The assessed pain threshold after acupuncture on the other hand was changed towards higher levels in women and unchanged in men. The individual variation was apparent in both women and men but larger in women. For statistical analysis of the variability for both group and individual related effects, the rank-based method by Svensson could be used. The present study indicates that evaluation of sensory and pain threshold response should be analysed separately in women and men.Variable results of pain alleviation in response to acupuncture have been reported, complicating its interpretation. Sources of variability are probably multi-factorial, including the contribution of gender related effects. Gender related variation in perceived pain has been discussed frequently, but documented effects of acupuncture referring to gender are sparse. Furthermore, factors such as operationalisation of the outcome variable and the statistical method for evaluation could also be sources of variability. When pain is regarded as subjective, the produced data should be treated as ordinal. The rank-based method by Svensson, taking the non-metric qualities of the ordinal data into account as well as the variability at the group and the individual level, is therefore an alternative. The present commentary aims to (1) evaluate changes in electrical sensory thresholds and electrical pain thresholds after low frequency electro-acupuncture separately in healthy women and men; (2) introduce and exemplify the method by Svensson in a user-friendly approach. To analyze the systematic patterns of change in thresholds, indicating evidence of treatment on a group level, the relative position (RP) and relative concentration (RC), were measured. The variation related to the individual, the relative rank variation (RV) was also measured. The results were divergent between women ( Some clinical trials that compared acupuncture with no treatment or other treatment modalities have been reported -6. HowevVariability in acupuncture treatments of pain is multi-factorial, such as clinical conditions, treatment strategies, study designs, outcome variables, instrument used and gender. Gender differences in pain evaluation have been discussed  but docuThe method used for statistical analysis, based on the operationalisation of the outcome variable like assessed pain intensity and its indicators, is important for the interpretation of the final results . Pain iswithout pre-defined assumption of distribution of the data other than the ordered structure, and is applicable to the raw data as it does not matter if they have linear, ordinal or dichotomy properties. Thus the results are valid and reliable for all types of ordered data even in small samples. Furthermore, the method gives the possibility of evaluating individually related changes separately from the systematic group related changes. When the objective of a study is evaluation of change, the interpretation of the systematic changes linked to the group effects gives support for evidence of the treatment, while the measure of individual variability illustrates the range of individual response.A quite recently developed statistical method, suitable for data from scale assessments with only the order known, could be used be used to evaluate changes, inter- and intra-observer agreements ,23. The In order to demonstrate how ordinal data can be analysed at both group and individual levels, we used an example to illustrate the changes of assessed thresholds in response to electro-acupuncture in participants of both genders.Students at the Karolinska Institutet who reported to be healthy participated in the study after their informed consent. The study protocol was approved by the Ethics Committee of Karolinska Hospital (dnr 01-169).®, Cefar Compex AB, Sweden) [PainMatcher instrument (Pain-Matcher Sweden) ,25 was u®, Sweden) placed at the acupoints ST36 and ST38 after deqi sensation was elicited. Acupoints ST36 and ST38 were selected because they are distant from the hand with minimal 'spinal segmental effects'. The current amplitude was increased until muscle contractions were seen and adjusted to below the level of perceived unpleasantness throughout the stimulation period of 20 minutes. Four threshold assessments were performed: (1) before EA, (2) during EA, (3) after EA for 20 minutes and (4) 10 minutes after ended EA.Low frequency electro-acupuncture, EA, (2 Hz) was used  with distributed current in alternating pulses . The electrodes were applied to the sterile disposable needles . The data of the assessed EST and EPT were ordinal and hence presented as the median, range and interquartile range (IQR) of the PM values.P-values less than 0.05 were considered statistically significant.The pattern of changes in the paired data of EST and EPT, before and ten minutes after EA, were shown in square contingency tables, and in scatter plots respectively. The main diagonals in the contingency tables, consisting of grey-shaded cells, and of dotted lines in the scatter plots, indicates no change in the respective threshold from the one occasion to the other. The proportions of participants with increased, unchanged and decreased thresholds values after versus before EA were calculated, as were the 95% confidence intervals (95% CI), in proportions of changed threshold levels between the two independent groups of women and men. The hypotheses of no change in assessed EST and EPT comparing values before and after EA were analyzed by the non-parametric sign test. For a further and more detailed evaluation of the variability  of EST and EPT levels, we used the rank-based statistical analysis method by Svensson ,26,27. I® Scandinavia AB, Sweden) was used for descriptive statistics and analysis with