guided FNAC. The largest nodule was aspirated in the presence of multinodular goiters; if that was not hypoechogenic, the largest hypoechogenic nodule was aspirated too. Hypoechogenic nodules containing small hyperechogenic granules—and/or those with ill-defined borders larger than 5 mm—were also aspirated.We used the following categories in cytological analysis: not diagnostic, benign, suspicious (including follicular tumors), and malignant. We also provided the subtype of the possible malignant disease with the last two categories.When FNAC raised the suspicion of MTC or a malignancy including MTC, the serum calcitonin level was determined.The clinical appearance was analyzed in the cases of all our patients with nodular goiter. The classification was a clinical suspicion of malignancy in the presence of recurrent palsy with causes determined only inside the thyroid, a hard nodule with an uneven surface except for cases which were caused by calcification, or a rapidly growing firm or hard thyroid mass except for cysts.Between 1991 and 1997, the serum calcitonin level was determined by radioimmunoassay (RIA); however, after this time period, we used an immunoradiometric (IRMA) calcitonin assay .4,601 patients underwent surgery which was indicated by a positive cytological report, a clinical suspicion of malignancy, compression signs, or because of the wish of the patient. Histologically diagnosed MTC cases were confirmed by a positive immunohistochemical reaction with calcitonin and chromogranin A: immunostaining of tumor cells with anticalcitonin and with chromogranin A was positive, while the results with antithyroglobulin test were negative in all 23 patients with the presence of MTC. All patients who were not operated on were participating in regular followup examinations with TSH, US every year, and repeated FNAC in case the nodule increased in volume by more than a third. 86.1% of these patients were present for at least one examination within 5 years. P < 0.05 (CF = 4.43) and P < 0.01 (CF = 9.75), respectively (The proportion of men in the MTC group (30.4%) is proved to be significantly higher than in the non-MTC group (12.9%) or in the benign nodular goiter group (9.2%), ectively . The aveP = 0.11) or in those with benign nodular goiter (4.1%), P < 0.001 (CF = 75.9). The clinical presentation raised the suspicion of malignancy in 10 of the 23 MTC cases including 1 patient with a benign FNAC result. Among 10 patients with a clinical suspicion of malignancy, 3 had palsy of the recurrent nerve with causes determined exclusively within the thyroid. Seven patients had hard nodules with an uneven surface, and 9 indicated a rapidly growing firm or hard thyroid mass among 19 patients with palpable nodules.The clinical presentation was more often indicative in MTC patients (43%) than in non-MTC patients  was the presence of multiple amorphous hyperechogenic foci within the nodule see . These fP = 0.04, chi-square test). See next section for further details. Results of cytological examinations were positive  in all 23 MTC cases except for 2 cases that occurred in the first 10-year period . It is iThere were 12 cases in the first decade and 9 cases in the second decade when the FNAC raised the suspicion of the possibility of MTC, although the final histopathological results did not prove the presence of MTC. The calcitonin level was <10 pg/mL in all cases except for two patients. In 12 of 21 cases, Hürthle-cell tumor caused differential diagnostic problems which were later verified histopathologically. In 4 other cases, the atypical lymphoid population caused issues.  The origins of the atypical cells on the smear were presented by a different diagnostic problem in the remaining 5 cases. The sensitivity of FNAC for diagnosing MTC was 74% (17/23), and the specificity was 99.6% , while the diagnostic accuracy was 99.5% .The calcitonin level was in the range 8–2,552 pg/mL with a median level of 277 in 19 preoperatively diagnosed MTC cases (including 2 patients whose disease was diagnosed only on the second occasion). In 1 patient with MTC with a distant metastasis, the calcitonin level measured with the RIA method was falsely negative (8 pg/mL). Three other patients demonstrated a calcitonin level <100 pg/mL . The calcitonin level was <10 pg/mL in 19 out of 21 cases where FNAC raised the possibility of MTC; however, the final histopathology ruled that option out. One false-positive calcitonin result (118 pg/mL with the RIA method) occurred in the case of a Hürthle-cell cancer and another case was noted in the case of an adenoid cystic cancer of a small salivary gland metastasizing to the thyroid (706 pg/mL with the IRMA method). The latter patient had chronic renal failure and was also hemodialyzed. Total thyroidectomy was performed in both cases. Detailed histopathological analysis did not detect either MTC or C-cell hyperplasia in these 2 cases. The serum calcitonin level became normal after thyroidectomy in both cases. It means that the extrathyroidal origin of the elevated calcitonin level can be precluded from the perspective.We encountered 5 patients during the first 10-year period, while the second period revealed only 1 further patient with the same problem.Patient 1. FNAC indicated colloid goiter.  The follow-up examination showed that the nodule had increased in size, and repeated FNAC pointed out the presence of MTC. Patient 2. FNAC resulted in a diagnosis of Hürthle-cell tumor. (The review of the smear indicated an error in interpretation.) The patient refused surgery. The nodule had increased in size after three years, and MTC was diagnosed cytologically. Patient 3. FNAC raised suspicion of malignancy. It might possibly be Hürthle-cell tumor or insular cancer as well.  The frozen section examination led us to the diagnosis of malignancy. The calcitonin level was high of 177 pg/mL after total thyroidectomy and histopathological diagnosis of MTC. Enlarged lymph nodes were detected in the medial compartment of the neck on the CT scan, and the patient was operated again. Patient 4. FNAC resulted in a diagnosis of Hashimoto's thyroiditis although the clinical presentation raised suspicion.  The patient underwent surgery. The frozen section diagnosis showed the presence of MTC. The surgical procedure was extended to the medial compartment of the neck. The final histopathology indicated MTC and Hashimoto's thyroiditis. Patient 5. FNAC indicated Hürthle-cell tumor. The final histopathology was Hürthle-cell carcinoma  and a 3 mm incidental focus of MTC. The postoperative serum calcitonin level was normal. The patient feels well and totally recovered 15 years after the surgical procedure. Patient 6. FNAC led us to the suspicion of malignancy.  The frozen section diagnosis indicated carcinoma with a high probability of MTC. The surgical procedure was extended to the medial compartment of the neck. The prevalence of MTC among our nodular goiter patients was 0.1% which is identical with published results without calcitonin screening  and lessDifferences in prevalence may be explained by the well-known goitrogenous effect of iodine-deficiency, which increases the number of benign nodules but not the malignant ones . The proThe question arises about the advantages and disadvantages that would have emerged if we had used calcitonin screening in all our cases or in all of the surgically treated nodular goiter patients.Two out of the 23 patients lost 3 years before the proper treatment was started. Nevertheless, the delay was not our responsibility in one of these cases as we advised surgery right after the false diagnosis of a Hürthle-cell tumor. Besides, there was another patient for whom reoperation could have been avoided. The question arises again: how many additional MTC cases would have been found if we had used calcitonin screening? If we extrapolate the 1 missed MTC that was larger than 5 mm in maximal diameter among the 4,601 operated patients compared to the 18,256 patients who were not treated surgically, this would have meant 4 more MTC cases. If we consider situations when we performed repeated FNAC in the presence of growing nodules and also the fact that no MTC cases occurred when the first and the second FNACs were negative, we can conclude that the appropriate number of clinically relevant missed MTCs were surely less than 4 cases. The situation differs concerning micro-MTCs. It seems to be obvious that we would have found more micro-MTC cases if we had performed calcitonin screening. Nevertheless, the clinical significance of occult MTC is not clear, therefore, we agree with other specialists that the occurrence of untreated occult MTC without morbidity or mortality should be considered in cost-effective models of routine serum calcitonin screening , 30. Our findings also proved that the calcitonin test became safer with time. Two false results  occurred with the RIA, while 1 false-positive emerged with the IRMA method. The latter occurred in a hemodialyzed patient with renal failure. Elevated calcitonin levels have been described in both cases in up to 25% of the former ones . AlthougIf we consider the advantages and the disadvantages, it seems possible that in our cases the disadvantages of calcitonin screenings may have exceeded the advantages of the method. It has been clearly demonstrated that the cost-efficiency of calcitonin screening is highly dependent on the prevalence of MTC and the specificity of FNAC. In a decision model for a hypothetical group of patients with a 0.78% prevalence of MTC, calcitonin screening seemed to be cost-effective . In the The aggressive clinical presentation was a typical but not specific sign of MTC; however, it led to the correct therapy in spite of the false-negative FNAC in the case of 1 patient.We have found one specific US sign for MTC which may be of practical relevance: the presence of irregular patchy hyperechogenic areas within hypoechogenic nodules—which were observed in 48% of our MTC cases—increased the possibility of MTC diagnosis more than 30 times. These foci are larger than 1 mm, and they are not so bright in contrast to microcalcifications. They also have an even patchy appearance, and the dorsal acoustic shadow is absent or narrower than the extent of the hyperechogenic focus. We agree with Gorman et al. that these foci are not simply coarse calcifications but correspond to deposits of calcium surrounded by amyloid .FNAC raised the possibility of a malignant disease in 21 cases out of the 23 (91% sensitivity). The average sensitivity for diagnosing MTC in our practice was 74%, though there was significant difference between our skills in the two time periods. The sensitivity was only 55% in the first 10-year period; however, it was 92% in the second 10-year period. As regards the opposite situation, we raised the possibility of MTC in 21 cases where the final histology was not MTC. The main problem in the cytological differential diagnostic was caused by Hürthle-cell tumors . On the Our results emphasize the role of the frozen sections in those patients where the FNAC resulted in the suspicion of malignancy which was not otherwise specified . The useAll our patients had sporadic form of MTC except for one person. The low proportion of familiar form may be explained by the fact that our MTC patients after surgery are treated in university centers, and the evaluation of relatives of familiar MTC patients is also done in these centers. To Summarize. The following protocol seems to be adequate in an iodine-deficient region with only a 0.1% prevalence of MTC: the performance of US-guided FNAC in all cases of hypoechogenic nodules, the testing of serum calcitonin only in cases where FNAC raised the possibility of MTC besides patients at risk of the familiar form of MTC, and also the performance of frozen sections in those cases where FNAC leads to the suspicion of malignancy which was not otherwise specified. It is even more important that thyroid FNAC should be carried out only by a cytologist with special interest and skills in this field for the evaluation of MTC. In evaluation teams where the experience of cytopathologists is limited, the widespread use of calcitonin testing demands consideration even if MTC is expected with a low prevalence rate.