the Sign test. For calculation of confidence interval of proportional change in the two groups the Computer software for Confidence Interval Analysis (CIA) [STATISTICA 8.0 (StatSoftis (CIA)  was usedis (CIA)  was usedP(X < Y)-P(Y < X).A systematic change, related to the group, appears as different marginal frequency distributions, heterogeneity, of the assessments seen in the contingency tables and plots Figure . Two typn and the number of categories of the scale is denoted m, thenIn the formulae below the number of individuals is denoted P (X1 <Yk <X0) - P(Y1 <Xk <Y0)The RC is calculated as the difference between the probabilities of the assessed threshold values being more concentrated to the central PM values between 1 to 99 than to the peripheral parts of the possible PM values after EA than before EA and vice versa thenM = minimum value of (xy p- xyp2) and (yx p- yxp2) provided 0 < (xy pand yxp) < 1where Possible values of RP and RC range from -1 to 1. A positive RP value indicates increased thresholds on the second occasion and the contrary holds for a negative RP value. A positive value of RC indicates that the assessed values are more concentrated towards the central parts of the scale on the second occasion than the first, while negative RC values reflects an adjustment towards the ends of the scale.The presence of systematic change is graphically illustrated with plotting the two sets of cumulative relative proportions of the marginal frequency distributions in the contingency tables against each other in a sort of a relative operating characteristic (ROC) curve starting at the  point with a main diagonal representing no change Figure . A non-znot explained by a systematic change, is evident from pairs of observations in the contingency tables of EST or in the scatter plots of the EPT data when dispersed from the expected pattern of change. To estimate the contribution of the individual variation to the pattern of change, firstly an augmented mean rank procedure is used. The augmented ranking is defined by the observations in the th cell of the contingency table according to values before EA (X) asA presence of individual variation in the pattern of change, i, j≤ m where the xij is the ijth cell frequency. The corresponding augmented mean ranks according to values after EA, Y, for 1 ≤ The measure of RV ranges from 0 to 1. The higher the values of RV, the more dispersed are the observations from the expected pattern of change . Strong A total of 23 women aged 27.7 (6.4) years and 22 men aged 28.6 (6.5) participated in the study. All subjects fulfilled the protocol with one women and one man failing to report the EST results.The median EST was reported by the women as 3  before and 3  after EA respectively. EST increase was reported by 5 (23%) of the 22 women, unchanged by 11 (50%) and a decreased EST by the remaining 6 (27%) women.The assessed median EST in the men were 5  before and 6  after EA respectively. EST increase was reported by 13 (62%) of the 21 men, unchanged by 4 (19%) and decreased by 4 (19%). The difference in proportions in women and men who reported EST increase was -39% , i.e. a greater EST increase was reported by the men than the women after EA.The pattern of change in EST, shown as the frequency of the reported PM values in the contingency tables, is denoted X before and denoted Y after EA Figure . The greiX, iY) respectively. Let i xand i ydenote the frequencies of the ith PM values of the individuals and C(X)i and C(Y)i denote the cumulative frequencies of the ith PM value of the two sets of marginal frequency distributions, X and Y. First the measure of systematic change in position, denoted relative position (RP) was calculated according to the formula (1) in the statistical sectionThe evaluation of possible systematic changes is based on the heterogeneity of marginal frequency distributions from the pairs of assessments before and after EA , i.e.Using the same pair of data the evaluation of the other type of systematic change in EST, we calculated relative concentration (RC) with formula M = minimum value of (pxy - pxy2) and (pyx - pyx2) provided 0 < (xy pand yxp) < 1where Correspondingly,The RP value was small, 0.02 and close to zero while there was a certain change in concentration, RC-0.14, towards the peripheral ends of the scale categories used. However, no evidence of significant systematic changes in assessed EST was found in the applied example; RP  and RC -0.14  since the confidence interval in both cases did cover the zero in the calculated systematic changes.The corresponding results of assessed EST in the group of men were RP 0.20  and RC -0.31 , indicating a systematic change towards higher EST and a spread of the PM values towards the end of the scale after EA as compared with before EA. The systematic change of EST in position among the men, RP 0.20, indicates a 20% greater chance that the EST is judged higher after EA compared with before as the opposite in a similar sample of healthy men. Correspondingly, the RC -31 indicates a 31% greater chance that the PM values of assessed EST among the men will be concentrated to the peripheral part of the scale after EA.The values of RP and RC close to zero indicate a lack of systematic changes for the group and produced a ROC curve close to the main diagonal as shown in Figure pure systematic change without any source of individual variation in the paired observations (i.e. the expected pattern of the systematic change), we used the rank-transformable pattern of change (RTPC) to pair off the two sets of marginal frequencies where all individuals kept their ordering relative to each other but may have changed the category The evaluation of individual changes in the applied threshold assessments are based on a ranking procedure in which the pairs of assessments are transformed into ranks, tied on the cells Figure , the augA Figure . The thr(4) where The measure of the individual changes, the relative rank variance is then defined by the square of the mean rank differences as in formula Then, according to Figures 5The pair of rank values for the four individual that have the mean rank differences of 9 respectively, means that these individuals contribute to a large extent to the heterogeneity in the group regarding assessed EST before and after EA as indicated by the significant measure of RV 0.23 . However, among the men the individual variation was considered small and negligible RV 0.03  indicating that the individuals among the men agreed in their opinion of changed EST.P < 0.001  before and 18  after EA. Twenty-one (21) of the 23 women (91%) reported the EPT as increased while two of them (9%) reported it as unchanged after EA. This change was considered significant, 1 Figure .The median EPT in the group of men was 19  before and 20  after EA respectively. Among the 22 men, 13 of them (59%) reported an increase of the EPT and the remaining 9 (41%) a decrease Figure . The difThe findings of systematic changes in EPT were also confirmed by the use of the Svensson method showing a systematic change in position, RP 0.39  in the women but not in the men, RP 0.09 (-0.06 to 0.24). The measure of relative concentration was similar in the both groups, RC 0.16  indicating a change of the PM values towards more central part of the scale, but without evidence of significant change Figure .The change referred to the individual variation was large with a wide CI in the group of women, RV 0.24 (95%CI 0.00 to 0.49) while it was smaller but obvious also among the men, RV 0.13 (95%CI 0.00 to 0.30).The exemplified study showed that changes of EST and EPT after EA vary in the groups of healthy women and men. In the female group, the EST was unchanged at the group level and was systematically changed among the men in both positions (increased) and in concentration  after EA. On the other hand, the EPT results showed the opposite pattern of change where the women responded with a systematic change towards increased EPT after EA while the men remained unchanged. The group related effect among the women was supported by the fact that 21 of the 23 responded with increased EPT after EA. Apart from this group related change, the women were dissentient on the number of categories when the threshold was increased, i.e. the individual variability was obvious. Among the men an individual variation was also apparent. The responses to electro-acupuncture may differ between healthy subjects and patients with different pain conditions. However, the observed variability in the reported findings indicates that effects of EA should be evaluated separately in women and men. Moreover, the treatment effects of EA may lead to more trustworthy results when the treatment is individually designed.Threshold assessments have been used in various studies of acupuncture applying different types of physical stimulation -31 with The assessment of thresholds may be a valuable complementary instrument, both in clinical and research work, for the evaluation of pain as the pain threshold levels are supposed to change as a consequence of neural plasticity in long-term pain conditions . Pain thThough challenging and difficult to assess and evaluate, the statistical evaluation of pain is of great importance to take into consideration. Otherwise important information could be missed, and the basic data for decision-making could be misleading. In the present study the applied statistical approach by Svensson ,23,26,27The present study indicates that evaluation of sensory and pain threshold responses to acupuncture should be analysed separately in women and men. For statistical evaluation of the variability for both group and individual related effects, the rank-based method by Svensson could be used.CI: confidence interval; CF: cumulative frequency; CRF: cumulative relative frequency; EA: electro-acupuncture; EST: electrical sensory threshold; EPT: electrical pain threshold; FREQ: frequency; IQR: interquartile range; PM: PainMatcher; RC: relative concentration; RP: relative position; RV: relative rank variance; RTPC: rank transformable pattern of change; ROC: relative operating characteristic; SD: standard deviation; ST: stomach; TENS: transcutaneous electrical nerve stimulation; Tot: total;The authors declare that they have no competing interests.TL conceived the idea of the manuscript. IL collected and analysed the data. Both authors contributed equally to the writing and finalising of the manuscript. Both authors read and approved the final version of the manuscript.


Two soft-agar methods for assaying chemosensitivity of human cancers in vitro were compared with respect to colony morphology, plating efficiency (PE) and chemosensitivity of human melanomas. In 9 xenografts and 9 patients' biopsy specimens Method A  gave considerably higher PE that Method B  and, in contrast to Method B, the number of colonies was proportional to the number of cells plated. Evidence was obtained that the observed differences in PE could be attributed to the low O2 concentration and the presence of rat red blood cells in Method A. Colony morphology was similar in the 2 assays. When cells from 4 xenografted melanomas were treated in vitro with DTIC, CCNU, vinblastine and abrin, and the inhibition of colony formation was assayed concurrently in the 2 soft-agar methods, the tumour cells appeared to be more sensitive to 3 of the drugs in Method B than in A. The results demonstrate that chemosensitivity data obtained with the 2 assays cannot be directly compared.