Arabidopsis thaliana plants infected with ORMV. Here, we tested whether the time of removing the infected leaves as well as viral titer have any effect on the degree of changes in HRF in systemic tissues. An increase in HRF in systemic non-infected tissues was more pronounced when the infected leaves were detached from the infected plants at 60–96 h post-infection, rather than at earlier time. Next, we found that exposure to higher concentrations of inoculum was much more efficient in triggering an increase in HRF than exposure to lower concentrations. Finally, we showed that older plants exhibited a higher increase in HRF than younger plants. We found that an increase in genome instability in systemic tissues of locally infected plants depends on plant age, the concentration of initial inoculums and the time of viral replication.In the past, we showed that local infection of tobacco leaves with either tobacco mosaic virus or oilseed rape mosaic virus (ORMV) resulted in a systemic increase in the homologous recombination frequency (HRF). Later on, we showed that a similar phenomenon occurs in  R-genes) that allow plants to recognize pathogen avirulence genes  or oilseed rape mosaic virus (ORMV) results in an increase in the frequency of homologous recombination HRF; . We demoArabidopsis thaliana. Infection of Arabidopsis plants with TMV was documented before  and ORMV showed that ORMV was more efficient . There was a strong positive correlation between the amount of virus used to initiate the infection and an increase in HRF (r = 0.726). The differences in HRF between plants infected with 100 and 200 ng were not significant so (P > 0.1).A higher viral titer is typically associated with faster systemic spread of the virus; the o plants . We hypoSince the previous experiment showed that the highest concentration of primary inoculums resulted in the highest increase in HRF, we hypothesized that an increase in HRF is more or less proportional to the amount of viral RNA initially encountered by plants. It was not clear whether viral replication was a determinant of the process. We hypothesized that if the virus is allowed to propagate in infected leaves for a longer period of time, we might observe a stronger induction of HRF. Since it was not clear whether the presence of virus in the systemic leaves used for the analysis of HRF would influence the results, we had to determine the time of systemic spread of the virus from the initial site of infection.Figure 2). Therefore, we concluded that the virus spreads systemically between 72 and 96 hpi.To test the speed of virus spread to the systemic leaves, we infected several groups of plants with ORMV (100 ng of per plant) and cut leaves at 24, 48, 72, and 96 hpi. We analyzed the presence of viral RNA in the systemic leaves by performing the quantitative real-time PCR (qRT-PCR). We found the presence of viral RNA in the sample from the 96 hpi group but not in the samples from the 72, 48, or 24 hpi groups . Remarkably, HRF did not significantly increase in the 12 and 24 hpi groups as compared to the control one (P > 0.1). To confirm these data, we performed a similar experiment with a different transgenic line, line 11 (C24 cultivar) carrying in the genome the β-glucuronidase-based recombination substrate. For this line, we used only three time points: 24, 36, and 72 hpi . A statistically significant increase in HRF was observed at 36 hpi (P < 0.05) and 72 hpi (P < 0.01) but not at 24 hpi (P < 0.1) as compared to the control. A significant increase in the HRF was also observed in several buffer-treated groups, especially in those in which buffer-treated leaves were removed at 72–96 h post-treatment. This experiment has confirmed that an increase in HRF in Arabidopsis plants occurs only when the virus is allowed to propagate in infected leaves for more than 24 h and showed that wounding stress potentiates the HRF increase.Next, we analyzed HRF in systemic tissues of plants infected with 100 ng virus when the infected leaves were removed at 12, 24, 36, 48, 60, 72, and 96 hpi. We found that an increase in HRF was the highest in the 60, 72, and 96 hpi groups, with the results being statistically similar among these three groups (P < 0.01) increase between 24 and 48 hpi . Viral titer picked at 72 hpi and the analysis at 96 and 120 hpi showed a significant decrease (P < 0.01 for both) as compared to that at 72 hpi. We hypothesized that a decrease in the viral titer in the infected leaves observed at 96 hpi could be due to systemic spread of the virus. Indeed, the analysis of ORMV RNA in non-infected tissues showed a substantial accumulation of virus at 96 hpi which increased at 120 hpi .Since no statistically significant increase was observed in the12 and 24 hpi groups, we hypothesized that an increase in HRF in systemic tissues requires certain viral replication in infected tissues. The analysis of viral concentration in the infected leaves showed a significant . Initially, we thought that the presence of the virus in the tissues would lead to a further increase in recombination, but as it has been shown above, it is not the case. It is possible that the system of recombination response saturates after plants are exposed to a certain amount of inoculums. Indeed, the influence of infection with 200 ng of virus was not different compared to 100 ng. Similarly, the 72 hpi group did not substantially differ from either the 60 hpi group or the 96 hpi group for that matter.It was surprising that an increase in HRF was not much greater in the 96 hpi group as compared to the 72 hpi group . The analysis of viral titers in the removed infected leaves (48 hpi) showed the highest accumulation of virus in the oldest leaves . This suggests that viral infection progresses faster in older leaves allowing for higher concentrations of virus. This, in turn, results in a higher increase in HRF in older plants.We next analyzed whether the HRF increase would also depend on whether plants were infected at younger or older age. We infected 2-, 3- and 4-week-old plants with 100 ng of virus and removed the infected leaves at 48 hpi. While analyzing HRF in systemic tissues 1 week post-infection, we found that an increase in HRF was the highest in the oldest plants . The levels of strand breaks also increased in the buffer control groups, presumably as a result of a response to mechanical wounding when plants were treated with buffer and carborundum. The level of strand breaks was always higher in the virus-treated groups as compared to the buffer-treated ones . When infected leaves were removed from plants at 48 hpi, a further accumulation of strand breaks was observed in non-infected leaves tested at 96 hpi, and the level of strand breaks was much higher than that in non-infected leaves at 48 hpi (data not shown). This suggests that there is an active process running in non-infected leaves that result in a further increase in the level of strand breaks.What could be the reason for an increase in HRF in unexposed tissues? It is possible that viral infection results in an increase in the number of strand breaks, perhaps through local changes in chromatin structure or/and changes in the expression of repair genes involved in homologous recombination. To test whether the levels of strand breaks change in infected and non-infected tissues, we analyzed the presence of strand breaks using the random oligonucleotide-primed synthesis (ROPS) assay. We found that the levels of strand breaks increased in infected and non-infected leaves at 48 and 96 hpi, with changes in the 96 hpi group being more dramatic  have a stronger effect on an increase in HRF.The paper by atic HRF . Similaratic HRF . SA is astrategy . Furtherstrategy . It can Arabidopsis results in transgenerational changes as it was observed in tobacco plants  that carries in the genome a single copy of the β-glucuronidase (GUS)-based recombination substrate (in vitro (GUS) or in vivo (LUC).For the experiments, we used two transgenic ubstrate  and lineubstrate . RecombiArabidopsis thaliana plants were germinated and grown in soil under 16 h day/8 h night at 22°C. Two leaves of A. thaliana plants were inoculated with different amounts of ORMV at the age of 3 weeks as previously described . The samples were purified using equal volumes (400 μl) of chloroform and 1-butanol. Viral particles were precipitated using separate volumes of 50 μl of 40% polyethyleneglycol 6000 and a 10% NaCl solution. The pellet was resuspended in a 1:50 dilution of phosphate buffer that lacked β-mercaptoethanol, and viral concentrations were quantified. The viral titer was estimated by measuring OD260 using a spectrophotometer. The titer was expressed in microgram of ORMV per 10 mg fresh weight. Each measurement was done in triplicates.Viral RNA was extracted, and virus concentration was analyzed as previously describe . In briet-test (two-tailed paired or non-paired) or a Single factor ANOVA. Statistical analyses were performed using the MS Excel software and Microcal Origin 6.0.In all cases, the average and standard errors were calculated. The statistical significance of the experiments was confirmed by performing either a Students The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Whilst regular exercise is advocated for people with type 1 diabetes, the benefits of this therapy are poorly delineated. Our objective was to review the evidence for a glycaemic benefit of exercise in type 1 diabetes.Electronic database searches were carried out in MEDLINE, Embase, Cochrane’s Controlled Trials Register and SPORTDiscus. In addition, we searched for as yet unpublished but completed trials. Glycaemic benefit was defined as an improvement in glycosylated haemoglobin (HbA1c). Both randomised and non-randomised controlled trials were included.Thirteen studies were identified in the systematic review. Meta-analysis of twelve of these (including 452 patients) demonstrated an HbA1c reduction but this was not statistically significant (standardised mean difference (SMD) −0.25; 95% CI, −0.59 to 0.09).This meta-analysis does not reveal evidence for a glycaemic benefit of exercise as measured by HbA1c. Reasons for this finding could include increased calorie intake, insulin dose reductions around the time of exercise or lack of power. We also suggest that HbA1c may not be a sensitive indicator of glycaemic control, and that improvement in glycaemic variability may not be reflected in this measure. Exercise does however have other proven benefits in type 1 diabetes, and remains an important part of its management. The current UK guidelines for exercise and physical activity are that all individuals undertake at least 150 minutes of moderate-intensity, or 75 minutes of high-intensity, aerobic physical activity per week. The currently recognised health benefits of exercise in type 1 diabetes have been reviewed previously. Here, we perform a systematic review and meta-analysis of randomised controlled and non-randomised parallel group trials of exercise training and glycaemic benefit in type 1 diabetes. We use glycosylated haemoglobin (HbA1c) as the measure of glycaemic control.EBSCO) up until August 2011. An updated search of Medline was performed up until May 2012. A variety of search terms were used for individuals with type 1 diabetes (population) and physical activity (intervention). We did not restrict our search to the outcome or study design to maximise the sensitivity of our search. Nor did we place any date or language restrictions. Our secondary search strategy included scanning bibliographies of the retrieved articles and searching for unpublished studies using key trial registries . The primary search strategy is outlined in the The electronic databases searched were MEDLINE (Ovid), Embase (Ovid), Cochrane library (Wiley) and SPORTDiscus  were only