The UK Medical Research Council (MRC) randomized trial of gastric surgery, ST01, compared conventional (D1) with radical (D2) surgery. Sample size estimation was based upon the consensus opinion of the surgical members of the design team, which suggested that a change in 5-year survival from 20% (D1) to 34% (D2) could be realistic and medically important. On the basis of these survival rates, the sample size for the trial was 400 patients. However, this trial was exceptional in the way that a survey of surgeons' opinions was made at the start of the trial, in 1986, and again before results were analysed but after termination of the trial in 1994. At the initial survey, the three surgeons from the trial steering committee and 23 other surgeons experienced in treating gastric carcinoma were given detailed questionnaires. They were asked about the expected survival rate in the D1 group, anticipated difference in survival from D2 surgery, and what difference would be medically important and influence future treatment of patients. The consensus opinion of those surveyed was that there might be a survival improvement of 9.4%. In 1994, prior to closure of the trial, and before any survival information was disclosed, the survey was repeated with 21 of the original 26 surgeons. At this second survey, the opinion of the trial steering committee was that 9.5% difference was more realistic. This was in accord with the opinion of the larger group, which remained little changed since 1986. The baseline 5-year D1 survival was thought likely to be about 32%, which corresponded closely to the actual survival of recruited patients. Revised sample size calculations suggested that, on the basis of these more recent opinions, between 800 and 1200 patients would have been required. Both surveys assessed the level of treatment benefit that was deemed to be sufficient for causing surgeons to change their practice. This showed that the 13% difference in survival used as the study target was clinically relevant, but also indicated that many clinicians would remain unwilling to change their practice if the difference is only 9.5%. The experience of this carefully designed trial illustrates the problems of designing long-term, randomized trials. It raises interesting questions about the common practice of basing sample size estimates upon the beliefs of a trial design committee that may include a number of enthusiasts for the trial treatment. If their opinion of anticipated effect sizes drives the design of the trial, rather than the opinion of a larger community of experts that includes sceptics as well as enthusiasts, there is likely to be a serious miscalculation of sample size requirements. © 2000 Cancer Research Campaign


Type A influenza viruses are important pathogens of humans, birds, pigs, horses and some marine mammals. The viruses have evolved into multiple complicated subtypes, lineages and sublineages. Recently, the phylogenetic diversity of type A influenza viruses from a whole view has been described based on the viral external HA and NA gene sequences, but remains unclear in terms of their six internal genes .In this report, 2798 representative sequences of the six viral internal genes were selected from GenBank using the web servers in NCBI Influenza Virus Resource. Then, the phylogenetic relationships among the representative sequences were calculated using the software tools MEGA 4.1 and RAxML 7.0.4. Lineages and sublineages were classified mainly according to topology of the phylogenetic trees and distribution of the viruses in hosts, regions and time.The panorama phylogenetic trees of the six internal genes of type A influenza viruses were constructed. Lineages and sublineages within the type based on the six internal genes were classified and designated by a tentative universal numerical nomenclature system. The diversity of influenza viruses circulating in different regions, periods, and hosts based on the panorama trees was analyzed.This study presents the first whole views to the phylogenetic diversity and distribution of type A influenza viruses based on their six internal genes. It also proposes a tentative universal nomenclature system for the viral lineages and sublineages. These can be a candidate framework to generalize the history and explore the future of the viruses, and will facilitate future scientific communications on the phylogenetic diversity and evolution of the viruses. In addition, it provides a novel phylogenetic view (i.e. the whole view) to recognize the viruses including the origin of the pandemic A(H1N1) influenza viruses. Type A influenza viruses can infect humans and many kinds of animals including birds, pigs, horses and some marine animals . The virIn the past century, type A influenza viruses have become highly diversified and complicated mainly through natural point mutations, cross-host transmission and genomic segment re-assortment among or within the subtypes, lineages or sublineages -47. ConsRecently, the panorama phylogenetic trees of type A influenza viruses based on their external HA and NA gene sequences were described, which could be used as the "maps" in tracing an influenza virus through phylogenetic analysis of the two genes . HoweverThe six internal genes of type A influenza viruses were important in phylogenetic analysis, as demonstrated below in tracing the origin of the pandemic influenza virus recently emerging in Mexico ,42. The Up to May, 20, 2009, 98261 sequences of type A influenza viruses were available in GenBank. More than half of them (61528) were from USA (36887), China , Australia (3444) and Canada (2949). Additionally, most of them (96248) were from humans (47958), birds (42282), pigs (4846) and horses (1162), and most of them (86254) were from the viruses isolated in or after the year 1990.Up to May, 20, 2009, 7189 PB2, 7226 PB1, 7074 PA and 7238 NP sequences (≥ 300 amino acid residues) as well as 7954 NS1 and 8605 MP sequences (≥ 150 amino acid residues) were available in GenBank. They were taken as the candidates of the representative sequences.2798  representative sequences were selected. Their designations and alignment were given in additional files Figures Figures The distribution of the main lineages of human , swine  and equine swine  influenza viruses in isolation places and isolation time were consistent among the six internal genes except that, as for the PB1 gene, subtypes H2N2 and H3N2 human influenza viruses were located in the avian lineage S2.1 rather than in the human lineage S2.3.The phylogenetic trees were also somehow different among the six internal genes, especially for avian influenza viruses. The most striking heterogeneity was that avian influenza viruses were largely divided into two lineages corresponding to the two hemispheres, respectively, based on the viral PB2, PB1, PA, NP and MP gene sequences Figures , 4 and 5Like the panorama phylogenetic trees based on the viral HA and NA genes , the oneFigures Figures Swine influenza viruses were also of high phylogenetic diversity. They could be divided into at least three major genotypes each of which were of multiple subtypes, as described below. In addition, pig infections with avian, human and equine influenza viruses were not rare, and a few swine influenza viruses such as A/swine/Quebec/4001/2005(H3N2) were strange in their gene sequences  and A/swine/England/WVL16/1998(H1N1). This genotype is equal to the sublineage S1.2.6, S2.2.6, S3.2.6, h1.1.3, S5.2.3, n1.1.7, S7.2.6, S8.2.2 of the relevant genes Figures  and 14, The third genotype is the re-assortant swine influenza viruses presumably emerging in the 1990s and circulating worldwide . It corrThe majority of the viruses from the first genotype were H1N1 and H1N2 subtypes of swine viruses. The majority of the viruses from the second and third genotypes were H3N2, H1N2 and H1N1 subtypes of swine viruses. A few H3N1 subtype isolates were also identified in the third genotypes. In addition, as showed by the aforementioned isolates in the third genotype, multiple subtypes of swine influenza viruses within the same genotype could circulate in the same region in the same year.The sublineage of the new AH1N1) influenza virus was situated at the top of the six panorama phylogenetic trees -like viruses in Figure The proposed nomenclature is easy to remember as it is of certain orders as given in Methods. This nomenclature, if widely accepted, should facilitate international communication on the evolution, ecology and epidemiology of type A influenza viruses through the unification of current miscellaneous nomenclatures which are not only ambiguous and misleading in some cases, but also covered only a part of the diversity of type A influenza viruses. For example, the so-called "North America lineage" swine influenza viruses actually circulated widely in North America and Asia in recent years.Influenza viruses are of high significance in human and animal health, and they are very complicated and continuously evolving. Therefore, it is desired to establish an ad hoc international expert group responsible to double check some special isolates, select the representative sequences, calculate the panorama phylogenetic trees, classify the lineages and sublineages and propose a universal nomenclature for influenza viruses and update them in time in the future. This is not that difficult as there have been many human or animal influenza reference laboratories . We recognize that our results are of reference value to the issues but also might be of some flaws.This study describes the first panorama analysis of the phylogenetic diversity and distribution of type A influenza viruses based on their six internal genes. It also proposes a tentative universal nomenclature system for the lineages and sublineages within the viral type. It provide a novel phylogenetic view (i.e. the whole view) to recognize the viruses including the origin of the pandemic A(H1N1) influenza viruses. It also presents a candidate framework to generalize the history and explore the future of the viruses.Till May 20, 2009, 7500-8800 sequences of each of the PB2, PB1, PA, NP, MP and NS genes of type A influenza virus isolates were available in GenBank. They were analyzed online using the web servers of Influenza Virus Resource in NCBI in sequence search, alignment and phylogenetic analysis ,54. For At least one representative from each of the lineages and sublineages was randomly selected with consideration of the distribution of the lineages and sublineages in hosts, isolation places and subtypes. That is to say, at least one representative was selected from those within the same sublineage provided they were distinct from the majority of the sublineage in the distribution of hosts, places, time or subtypes, and also at least one representative was selected from the majority. Most lineages or sublineages of avian influenza viruses are of multiple HA or NA subtypes, and so their representatives were selected with little consideration of subtype. On the other hand, most lineages or sublineages of human, swine or equine influenza viruses are of limited HA or NA subtypes, and thus at least one representative