included if they were given to both intervention and control arms. We included both randomised and non randomised parallel controlled trials.The primary outcome measure was change in HbA1c, with HbA1c data extracted before and after the intervention. In those studies where complete HbA1c data was not reported, we contacted the authors for clarification. Our secondary outcome measure was adverse events (hypoglycaemia).Initial selection was based on title and abstract. The original article was obtained where it was unclear whether the study met our inclusion criteria. Two reviewers (AK and MC) selected the studies independently. Where there was disagreement between the two reviewers, resolution was through a meeting with PN and KN. Reasons for exclusion were documented and are available from the authors on request. Where necessary, foreign language papers were translated.A data extraction and quality assessment form was developed based on the template suggested by Centre for Review and Dissemination (CRD) for systematic reviews in their guidelines Each of the trials measured the outcome (HbA1c) both before and after the intervention period, and for both control and intervention arms. Trials did not report paired difference summary statistics and so we could not obtain paired difference measures. Rather, it was necessary to extract information on mean, standard deviations, and number of participants both before and after the intervention period and for both intervention and control arms for each trial. For trials which reported the standard error of the mean (SEM), we converted this to a standard deviation (SD) by multiplying by the square root of the number of participants in that arm. Any studies that only reported median values without any measure of variation (such as inter-quartile range) could not be included in the analysis.For any trials which were multiple intervention arm trials we extracted and summarised the trial information for each arm. We then combined the intervention arms into a single combined arm by estimating the weighted mean values (both before and after) and estimating the weighted SD using the usual pooled standard SD From the estimated mean and SD values for HbA1c both before and after the intervention period, we then estimated the mean difference (after-before) and SD in both the control and intervention arms. It was necessary to estimate the SD for these change effects as no estimates of correlation (or SDs of changes) were reported in the original trial reports. We used a conservative estimate for the correlation (0.5) as advocated All trials reported on the outcome HbA1c. However, because HbA1c-DCCT standardisation only occurred in 1997, we summarised treatment effects using the standardised mean difference (SMD) of Hedges adjusted g Clinical, methodological and statistical heterogeneity were explored. Clinical heterogeneity was explored by considering the clinical diversity of the study participants in terms of their age and study duration. Methodological heterogeneity was explored by exploring variations in randomised and non randomised studies. Statistical heterogeneity was tested using Cochran’s Q, a statistic based on the chi-square test and quantified by the I-square test. Results were then pooled across studies using a random effects meta-analysis  using the generic inverse variance method. We pooled over all studies within the subgroups of child and adult studies. We further investigated the effect of age and duration of intervention by including the pooled (over control and intervention arms) weighted mean age and duration as covariates in a meta-regression model. Residual levels of heterogeneity were investigated by comparing I-squared values before and after adjustment for covariates (age and duration).Risk of small study bias (or publication bias) was explored using the contour enhanced funnel plot, stratifying by age of study population (adult or child) and was further evaluated by the Egger test.The database searches identified a total of 3,740 potentially relevant papers . One furOf the thirteen included studies, eight were randomised controlled trials. Randomisation procedures were not reported in any of these studies. In one study the reported randomisation method  resulted in it being classified as non-randomised parallel group trial for the purposes of this review. The study by D’Hooge et al The remaining 12 studies (452 patients) were included in the meta-analysis. The overall, pooled data showed a non-significant SMD reduction in HbA1c of −0.25% with exercise training  and for non-randomised studies −0.25 (95% CI −0.59 to 0.09). The degree of heterogeneity observed in the randomised studies was I-squared = 66.4% whereas this was lower in the non-randomised studies (I-squared = 33.4%).Meta regression of HbA1c against length of study duration suggested a non-significant association between HbA1c reduction and longer intervention , which may have contributed to the heterogeneity of study results. However, ADA guidance on exercise for people with type 1 diabetes is limited only to duration and intensity There are a number of potential explanations for the findings of this meta-analysis. Firstly, the programmes of exercise may not have been of sufficient duration. This is supported by our sub-analysis which shows a trend for HbA1c reduction with longer duration of intervention. Based on pooled data from these studies, and assuming that the rate of glycaemic benefit persists in a linear fashion, we estimate that studies of greater than 25 weeks duration would be needed to obtain an HbA1c reduction in the region of 0.5%. This has implications for the design of future trials. Secondly, as has been demonstrated in some studies of type 2 diabetes, HbA1c has been used as a measure of glycaemic control in our analysis but this may not be the most appropriate measure of glycaemic control. To illustrate, glycaemic variability has been suggested to contribute to the development of microvascular complications in type 2 diabetes. In contrast to our results, a recent meta-analysis by Tonoli et al Overall there is a lack of large well-conducted studies on glycaemic benefits of exercise in type 1 diabetes. Our systematic review identified thirteen studies, from which data on 452 patients has been used for analysis. In contrast, a recent meta-analysis of exercise intervention in type 2 diabetes (which did detect an HbA1c lowering effect) analysed 47 RCTs including more than 8500 patients. designing larger trials lasting at least six monthstrial design that is randomised and well controlled (with matched type 1 diabetic subjects and recording of dietary intake)examining the effect of exercise on glycaemic variabilityexamining the effect of exercise intensity, and the incorporation of a dietary programme on glycaemic benefitexamining the effect of age and duration of diabetes on glycaemic benefitWhilst this meta-analysis did not detect a glycaemic benefit to exercise, there are other well defined benefits in type 1 diabetes. These include reduction in macrovascular risk, mortality, and improvement in wellbeing. Figure S1Funnel plot testing for publication bias.(TIF)Click here for additional data file.Appendix S1Search Strategy.(DOCX)Click here for additional data file.


Studies investigating the outcome of conservative scoliosis treatment differ widely with respect to the inclusion criteria used. This study has been performed to investigate the possibility to find useful inclusion criteria for future prospective studies on physiotherapy (PT).A PubMed search for outcome papers on PT was performed in order to detect study designs and inclusion criteria used.Real outcome papers  have not been found. Some papers investigated mid-term effects of exercises, most were retrospective, few prospective and many included patient samples with questionable treatment indications.There is no outcome paper on PT in scoliosis with a patient sample at risk for being progressive in adults or in adolescents followed from premenarchial status until skeletal maturity. However, papers on bracing are more frequently found and bracing can be regarded as evidence-based in the conservative management and rehabilitation of idiopathic scoliosis in adolescents. Scoliosis is a three dimensional deformity of the spine and trunk, which may deteriorate quickly during periods of rapid growth -3. AlthoWhile children grow until they have fully matured, there are certain times with more or less growth during childhood and adolescence and curvature progression is more or less probable during different phases of growth ,2 Figur. The ‚baIn patients with idiopathic scoliosis during adolescence, the risk for being progressive can be calculated using the formula by Lonstein and Carlson . Based oWhile premenarchial girls at average have Risser 0, the Risser sign arises after the onset of menarche/voice change (in boys). A 14 year old girl usually has Risser 3, sometimes 4, a 15 year old girl usually has 4, sometimes 5.A 10 year old girl with 20° and the first signs of maturation before the onset of menarche is usually Risser 0. Therefore, the progression factor is 2, indicating a risk for being progressive of 90%.A 15 year old girl with 20° usually is 2.6 years postmenarchial with Risser 4. Therefore, the progression factor in this case is 0.53, indicating there is no more risk for being progressive and that there is no more indication for further treatment.Physiotherapy, corrective bracing and spinal fusion surgery are the treatment modules currently applied in the treatment of scoliosis . While tFew prospective controlled studies (Level II) on bracing started in immature patient samples and ended after cessation of growth -9 the stPurpose of this Pub Med review was to analyze the data provided by Pub Med on physiotherapy in patients with scoliosis as well as the materials already presented in systematic reviews ,12 focusA PubMed (and hand search of papers cited in previous reviews) for outcome papers on PT was performed in order to detect study designs and inclusion criteria used for studies on physiotherapy. Retrospective controlled studies (Level III), prospective controlled studies (Level II) and randomized controlled studies (Level I) with untreated controls having scoliosis were taken into account, but also other study designs were recorded.The search  was performed for manuscripts using the mesh terms "scoliosis AND physiotherapy/exercises/exercise. The inclusion criteria were as follows:- Patients: Diagnosis of AIS (Adolescent Idiopathic Scoliosis) in adolescence and adulthood, confirmed through X-rays; we focused on patients in growing age;- Experimental intervention: patients treated exclusively with physiotherapy, without any other associated intervention;- Control group: any kind of scoliosis patients with observation only;- Outcome measures: only Cobb degrees: results could be reported in absolute terms or as percentage of patients improved/worsened;- Study design: any study design to be able to find hidden controlled trials.Previously published reviews  were scrutinized. Targeting at a proper treatment indication the risk for being progressive of the average patient from the studies was calculated according to the formula published by Lonstein and Carlson , wheneveIn case the Risser stage of the average patient was not available, an estimation of the Risser was performed according to the average age of the group using the data available in literature -15. As tFor adult patients with scoliosis, the focus was laid upon controlled studies with an untreated control group with a follow-up of > 5 years as the slow progression in adulthood will usually not exceed the margins of the technical error of 5° usually calculated for Cobb angle measurements .193 papers displayed when the terms ‚Idiopathic Scoliosis AND physiotherapy' were entered. 167 papers displayed when the terms ‚Idiopathic Scoliosis AND exercises' were entered and 139 papers displayed when the terms ‚Idiopathic Scoliosis AND exercise' were entered. In the majority of the papers physiotherapy was not the main or only treatment. Many papers were found on bracing, other papers did not account for the Cobb angle as an outcome parameter.Hand search was performed for studies mentioned in papers, especially on the review papers found ,12.Real outcome papers (start of treatment in immature samples/end results after the end of growth) have not been found. Some papers investigated mid-term effects of exercises, most were retrospective, few prospective and many included patient samples with questionable treatment indications.Most of the studies had patient samples not meeting the treatment indications as proposed within the guidelines  compared two different unproven concepts against each other .There was no paper with an adult sample of patients treated by physiotherapy compared to untreated controls.One study , questioTwo other short-term controlled studies have been published, stating that surgical treatment was superior to conservative treatment ,19 and cOther studies published recently did not study the change of Cobb angle -22.No paper was found concerning patients at risk for being progressive, followed to skeletal maturity under physiotherapy treatment alone. Claims made to regard physiotherapy as an evidence based method of treatment, are therefore, scientifically unjustified ,12 Roman Roman12 The only evidence on Level II is found in the immature sample (n = 94) from the prospective controlled study from our group . HoweverAnother paper comparing two unproven groups of physiotherapy  appears to be more effective [Nevertheless, the authors claim in their conclusion: ffective .This study included seventy-four consecutive outpatients with adolescent idiopathic scoliosis, mean 15 degrees (standard deviation 6) Cobb angle, 12.4 (standard deviation 2.2) years old, at risk of bracing who had not been treated previously .Italian girls with an average age of 12.4 years surely can be estimated as being postmenarchial with a Risser 1 at least. When calculating the average patient from this sample using the Lonstein and Carlson formula  with a cThis study is only one example of documented malpractice and unfortunately literature is full of samples not needing any treatment, but claims have been made from these studies that physiotherapy would be of benefit.23.4-3 × Risser 2 /13.2 = 1.32, which makes less than 40% chance for progression with a clear indication for observation, only) As these girls surely are in the descendent phase, the prognosis is getting better every day and as a curve of < 30° is very unlikely to progress after cessation of growth, there is surely no urgent indication for treatment and no real risk for the need of brace treatment).The authors have also published a retrospective study  includinThe Chinese RCT  has a paThe problem of treating mature patients and claiming beneficial outcomes is also evident in bracing  Figure .As can be seen in Table The studies by Weiss 2003, Mollon and Rodot 1986 and Ducongé 2002 ,29,34 wiNo controlled paper with adult patients at risk for progression (curvatures exceeding 35°)  has beenAccording to the findings from this review, studies on physiotherapy in idiopathic scoliosis patients have the following shortcomings:wrong treatment indications- lack of risk for progression- lack of comparability- lack of homogeneity- Prospective study designs should not be overestimated when the material within the study can be inappropriate . In studIt is also important to see the current evidence for physiotherapy during growth within the context of the other module of conservative treatment such as bracing. The only paper presenting at least some evidence for physiotherapy is the prospective controlled paper from my previous working group , howeverRecent papers present samples not followed up by x-ray investigations and therefore could not be compared to other studies from this review -22. HoweThus overtreatment seems to be an important issue in the studies on conservative management of scoliosis.There are a few studies on conservative treatment of adult scoliosis patients published recently -19, but A similar paper, published in 1995  was alsoStudy design may be prospective controlled, however as the problems (complications) of spinal surgery arise after many years, mostly with a lifetime risk of 40-50% ,46, a foAn agreement of the scientific community on common inclusion criteria for future studies on PT seems necessary. We suggest the following: (1) girls only, (2) age 10 to 13 at the first signs of maturation (Tanner II), (3) Risser 0-2, (4) risk for progression 40-60% according to Lonstein and Carlson.It would be even better to only include patients from the ascendant phase of the pubertal growth spurt: (1) girls only, (2) age 10 to 12 at the first signs of maturation (Tanner II), (3) Risser 0, (4) risk for progression 40-60% according to Lonstein and Carlson.However, the problem would be that the number of the patients included would be very limited as has been shown in a prospective controlled paper on bracing using this kind of inclusion criteria . UsuallyThe postural correction plays a major role in physiotherapy like in bracing -9,47 treThere is still no evidence that physiotherapy exercises can decrease the progression of scoliosis in immature samples with idiopathic scoliosis (with significant Cobb angles > 15 degrees) and thus the correction by braces is emphasized. However, physiotherapy exercises should be regarded as a complement to bracing concerning postural control during activities of daily living (ADL) . PosturaPT may have a beneficial effect on the patient with idiopathic scoliosis as this has been demonstrated in many pre-/post cohort studies , however- Most of the studies included patients not yet or no more at risk for being progressive.- Additionally, the papers on adults with scoliosis  have a follow-up period too short to draw any conclusions as complications of surgery in most of the cases appear more than 5 years after surgery.- There was no outcome paper on PT in patients with idiopathic scoliosis at risk for being progressive followed from the premenarchial status until skeletal maturity. Therefore, only bracing can be regarded as being evidence based in the management of scoliosis patients during growth.- There is little evidence that PT might have beneficial effects on spinal curvatures. Specific exercises like Side Shift or Schroth could be favoured.- Future studies on physiotherapy in idiopathic scoliosis should only be accepted when they follow the inclusion criteria as presented within this paper.The author is advisor of Koob-Scolitech, Abtweiler, Germany.


Plasmodium falciparum, the parasite that causes the most serious form of malaria, antifolate drugs inhibit the function of essential enzymes in the folate pathway. However, a handful of mutations in the gene coding for one such enzyme, dihydrofolate reductase, confer drug resistance. Understanding how evolution proceeds from drug susceptibility to drug resistance is critical if new antifolate treatments are to have sustained usefulness.Patterns of emerging drug resistance reflect the underlying adaptive landscapes for specific drugs. In We use a transgenic yeast expression system to build on previous studies that described the adaptive landscape for the antifolate drug pyrimethamine, and we describe the most likely evolutionary trajectories for the evolution of drug resistance to the antifolate chlorcycloguanil. We find that the adaptive landscape for chlorcycloguanil is multi-peaked, not all highly resistant alleles are equally accessible by evolution, and there are both commonalities and differences in adaptive landscapes for chlorcycloguanil and pyrimethamine.Our findings suggest that cross-resistance between drugs targeting the same enzyme reflect the fitness landscapes associated with each particular drug and the position of the genotype on both landscapes. The possible public health implications of these findings are discussed. Plasmodium falciparum, the causative agent of the most virulent form of malaria, has demonstrated an astounding ability to rapidly evolve resistance to many different classes of drugs. Drug resistance in malaria is thus a zealously investigated topic, covering everything from developing methods to detect emerging drug resistance to designing drugs that may prolong usefulness in the face of rapidly evolving resistance. Discovering the adaptive landscape of drug resistance provides another way to gain an understanding of how antimalarial drug resistance evolves and how the course of evolution is shaped by molecular forces.One class of antimalarials, the antifolates, works by competitively inhibiting enzymatic action in the folate pathway. Within the folate pathway the enzymes dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) are most often targeted for inhibition. DHFR performs the essential task of reducing dihydrofolate to tetrahydrofolate pfdhfr, the P. falciparum gene coding for dihydrofolate reductase (DHFR) that may be compounded by concurrent resistance in DHPS , 51 , 59 , 108 , and 164  are known to greatly increase antifolate resistance in vitro studies show that a triple mutant N51I/C59R/S108N increases resistance to pyrimethamine 80 fold and resistance to chlorcycloguanil 40 fold P. falciparum strains carrying these same mutations (N51I/C59R/S108N) are associated with high rates of treatment failure using sulfadoxine-pyrimethamine but remain treatable by chlorproguanil-dapsone Moreover, the laboratory findings that show genotypes containing one or more of these mutations to confer antifolate resistance correlate with the molecular markers associated with resistance to one or multiple drugs in the field. Thus, pfdhfr in yeast pfdhfrpfdhfr have been described between the antifolates pyrimethamine and WR99210 Although much is known about the genes and mutations leading to chlorcycloguanil resistance, genetics in malaria parasites remains technically challenging thus limiting the scope of research possible in live parasites. The development of heterologous expression systems for More recent studies have sought to understand the evolutionary trajectories that may lead to antifolate drug resistance in this system, focusing on resistance to pyrimethamine. The evolution of pyrimethamine resistance has been modeled at the molecular level in yeast pfdhfr gene to chlorcycloguanil in a yeast system in order to explore the evolutionary landscape underlying chlorcycloguanil resistance. We assayed all possible combinations of 6 mutations at 5 amino acid residues  and explored the evolutionary pathways accessible from the drug-susceptible wildtype allele to strongly resistance conferring mutant alleles. We also consider the implications of chlorcycloguanil resistance in the light of known patterns of pyrimethamine resistance caused by mutations in the same codons.The aim of the current study is to use the relative resistance of mutant alleles of the pfdhfr as expressed in a yeast system Of the 48 alleles constructed, 29 were viable and able to grow in the absence of metabolic supplement . PreviouThere is no discernable relation between the number of mutations in an allele and its viability .. The A1The mutation A16V, which exhibits a non-viable phenotype when present on a wildtype background, is associated with drug resistance on a background containing S108T, and in this background A16V increases chlorcycloguanil resistance 10,000 fold.In order to rank the four most highly resistant alleles for purposes of the simulations, we estimated the IC25 and IC2 values using