was selected from each of the subtypes, if available. Human and equine influenza viruses are largely of little geographical difference, and so their representatives were selected with little consideration of isolation places. However, avian influenza viruses isolated in the Eastern Hemisphere  were usually different from their counterparts isolated in the Western Hemisphere (North and South America), and thus at least one representative was selected from each of the hemispheres, if available. In addition, at least one representative was selected from those of similar distribution within the same sublineage provided that they were distant from others . RepresGenetic distances among the representative sequences were calculated using the model of nucleotide maximum composite likelihood including transitions and transversions using the software MEGA 4.1 . The subRepresentative sequences were aligned using the software of Clustal X , and theAll the sequences of each of the six internal genes of the viruses isolated from a certain host or in a certain period available in GenBank were sought as the test sequences. Their phylogenetic diversity was analyzed online using the web servers of Influenza Virus Resource in NCBI ,54, and The phylogenetic relationships among the representatives were also calculated using RAxML version 7.0.4 with General Time Reversible (GTR) substitution matrix according to the maximum likelihood (ML) model ,58, to eLineages and sublineages were classified mainly based on the topology of the phylogenetic trees, i.e. each lineage and sublineage should be of a relatively separate cluster in the trees. Distributions of the viruses in hosts, regions and time were also considered in the lineage and sublineage classification. For example, a branch within the trees might be classified as a sublineage provided it is of a distinct host even if it was of a low bootstrap value.Designation of the lineages began with the segment number which was followed by a point and another number (e.g. S2.1 represents the first lineage of the second segment corresponding to PB1 gene), and designation of the sublineages began with the lineage designations followed by a point and a number (e.g. S2.1.1 represents the first sublineage within the lineage S2.1). The numbers within the designations of the lineages and sublineages were favorably in the order from the major to minor ones in terms of the numbers of representative sequences, and in the order of avian ones followed by human, swine, equine ones in terms of hosts, and in the order of the Western Hemisphere followed by the Eastern Hemisphere in terms of isolation places, and in the order from the past to nowadays in terms of isolation time.The authors declare that they have no competing interests.CJM was responsible for design and coordination of the study as well as writing the manuscript. CWC was responsible for analyzing the data and revising the manuscript. All others were responsible for sequence analysis. All authors read and approved the final manuscript.The designations and alignment of the representative sequences of PB2 gene. The data could be read out with NotePad and the Mega software, and gaps are showed with hyphens.Click here for fileThe designations and alignment of the representative sequences of PB1 gene. The data could be read out with NotePad and the Mega software, and gaps are showed with hyphens.Click here for fileThe designations and alignment of the representative sequences of PA gene. The data could be read out with NotePad and the Mega software, and gaps are showed with hyphens.Click here for fileThe designations and alignment of the representative sequences of NP gene. The data could be read out with NotePad and the Mega software, and gaps are showed with hyphens.Click here for fileThe designations and alignment of the representative sequences of MP gene. The data could be read out with NotePad and the Mega software, and gaps are showed with hyphens.Click here for fileThe designations and alignment of the representative sequences of NS gene. The data could be read out with NotePad and the Mega software, and gaps are showed with hyphens.Click here for fileThe original tree with virus designations of PB2 gene of type A influenza viruses. The figure is corresponding to Figure Click here for fileThe original tree with virus designations of PB1 gene of type A influenza viruses. The figure is corresponding to Figure Click here for fileThe original tree with virus designations of PA gene of type A influenza viruses. The figure is corresponding to Figure Click here for fileThe original tree with virus designations of NP gene of type A influenza viruses. The figure is corresponding to Figure Click here for fileThe original tree with virus designations of MP gene of type A influenza viruses. The figure is corresponding to Figure Click here for fileThe original tree with virus designations of NS gene of type A influenza viruses. The figure is corresponding to Figure Click here for fileThe panorama tree of HA gene of subtype H1 influenza viruses. The tree was calculated using the methods reported previously [eviously . Some clClick here for fileThe panorama tree of NA gene of subtype N1 influenza viruses. The tree was calculated using the methods reported previously [eviously . Some clClick here for fileThe panorama tree of PB2 gene of type A influenza viruses calculated using the maximum likelihood model. The figure is corresponding to additional file 7 using the same sequence dataset.Click here for file