the non-linear regression values obtained from the same analysis used for estimating the IC50s (see Table S3 in The 83 evolutionary trajectories that were accessible during the simulations are summarized in Table S5 in The single most often accessed trajectory (00000-00010-00110-00111) is observed on all 750 landscapes, and accounts for 20% of all realized trajectories. The most frequently followed trajectory to the double mutant 10020 (A16V/S108T) is 00000-00020-10020, which is followed in about 6% of simulations. The most frequently followed trajectory to the quintuple mutant 11121 (A16V/N51I/C59R/S108T/I164L) is 00001-00021-01021-01121-11121 and accounts for approximately 2% of realizations.There is a long-standing and continuing interest in resistance to antifolates and the level of cross-resistance observed between different antifolates Common to pathways of pyrimethamine and chlorcycloguanil resistance are the triple mutant 00111 (C59R/S108N/I164L) and the quadruple mutant 01111 (N51I/C59R/S108N/I164L), both of which increase resistance significantly in the presence of either compound. In contrast, the beneficial effect of the double mutant 10020 (A16V/S108T) differs greatly in magnitude between pyrimethamine (two fold increase) and chlorcycloguanil . Evolutionary analysis showed that this double mutant constitutes a fitness peak on the chlorcycloguanil landscape, and this submaximal fitness may be reached in only two mutational steps. In contrast, 10020 is an allele on the pyrimethamine landscape that is so rarely traversed that it does not show up in simulations. This example serves to illustrate the importance of understanding not just the genetic basis of resistance, but also the fitness landscape in the presence of a particular selective agent, in this case the specific antifolate drug.in vitro resistance assays and in silico landscape simulation certainly exists.Looking at practical applications of doing such simulations for more than one drug, one could imagine using genotypic information of the alleles currently present in parasites infecting a patient  and establishing if whether those alleles are fitness peaks on second line drugs that are being considered. In other words, in a scenario where chlorcycloguanil has been the major selective force and the presence of the 10020 allele has led to strong drug resistance, pyrimethamine would still be effective. However, if chlorcycloguanil resistance is conferred by amino acid substitutions of the 00111 type, the occurrence of a single mutation to 01111 would render both drugs ineffective. Of course, further systematic corroboration with clinical data is necessary before this scenario can safely determine care for human patients, but the possibility for the eventual fruitful application of Interestingly, all four of the alleles with the highest IC50s include the I164L mutation. The addition of I164L on certain backgrounds is associated with as much as a 1000-fold increase in resistance to antifolates, including chlorcycloguanil Our findings further demonstrate that, based on the chlorcycloguanil landscape in yeast, I164L is not necessarily a late arising mutation. This likely reflects a fundamental difference between the adaptive landscapes for pyrimethamine and chlorcycloguanil and underscores the importance of drug specific patterns of evolution. Simultaneously we recognize that I164L may not be as readily found as a single mutation in nature due to some additional fitness deficit that is not related to selection by pyrimethamine use but which we cannot determine in this context. It has already been observed that early mutations may entail some loss of fitness under a regime where no drug pressure is present but fitness is later restored by compensatory mutations The second most often accessed endpoint is the quintuple mutant 11121 (A16V/N51I/C59R/S108T/I164L), an allele not generally observed in clinical settings. Conceivably this may simply be due to a smaller sample size for alleles associated with chlorcycloguanil resistance. Chlorcycloguanil is not and has not been used as widely as pyrimethamine, and there is less data available on resistance mutations in the field apart from clinical trials. However, there are also intrinsic explanations for this observation. As two more mutations are required to reach this allele, it may be expected to occur more infrequently than a triple mutant given that mutations at all positions are equally likely. Alternatively, the relative dearth of pathway realizations leading to the quintuple mutant 11121 may be a reflection of the negative interaction between the mutations found in this allele. This allele contains the mutation S108T, which is much more rarely observed than S108N and usually occurs in combination with A16V The order in which mutations occur may thus play a critical role in determining whether a particular path leading to 11121 is accessible by evolution, or whether a fitness valley makes this trajectory impassable. Accessing a path of increasing resistance to the triple mutant 00111 (C59R/S108N/I164L) does not have the same problem, as this allele contains mutations that ”mix” much better. Due to the effect of such epistatic interactions there may be a more limited number of viable alleles available as options when traversing the landscape from 00000 to 11121.10101-10121-11121). Unlike the alleles on the more frequently traversed pathways to the fitness peak at 00111 (C59R/S108N/I164L), the allele 10101 is not one generally found in clinical settings. The trajectory analysis thus shows that the absence of this allele in nature is likely due to the unfavorable location of this point in sequence space, having both a low probability of realization and not occurring as the end point to a pathway, rather than due to higher susceptibility to drug treatment.Interestingly, 10121 (A16V/C59R/S108T/I164L) and 10101 (A16V/C59R/I164L) exhibit high IC50s, indistinguishable from those of the triple mutant 00111 (C59R/S108N/I164L) and the quintuple mutant 11121 (A16V/N51I/C59R/S108T/I164L), but these alleles are not accessed as endpoints in the simulations. In the case of 10121 this is probably due to the adjacency in sequence space of the quintuple mutant 11121, which was assigned a higher rank based on our estimations. However, in all our simulations, 10101 (A16V/C59R/I164L) appears only once, and then as an intermediate step on a pathway leading to 11121  increased resistance to the drugs 225-fold and 48-fold, respectively in vitro the presence of this triple DHFR mutant at codons 108, 51, and 59 (combined with a double DHPS mutant) increased drug resistance to both antifolates, this combination of mutations was only associated with clinical treatment failure with sulfadoxine-pyrimethamine but not with chlorproguanil-dapsone The results of the evolutionary trajectory analysis further complement studies showing incomplete cross-resistance between chlorcycloguanil and pyrimethamine. In an P. berghei in mice showed that there is some cross-resistance between parasites exposed to chlorcycloguanil and pyrimethamine, with exposure to chlorcycloguanil inducing broader resistance than pyrimethamine In 1968 a study of the parasite in vitro system in a relatively short period of time as compared to tracking trends in the field. Scientists and clinicians may then have advance notice of the mutational progression in trajectories that are likely to lead to the exhibition of high drug resistance allowing them to use their limited resources in a more focused way as they survey for the emergence of drug resistance. Transgenic systems such as the one used in this study cannot address all the complexities involved in the evolution of drug resistance in clinical settings. An adaptive landscape established in a yeast system may thus not directly apply to natural populations of P. falciparum. Rather it provides a tractable way of exploring the processes governing patterns and constraint of drug evolution in nature and serves to inform further studies in the field.Knowledge regarding the progression of evolutionary trajectories can be determined in an dhfr knockout strains may be rescued by replacing the endogenous yeast gene with the P. falciparum gene. This creates a yeast strain that is sensitive to antimalarial drugs targeting DHFR and thus allows the study of the impact of mutations on drug sensitivity in a tractable manner. Genetic manipulation and drug resistance assays in P. falciparum remains technically challenging and thus in vitro experimentation can be very valuable if we keep in mind that this heterologous system may not in all cases accurately reflect the in vivo system. The construction of the Saccharomyces cerevisiae TH5  strains containing mutant Plasmodium falciparum dhfr (pfdhfr) alleles is described in detail elsewhere dhfr gene of P. falciparum were constructed on the same genetic background using site-directed mutagenesis . Mutations were confirmed by sequencing. We transformed the yeast with each of the 48 alleles on a shuttle vector using the EZ Yeast Transformation Kit . The presence of the GR7 vector was selected for by growing transformed colonies on tryptophan dropout media (SC trp−). The GR7 shuttle vector and the TH5 strain of S. cerevisiae were generously provided by Carol Sibley and were specifically engineered for use in in vitro assays to study antifolate resistance in PlasmodiumYeast −8 M, 10−7 M, 10−6 M, 10−5 M, and 10−4 M each. Chlorcycloguanil was diluted in ethanol to a final concentration of 1% ethanol. Chlorcycloguanil was generously provided by Jacobus Pharmaceuticals . Growth rate and resistance experiments were conducted on a Bioscreen C microbiological workstation (Thermo Labsystems). Growth rates were measured by spectrophotometry at 600 nm at 30°C in 15 minute increments over a period of 72 hr.Growth rates and drug resistance assays were conducted as described previously The half-maximal inhibitory concentration (IC50) for each strain was determined using non-linear regression analysis in the open-source statistical environment R Our evolutionary model assumes that the time between mutations arising is much longer than the time to loss or fixation of new mutations, which allows the fixation of at most one mutation during each interval of time. At each interval, all mutational neighbors that will result in an increase in fitness are considered, including reversion to a previously fixed mutation in vitro system where some intricacies of in vivo systems may not be discernable but strong and important effects may still be observed in well planned experiments.We simulated landscapes and trajectories in this manner as it allows the probability estimates to account for uncertainty in our resistance measurements. Many trajectories thus occur on landscapes that have low-probabilities, i.e. the landscape itself has a low likelihood of realization. We therefore only report a consensus of trajectories that occur on at least 85% of landscapes. Doing so allows us to identify the strongest trends which is appropriate as in an 5 independent computer simulations yielding a complete mutation-selection trajectory were carried out.Using the IC50 values for the different mutant alleles, we ran computer simulations to determine the evolutionary trajectories most often accessed. Each initial condition consisted of a fitness landscape constructed by choosing a set of IC50 values, one for each genotype, by sampling one value at random from a normal distribution with the same mean and variance of IC50 as estimated for each genotype. For each of 750 such fitness landscape so constructed, 10File S1This chart shows the growth rates of all viable alleles in the absence of drug. The growth rates were determined by measuring cell density at OD600.(DOCX)Click here for additional data file.


Advances in technology allowed the development of a novel smoking cessation program delivered by video messages sent to mobile phones. This social cognitive theory-based intervention  used observational learning via short video diary messages from role models going through the quitting process to teach behavioral change techniques.  The objective of our study was to assess the effectiveness of a multimedia mobile phone intervention for smoking cessation. A randomized controlled trial was conducted with 6-month follow-up. Participants had to be 16 years of age or over, be current daily smokers, be ready to quit, and have a video message-capable phone. Recruitment targeted younger adults predominantly through radio and online advertising. Registration and data collection were completed online, prompted by text messages. The intervention group received an automated package of video and text messages over 6 months that was tailored to self-selected quit date, role model, and timing of messages. Extra messages were available on demand to beat cravings and address lapses. The control group also set a quit date and received a general health video message sent to their phone every 2 weeks.P = .8). Feedback from participants indicated that the support provided by the video role models was important and appreciated.The target sample size was not achieved due to difficulty recruiting young adult quitters. Of the 226 randomized participants, 47% (107/226) were female and 24% (54/226) were Maori . Their mean age was 27 years (SD 8.7), and there was a high level of nicotine addiction. Continuous abstinence at 6 months was 26.4% (29/110) in the intervention group and 27.6% (32/116) in the control group  While smoking prevalence has been declining in many countries ,2 high pSmoking quit rates are low even where intensive behavioral and pharmacological support is available , althougOur successful text messaging smoking cessation program  was receA randomized controlled trial was undertaken between November 2007 and August 2009 to determine whether a video-based smoking cessation intervention delivered via mobile phone was effective at increasing smoking cessation rates compared with a control group over a 6-month period. In this paper we describe this trial, and reflect on challenges faced in recruitment that undermined its capacity to adequately test the effectiveness of the intervention.Recruitment was targeted at young adults (16-25 years) and particularly toward young Maori. The study was advertised extensively via radio, internet, mobile phone (to those who had signed up for such a service), paper-based and online magazines, Maori-specific media of all types, local and national newspapers, and media releases to national media outlets. Advertisements were placed in tertiary education institutions , primary health care services, smoking cessation services, large employer health promotion programs, and posters or leaflets at cafes, bars, and sports grounds.Participants were eligible if they were at least 16 years of age, smoked daily, and wanted to quit. Participants were required to have a mobile phone that was capable of receiving video messages. The video messages were sent as a text message with a universal resource locator (URL) address in the text. Participants highlighted the URL to trigger automatic downloading and playing of the video on the phone  and reply to a consent text message with the words “I consent.” Consenting participants were directed to complete baseline data collection on an online form. On submission of this information, computer randomization allocated participants to an intervention or control group, using stratified minimization for age , ethnicity , and level of nicotine dependence .Both groups nominated a quit day (QD) on which they aimed to stop smoking that was between 1 and 3 weeks from randomization. Participants also nominated two time periods (in a 24-hour clock) during which they wished to receive the mobile phone messages.Those in the intervention group were also directed to an online brief description and photograph of the six role models (three of whom were Maori) and asked to select one person from whom they would receive messages . The steps in the development of the intervention have been described in detail elsewhere . In brieThe intervention was arranged into a chronological schedule of mobile phone messages that included the role model videos, text messages , and other video messages . Additional features included a website for intervention group participants that allowed them to review video messages they had been sent (and rate them if desired), change their selected time periods, and change (or add to) their selected role model. Intervention group participants could also ask for extra support messages on demand by texting keywords to the study shortcode (four-digit number). Texting “crave” and the context  would result in the immediate automated sending of an appropriate video or text message on how to beat cravings within that context. Texting “relapse” would result in three messages over the next 90 minutes to motivate to keep going with the quit attempt and suggest ways of getting extra support. The control group participants received one video message every 2 weeks with general health messages and reminders about the study for 6 months. The primary outcome for the study was continuous abstinence as defined by the Russell standard , which aSmoking status was verified on a random sample of 10% of eligible participants prior to randomization. Verification of quitting status was attempted in all participants reporting continuous abstinence at 6 months using salivary cotinine reading on a mailed-out and returned NicAlert  test-strip pack. Salivary cotinine has a half-life of 15-40 hours and is able to distinguish smokers from nonsmokers using a cutoff of 10 ng/mL of cotinine  . Two staThe nature of the intervention ensured the study could only be single blinded – that is, participants were aware of which group they were allocated to. However, most data were collected via web-based forms completed by participants, and researchers involved in data collection, particularly outcome assessment, were blind to allocation.P = .05. This included a loss to follow-up of 20%. All statistical analyses were performed using SAS version 9.1.3 , all statistical tests were two-tailed, and a 5% significance level was maintained throughout the analyses. The main analyses were based on the intention-to-treat principle as recommended for cessation studies [Initial calculations indicated that a target sample size of 1300 participants would detect a relative risk of 1.75 for a control group 6-month quit rate of 8.5% (intervention group quit rate of 15%), with 90% power at  studies , where pParticipants were recruited into the study between November 2007 and February 2009, and this proved much more difficult than expected. We attempted multiple sequential “waves” of recruitment efforts via new and multiple sources. However, each wave did little to change the overall recruitment rate. The study catchment area was also increased sequentially from the Auckland region (population approximately 1.4 million), to the Northern Region of the North Island (population approximately 2.3 million), to the whole of New Zealand (population 4.1 million). Initial incentives of monthly prize draws of new third-generation (3G) phones were deemed insufficient to attract new participants, and reimbursements to all participants for their time and participation were later added. Due to these problems, and the costs involved in recruitment, we decided to close the study to recruitment with 226 randomized participants.                 P = .8). Of the 61 participants reporting continuous abstinence at 6 months, 10 were either noncontactable or stated they had relapsed since the end of the study period (when they had claimed to have quit) and therefore could not undergo verification of quitting status. The remaining 51 were sent NicAlert test-strip packs and were contacted repeatedly to return the strips. Fourteen quitters in the intervention group (48% of 29) returned the strip and seven (24%) were confirmed as nonsmokers. Fifteen quitters in the control group (47% of 32) returned the strip and 11 (31%) were confirmed as nonsmokers.                No significant difference was found between the groups in the intention-to-treat point prevalence abstinence , which was recorded at three time points and is shown in At 6 months those who reported quitting were asked to rate their confidence in being able to stay quit, and those who had relapsed were asked to rate their confidence in being able to quit again. There were no significant differences between the intervention and control group mean scores at any of these points (data not shown). P = .3) stated they did not attempt to quit at all, but the majority of respondents in both groups made multiple quit attempts. In the intervention group 17 of 69 respondents (25%) and in the control group 26 of 68 respondents (38%) (P = .2) had used pharmacological quitting support  at any stage in the 6-month study period.At 6 months all participants were asked how many quit attempts they had made during the study period. In the intervention group 7/73 respondents (9.6%) and in the control group 4/81 (4.9%) ; those whose comments related to the program ; and those who said all of it (5/59).When asked what they disliked most about the program, 20/49 said they disliked nothing, six complained of some sort of technical issue, and seven did not feel the content was right or did not relate to the models. Five said there were too many messages, one said the messages reminded them to smoke, and one had the  perception they were being charged for messages. The most common suggestions to improve the program were around having more personal (human) contact, individually or via support groups or internet social networking.A report from the intervention program confirmed that 29/110 participants (26.4%) had used the text “crave” function and 18/110 (16.4%) the text “relapse” function.Equal numbers of participants in each group (n = 4) reported having a motor vehicle accident during the study period where the participant was the driver. In the control group one such accident occurred while the participant was using their mobile phone, one within 5 minutes of receiving a message, and two while the participant was smoking, whereas none of the accidents in the intervention group were reported as being temporally related to mobile phone use or smoking.This study is the first to have developed and trialed a smoking cessation intervention delivered via video messaging on mobile phones. We found no significant differences in quit rates between the intervention and control groups . However, the trial was substantially underpowered due to our failure to recruit sufficient participants to reach the desired sample size and the higher than expected self-reported control group quit rate. In fact, quit rates in both groups were high compared to New Zealand’s quitline quit rates of 17% (6-month continuous abstinence) and 10% in the 18- to 24-year age group, but similar to those reported in our previous study of a text messaging cessation intervention . TherefoThe strengths of the study include a study design in accordance with CONSORT guidelines and the strict definitions and analysis of smoking abstinence outcomes. We also used theory on which to base the intervention: this has been shown to be important in technology-based health behavior change  and ensuThe obvious limitation of the study is the suboptimal recruitment. There are several potential reasons for this, which present challenges to be addressed in future trials. First, our recruitment efforts were targeted at adolescents (16 years and over) and young adults. We found that, despite indicating their interest in quitting, most young people were not actually ready to commit to a cessation intervention. This has been demonstrated elsewhere in focus groups and surveys of young people -32. The Second, the costs of messaging and advanced technology may have proved a barrier for some. At the time of recruitment, New Zealand mobile phone data charges  were expensive. We spoke to two participants who were wary of being charged (despite being advised the program was free) and there may have been more who did not register for this reason. Also many people were unaware whether their mobile phone could receive video messages. These factors may have dissuaded people even registering their interest and therefore we have no information on their relative importance in our recruitment. However, if poor recruitment was related to a wariness of new multimedia messaging, we feel that this will have been short-lived: in our current trial of a multimedia mobile phone program to prevent adolescent depression we have recruited 1200 participants over 30 school weeks.Thirdly, plans to incentivize participation were hampered by several factors. Monetary incentives are considered to be effective in encouraging participation of adolescents in research , so we ptx2quit program went live in New Zealand in June 2008 with national promotion by Quitline, and recruited approximately 4000 participants in the following 12 months [Finally, and somewhat ironically, the text messaging cessation program trialed in our own earlier study  may haveMobile phones are increasingly being used globally in health services as a means of more frequent and convenient contacts with health providers ,40, remoThis study adds to this body of knowledge by demonstrating the feasibility and participant appreciation of video messages via mobile phones to provide observational learning and support for healthy behavior change. It is also of note that participants were happy to complete research procedures such as consent and data collection by mobile phone. Indeed we achieved higher response rates to text message questions (217/226 or 96% response rate to a question about confidence at QD and 170/226 or 75% response rate to a smoking status question at 12 weeks post-QD) than to our online data collection forms (despite text message reminders to complete them).  In conclusion, this trial struggled to recruit participants, in particular young adults who wanted to quit smoking. This may explain the failure to show an effect of the intervention, or it may be that the complex theory-based intervention is no more effective than simple less-frequent video messages from researchers. However, there was sufficient positive feedback about the support obtained by observing the role models in the program to warrant further investigation in this area. Further research should explore the effect of this role model-based mobile phone smoking cessation intervention for older adults – a group that are perhaps more serious about stopping smoking and are becoming higher users of newer mobile phone technology.