Vertebral endplate signal changes (VESC) are more common among patients with low back pain (LBP) and/or sciatica than in people who are not seeking care for back pain. The distribution and characteristics of VESC have been described in people from clinical and non-clinical populations. However, while the clinical course of VESC has been studied in patients, the natural course in the general population has not been reported. The objectives of this prospective observational study were to describe: 1) the distribution and characteristics of VESC in the lumbar spine, 2) its association with disc degeneration, and 3) its natural course from 40 to 44 years of age.Three-hundred-and-forty-four individuals (161 men and 183 women) sampled from the Danish general population had MRI at the age of 40 and again at the age of 44. The following MRI findings were evaluated using standardised evaluation protocols: type, location, and size of VESC, disc signal, and disc height. Characteristics and distribution of VESC were analysed by frequency tables. The association between VESC and disc degeneration was analysed by logistic regression analysis. The change in type and size of VESC was analysed by cross-tabulations of variables obtained at age 40 and 44 and tested using McNemar's test of symmetry.Two-thirds (67%) of VESC found in this study were located in the lower part of the spine (L4-S1). VESC located at disc levels L1-L3 were generally small and located only in the anterior part of the vertebra, whereas those located at disc levels L4-S1 were more likely to extend further into the vertebra and along the endplate. Moreover, the more the VESC extended into the vertebra, the more likely it was that the adjacent disc was degenerated. The prevalence of endplate levels with VESC increased significantly from 6% to 9% from age 40 to 44. Again, VESC that was only observed in the endplate was more likely to come and go over the four-year period compared with those which extended further into the vertebra, where it generally persisted.The prevalence of VESC increased significantly over the four-year period. Furthermore, the results from this study indicate that the distribution of VESC, its association with disc degeneration and its natural course, is dependent on the size of the signal changes. Vertebral endplate signal changes (VESC) have been proposed to be a cause of low back pain (LBP),2. In a In a descriptive study of VESC, Modic et al investigated 474 patients referred for lumbar Magnetic Resonance Imaging (MRI).The distribution and characteristics of VESC in the lumbar spine have been described in patients with non-specific LBP and/or sciatica ,6-9, in The natural course of VESC has been investigated in longitudinal studies of patients with LBP and/or sciatica,6,9,16 aTo our knowledge, the distribution and characteristics of VESC have not been described in the general population. The aims of this study were to describe the distribution and characteristics of VESC in the lumbar spine, its association with disc degeneration, and its natural course from the age of 40 to 44.In this prospective observational study, people sampled from the Danish general population were MRI-scanned at the age of 40 years (in 2000/2001) and 44 years (in 2004/2005). Details of this cohort have been described previously. PermissMRI was performed with a 0.2 T MRI-system . A body spine surface coil was used with the participants lying in the supine position. The following sequences were used:• A localizer sequence of five images, 40/10/40 degrees (TR/TE/flip angle) consisting of two coronal and three sagittal images in orthogonal planes• Sagittal T1-weighted spin echo, 621/26 (TR/TE), 144 × 256 matrix, 300 mm. field of view, 11 slices of 4 mm. thickness, 2 acquisitions, 6 min. 1 sec. scan time• Sagittal T2-weighted turbo spin echo 4609/134 (TR/effective TE), 210 × 256 matrix, 300 mm. field of view, 11 slices of 4 mm. thickness, 2 acquisitions, 8 min. 42 sec. scan time• Axial T2-weighted turbo spin echo 6415/134 (TR/effective TE), 180 × 256 matrix, 250 mm field of view, 15 slices of 5 mm. thickness, 2 acquisitions, 7 min. 49 sec. scan time. Slices were placed in the plane of the five lower discs.The MRI evaluation was performed by an experienced radiologist (JSS), the first author (TSJ) and a second chiropractor (Chiro2) using standardized evaluation protocols,20. JSS The inter observer reproducibility of disc height and disc signal between JSS and a second radiologist, has previously been reported to be 0.59 and 0.66, respectively. The traChiro2 is a chiropractic radiologist who has been trained in radiology in a three year full-time residency program (DACBR) and in three Fellowships in musculoskeletal radiology and neuroradiology. The training of Chiro2 in this study was carried out by TSJ under supervision of the radiologist. After introduction to the evaluation protocol, 18 cases from the study cohort were read in a joint session in order to reach consensus. After consensus was reached, 38 image sets from the study cohort were evaluated independently by TSJ and Chiro2. The Kappa value for inter-observer reproducibility between the two readers for the 38 cases was 0.51, which was lower than the limit of 0.6 that was predefined as acceptable. Communication on the reasons for disagreement was undertaken and a new round of consensus readings of 8 cases was performed. Nine weeks after the first reproducibility study, the same 38 cases and 12 new cases were evaluated independently by the two chiropractors. The results from the chiropractors evaluations were compared to the radiologists original readings. The Kappa