It is expected that by the end of 4 years of medical training, medical students should be comfortable with the teaching and use of asthma and allergy devices. We sought to determine third-year medical students’ comfort levels in the use of these devices and evaluated the effectiveness of a medical device training seminar.65 third-year medical students participated in an asthma and allergy device training seminar provided by Windsor Allergy and Asthma Education Centre during their Pediatrics core rotation. The students’ comfort levels with the use of 7 devices were self-graded on a scale of 1-10 prior to training, and then again immediately after. Students’ interests in either medical or surgical specialties, presence of asthma and/or allergy conditions in the students, and stages of clinical training were collected. Mean comfort level scores before and after the training were compared using a paired t-test.Prior to the seminar, mean comfort level scores ranged from 1.78 to 3.66 for each medical device. Scores were consistently low, regardless of the students’ interests in particular specialties. Mean scores ranged from 8.65 to 9.15 after the seminar, which represented a significant increase for every device (p<0.05).Third-year medical students were not comfortable with the use of asthma and allergy devices. A medical device training seminar increased the trainees' comfort level and should be considered as a regular part of the clinical training curriculum. Further study is warranted to determine whether the improved comfort level is retained at the end of 4-years of medical training.


The clinical features of type 2 diabetes may differ depending on whether first depression episode precedes or follows the diagnosis of diabetes.Type 2 patients from the observational community-based Fremantle Diabetes Study Phase II underwent assessment of lifetime depression using the Brief Lifetime Depression Scale  supplemented by information on current depression symptoms  and use of antidepressants. Patients were categorized as never depressed (Group 1), having had depression before diabetes diagnosis (Group 2), diagnosed with depression and diabetes within 2 years of each other (Group 3) and having depression after diabetes diagnosis (Group 4).Of 1391 patients, 20.8% were assigned to Group 2, 6.0% to Group 3 and 14.5% to Group 4. In Group 2, depression occurred a median 15.6 years before diabetes onset at age 37.2±14.7 years. These patients had similar clinical characteristics to never depressed patients except for reduced self-care behaviours and having more symptomatic peripheral arterial disease. In Group 4, depression occurred a median 9.9 years after diabetes onset at age 59.8±13.0 years. These patients had long duration diabetes, poor glycaemic control, more intensive management and more diabetic complications. Group 4 patients had more current depression than Group 2 but were less likely to be receiving antidepressants.The clinical features of depression and type 2 diabetes are heterogeneous depending on their temporal relationship. There may be corresponding differences in the pathogenesis of depression in diabetes that have implications for diagnosis and management. Both type 2 diabetes and depression contribute substantially to the global burden of disease. They coincide more often than expected The mechanisms underlying these associations remain poorly understood. Depression is potentially diabetogenic because of lax self-care behaviours and associated physical inactivity and obesity vice versa, e.g. diabetes onset or diabetic complications could occur at an earlier age in those with previous depression. Because of a paucity of published data relevant to this hypothesis, we assessed lifetime depression and age at first depressive episode in type 2 participants in the Fremantle Diabetes Study Phase II (FDSII) In view of these considerations, we hypothesized that there would be potentially important clinical differences in diabetes onset, management and outcomes depending on whether depression preceded diabetes or Written informed consent was obtained from the participants of this study. The individual studies described below were approved by the Human Research Ethics Committee of the South Metropolitan Area Health Service of Western Australia.The FDSII is an ongoing, observational study of known diabetes conducted in a postcode-defined geographical area surrounding the port city of Fremantle in the Australian state of Western Australia. The recruitment period for FDSII was between 2008 and 2011. Patients with diabetes were identified using available hospital, clinic and family practice patient lists, widespread local advertising  and mail-outs to registrants of the Australian National Diabetes Supply Scheme then invited to participate in a longitudinal study after confirmation of the diagnosis of diabetes. Details of these procedures as well as sample characteristics including classification of diabetes types and non-recruited patients have been published elsewhere Each participant underwent a baseline assessment that comprised a comprehensive questionnaire, physical examination and fasting biochemical tests Current depression was assessed using the 9-item Patient Health Questionnaire (PHQ-9), a brief self-assessment of depressive symptoms that is valid and reliable with high sensitivity and specificity in both primary care and in community studies of diabetes Complications were ascertained using standard criteria For FDSII, a novel questionnaire was devised by one of the investigators experienced in developing psychiatric instruments (SES), to assess lifetime prevalence of depression and age at time of first episode see . This quThe BLDS was validated in a convenience sample of 27 FDSII participants, including enrichment with those suffering current depression. Each underwent an additional assessment using the Structured Clinical Interview for DSM-IV (SCID) Affective Disorders Module administered by an experienced psychiatrist (SES) who was blind to the BLDS and PHQ-9 responses. Using SCID diagnosis as the gold standard, the BLDS criteria had 92.5% sensitivity and 100% specificity; all but one patient with a diagnosis of lifetime depression based on the SCID assessment also had depression on the BLDS. Another 21 patients completed the BLDS a second time one week after the first with a Kappa statistic of 0.72 for diagnosing lifetime depression and intraclass correlation coefficient of 0.80 for the total score.Participants were classified into four groups based on the relative timing of diabetes and first depression onset defined using BLDS, supplemented as described below by the presence/absence of current depression and antidepressant medication history. Group 1 were those who had never experienced depression. Group 2 comprised participants diagnosed with depression >2 years before diabetes onset. Group 3 were diagnosed with both depression and diabetes within 2 years to allow for potential uncertainty surrounding the timing of diagnosis. Group 4 comprised participants diagnosed with depression >2 years after diabetes diagnosis. Patients who screened negative on the BLDS but positive on the PHQ-9 were considered to have new-onset depression.There were 71 FDSII participants taking antidepressant medications who screened negative for both lifetime and PHQ-9 depression. Two experts  independently assessed whether the antidepressant medication was prescribed for depression or other reasons with consensus when there was initial disagreement. Forty-one were considered to be have been prescribed treatment for depression and were assigned to groups based on the date of first prescription  while 23 were considered to have been treated for other conditions  and seven were excluded with potentially unreliable responses (unclassifiable).t-test, and for non-normally distributed variables by Mann-Whitney U-test. Multiple comparisons were by Fisher's exact or chi-squared tests for proportions, one-way ANOVA for normally-distributed variables, and the Kruskal-Wallis test for non-normally distributed variables. To further examine differences between groups 1 and 2, a nested case-control study was conducted with each group 2 case matched by age, sex and diabetes duration to a control from group 1 in a 1∶1 ratio. A two-tailed significance level of P<0.05 was used throughout.The computer package IBM SPSS Statistics 19  was used for statistical analysis. Data are presented as proportions, mean±SD, geometric mean (SD range), or, in the case of variables which did not conform to a normal or log-normal distribution, median . For independent samples, two-way comparisons for proportions were by Fisher's exact test, for normally distributed variables by Student's P = 0.80) and had similar diabetes duration , but they were more likely to be female , less educated , not fluent in English , and to be Aboriginal (28.1% vs 4.2%) while being less likely to be of Anglo-Celt ethnicity .Of the 1551 patients with type 2 diabetes  recruited to the FDSII, 1443 (93%) completed the PHQ-9, 1399 (90%) completed the BLDS, and 1391 (90%) had sufficient data for classification. The 160 non-completers were the same age as the completers , 20.8% had depression that preceded diabetes (Group 2), 6.0% developed depression within 2 years of diabetes diagnosis (Group 3) and 14.5% had depression that followed diabetes onset . When considering patients taking antidepressants without current depressive symptoms (who could be considered to have treated or controlled depression), there were also significantly fewer of these in Group 4 than in Group 2 .Key diabetes and depression-related features of the four groups are summarised in Sociodemographic and clinical features of the four groups are summarised in The main finding of the present study was that there were substantial clinical differences between patients with type 2 diabetes and a history of depression depending on whether depression pre-dated diabetes or first developed after the onset of diabetes. In Group 4, depression first occurred an average of a decade after the onset of the diabetes and was associated with poor glycaemic control despite intensive diabetes management and considerable morbidity from microvascular and macrovascular complications. All conventional chronic complications, except for stroke, were more prevalent in this patient group, and the associated disease and treatment burden was likely to be considerable. In Group 2, the first episode of depression was reported to have occurred a median 15 years before the onset of diabetes and there were few differences compared with never-depressed patients with no difference in diabetes onset and most chronic complications. Nevertheless, this patient group also had several adverse prognostic features that have been associated with depressive disorders We created an additional category, Group 3, in which both diabetes and depression developed at around the same, for methodological reasons. This group had clinical features that were intermediate between the other depression groups but comparisons are difficult given the small group size and differences in age and diabetes duration when they were studied. It is conceivable that psychological vulnerability explains why some patients become depressed at or soon after the diagnosis of diabetes and the planned longitudinal follow-up of the cohort should help clarify the clinical picture in this situation.There were also between-group differences in relation to current depressive symptoms. Group 2 patients had less current depression than the other two groups. This may have been, in part, because of successful diagnosis and treatment. Proportionally more of this group were on antidepressants without reporting current depression symptoms, presumably indicating a prior response to initial and maintenance therapy. In addition, proportionally more of the patients in this group who had current depression symptoms were taking antidepressants, indicating that they had already been diagnosed. This is in contrast to Group 4 patients who had the highest prevalence of major and minor depression yet the lowest rate of antidepressant treatment. Mood disorders that first occur in the context of established diabetes appear to be more difficult to detect than at other times and these patients have a higher risk of remaining under-diagnosed and undertreated.The strengths of the present study include the comprehensive nature of the clinical assessment conducted in a large community-based cohort and the use of the well-validated PHQ-9 depression assessment instrument. The limitations include the cross-sectional nature of the analysis, the reliance on patient self-reports for depression assessments including the use of a novel scale for assessing lifetime depression given the absence of a suitable, alternative instrument. The PHQ-9 has high negative predictive value but a positive predictive value of around 50% There is limited information on the course of depression in diabetes and most existing prospective studies have been conducted in small, clinic-based samples and aimed to assess depression incidence and causes The present findings have clinical implications for the diagnosis and management of depression in diabetes. Enquiring about lifetime depression histories appears worthwhile when type 2 diabetes is diagnosed. Patients with a prior history of depression would be expected to benefit from targeted programs aimed at preventing future depression episodes In conclusion, there are substantial differences in the clinical features of both diabetes and depression depending on whether depression predates diabetes or first occurs in established diabetes. Differentiating these temporal patterns may assist future studies of the aetiology, diagnosis and management of depression in patients with type 2 diabetes.Table S1Brief Life-time Depression Scale that was developed, validated and used to assess lifetime depression.(DOCX)Click here for additional data file.


Neck and shoulder complaints are common among employees in sedentary occupations characterized by intensive computer use. Such musculoskeletal pain - which is often associated with restricted range of motion and loss of muscle strength - is one of the most common conditions treated by physical therapists. The exact mechanism of neck pain is rarely revealed by clinical examination and the treatment has varied from passive rest to active treatments. Active treatments have often been divided into either training of the painful area or the surrounding musculature avoiding direct training of the painful area. Our study investigates the effect of the latter approach.A randomized controlled trial of 10 weeks duration is currently being conducted. Employed office workers with severe neck-shoulder pain are randomized to 3 × 20 min shoulder function training with training supervision or to a reference group receiving advice to stay physically active. Shoulder function training primarily focuses on the serratus anterior and lower trapezius muscle with only minimal activation the upper trapezius.An announcement was sent to the administrative section of the university including jobs characterized by intensive computer work. The first 100 positive replies entered the study. Among these inclusion criteria were pain intensity in the neck/shoulder of at least 3 on a 0-9 scale. Exclusion criteria were cardiovascular disease, trauma, hypertension, or serious chronic disease. Before and after the intervention period the participants replied to a questionnaire about musculoskeletal disorders and work disability, and underwent a standardized clinical examination of the neck and shoulder girdle. Further, on a weekly basis the participants log pain intensity of the neck and shoulder during the previous week.The primary outcome measure is pain in the neck and shoulders at week 10 based on the weekly pain registration and results from the clinical examination. Secondary outcomes are strength and work disability.NCT01205542ClinicalTrials (NCT):  Neck and shoulder complaints are common among employees in sedentary occupations characterized by intensive computer use. ChronicPhysical exercise is a cornerstone in health and well-being. An incrWe are currently conducting a randomized controlled trial in Denmark. The trial duration is September 2010 to December 2010. The participants were recruited from a large university.All of the participants gave their written consent to participate in the study. The local ethics committee approved the study protocol (H-C-2008-103), which qualified for registration in the ClinicalTrials.gov, number NCT01205542An announcement with a short introduction and invitation text, together with a link to an internet-based questionnaire was send to office workers from the administrative section of the university. When 100 had replied positive regarding participation to the questionnaire we closed for further recruitment based on a priori power calculations and drop out estimates. Out of the 100 responders 8 subsequently declined to participate in the study.Table Inclusion criteria were pain intensity in the neck/shoulder of at least 3 on a 0-9 scale.Exclusion criteria were a) hypertension  or cardiovascular diseases , b) symptomatic herniated disc or severe disorders of the cervical spine, c) postoperative conditions in the neck and shoulder region, d) history of severe trauma, and e) pregnancy, f) other serious disease.Using a computer generated random numbers table, the remaining 47 participants were randomly allocated to training or control. Gender and age was used as stratification variables. Subsequently, participants were informed via email about group allocation. A flow-chart of the process is shown in Figure The training-group was allocated a total of one hour training per week for 10 weeks during working-hours. Experienced instructors assist all of the training sessions. The reference group was not offered any physical training, but encouraged to stay active, and they replied to the same questionnaires as the training-group.The training-group performed shoulder function training with exercises which have shown to activate the serratus anterior and lower trapezius muscles to a high extend but with only a low activation of the upper trapezius. The exercises used have been selected on the basis of a yet unpublished study and the two main exercises -push-up plus and press-ups - are pictured in Figure The participants received a training diary for registering training sessions. We encouraged participants to train with an instructor during a specified time period together with their colleagues. Instructors taught the participants how to perform the exercises, and helped with exercise adjustment when needed. The instructors focused on positive feedback to maintain motivation.We applied an email-based questionnaire including e.g. the Standardized Nordic questionnaire for musculoskeletal disorders, stages Musculoskeletal pain symptoms of the neck, shoulder, arm, hand, and back were evaluated using scales concerning both intensity and duration of symptoms. Participants replied to the questions "How many days have you had trouble in [body part] during the last three months?"  for symptom duration, and "On average, how intense was your pain in [body part] during the last three months on a 0-9 scale?" (0 means no complaints and 9 means pain as bad as it can be) for symptom intensity. Answers to the question that concerned symptom duration were recoded as follows: 0 days = 0, 1-7 days = 4, 8-30 days = 19, >30 days = 60, everyday = 90[yday = 90. FurtherStages of change in relation to physical activity was in this study assessed by a questionnaire originally presented by Marcus and colleagues in 1992[s in 1992. QuestioProductivity was rated on an 11-step ordinal scale: "How do you perceive your overall productivity the last 4 weeks?" The rating went from 0 (the worst a worker could do) to 10 (the best a worker in the same job could do)[could do).work disability at baseline and follow-up by the work module of the Disability of the Arm, Shoulder and Hand questionnaire (DASH): "In the past week did you have any difficulty:" 1) "using your usual technique for your work?", 2) "doing your usual work because of arm, shoulder or hand pain?", 3) "doing your work as well as you would like?", 4) "spending your usual amount of time doing your work?". Participants replied on a 5-point Likert scale from "No difficulty" to "Unable". The DASH score was normalized on a scale of 0-100 [Participants rated ng by 25).A physiotherapist performed a thorough clinical examination of the neck and shoulder girdle of the participants as previously described in detail. This inTesting of physical capacity was performed at baseline and 10-week follow-up in both groups.Pressure Pain Threshold (PPT) was measured at 4 sites using a standardized procedure[procedure. Muscle Maximal muscle strength was assessed by a maximum isometric shoulder protraction test and shoulder elevation test against a pair of strain gauge dynamometers at baseline and at the end of the intervention (10 weeks).Muscle activation during strength testing was assessed through surface electromyography (EMG). Electrodes were placed according to SENIAM recommendations on the skin over the following muscles: 1) upper trapezius, 2) middle trapezius, 3) lower trapezius, 4) upper serratus anterior and, 5) lower serratus anterior.The primary outcome is change in pain of the neck and shoulders at 10 weeks. Secondary outcomes include the other measures mentioned above. We will firstly analyze the data according to the intention-to-treat principle and secondly, per protocol. We will use repeated measures analysis of variance. The following null-hypothesis will be tested;1) There is no difference between the training group and reference group for the change in neck/shoulder pain from baseline to week 10.Power analyses performed prior to the study showed that - to reject the null-hypothesis of equality - we should include 20 participants per group  for 80% power to detect a change in pain of 1,5 on a 0-9 scale between groups.The authors declare that they have no competing interests.GS, MKZ, OSM, LLA and CHA were responsible for the research design. CHA drafted the paper, and all co-authors made significant contributions to drafting the protocol. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/12/14/prepub