value for inter-observer reproducibility between the two chiropractors and the radiologist was 0.81 which was above the cut-point of 0.6 for having acceptable agreement.For the purpose of the present study, the type, location, and size of VESC were evaluated for each lumbar endplate (L1 – S1). The type of VESC was graded for each lumbar endplate (L1-S1) as either type 1, type 2, or type 3 as described by Modic et al. If moreThe location of VESC was defined as involving: 1) the central part of the endplate, 2) the anterior part, 3) the posterior part, 4) the lateral parts, or 5) two or more of the previous locations. The size of VESC was defined as the maximum cranio-caudal extension: 0 = No VESC, 1 = observed in the endplate (EP) only, 2 = <25% of vertebral body height, 3 = 25% to 50% of vertebral body height, and 4 = >50% of vertebral body height as seen on the sagittal images ,22 or reThe MRI evaluation protocols used in the present study have been shown to have substantial to almost perfect reproducibility in relation to the evaluation of disc signal, disc height, and VESC with Kappa values for intra- and inter-observer reproducibility ranging from 0.77 to 1.0 and 0.72 to 0.91 respectively,20.An analysis was performed on people who participated in the study at both the age of 40 and 44 (responders) and those who only participated in the original study at age 40 (non-responders). The proportions of the following baseline variables were analysed at age 40: gender, presence of VESC, disc contour, disc degeneration, spondylolisthesis, LBP, heavy smoking, heavy physical workload, Body Mass Index, highest educational level, employment status, and the Back Beliefs Questionnaire score. The only significant difference between non-responders (n = 68) and those who participated in the follow-up study was their employment status. Nineteen percent of the non-responders were unemployed as compared with only seven percent among those who participated in the follow-up study.Descriptive data were obtained for the VESC variables at both ages. The characteristics and distribution of VESC were analysed by frequency tables and cross-tabulations. Difference in prevalence of VESC at the two points of time was tested with McNemar's test of symmetry. The association between VESC and disc degeneration was analysed using logistic regression analysis. The change in type and size of VESC was analysed by cross-tabulations of the same variable obtained at age 40 and 44.Three hundred and forty four people aged 44 years (161 men and 183 women), of the original cohort of 412 individuals aged 40 (83%), had an MRI at both time points and were therefore included in this study.At the age of 40, 133 (39%) of the 344 people had a total of 237 endplate signal changes  of 1720 discs evaluated at baseline. Disc degeneration was more common at disc levels with VESC (64%) than at levels without VESC (17%) p < 0.0001. Moreover, there was an association between disc degeneration and the size of VESC, as the proportion of discs with degeneration increased from 53% for VESC that was only observed in the endplate, to 100% for those that extended more than 50% into the vertebra, p < 0.0001 . The number of people with VESC of any type or size, increased from 39% to 49%  were type 1, the remainder being either type 2 or mixed type  the prevalence of VESC increases from the age of 40 to the age of 44 and 2) the distribution over vertebral levels, presence of disc degeneration and the natural course of VESC is dependent on the size of the signal changes.The increase in the prevalence of VESC over a four-year period confirms previous observations that there is a positive association between age and the prevalence of VESC ,4,9,10.As stated in the introduction, other studies suggest that there is a difference in the size and distribution of VESC in patients with LBP compared with people without LBP,9,12. InThe positive association between the size of VESC and the presence of disc degeneration raises the question as to whether or not VESC is a response to advanced and/or accelerated disc degeneration. In support of this theory, two prospective studies of patients with sciatica treated non-surgically and surgically, have reported a large increase of type 1 changes over periods of 14 and 24 months respectively,7. FurthRegarding the natural course of VESC, the results from this study show that the signal changes that were only observed in the endplate were those that tended to be transient, whereas those that extended beyond the endplate were more likely to persist over a four-year period. The most straightforward explanation for this is that the smaller the lesion, the easier it is for the body to heal itself. A related explanation could be that people in whom the VESC progress over time 1) are more prone to injury, 2) have a greater inflammatory response to injury, 3) have poorer regenerative abilities, and/or 4) have one or more of the lifestyle factors associated with VESC (i.e. smoking).There are factors in the present study that could have influenced the reported prevalence of VESC and need to be addressed. The evaluation protocol used in this study described all VESC regardless of size, whereasIn relation to the high prevalence of type 1 changes, all MRI scans in the cohort study were performed on a low-field MRI unit (0.2 T). It is known that the contrast between different types of tissues are visualised differently at low-field scanners and high-field scanners. Therefore, it is possible that there are differences in the way that the different types of VESC are visualised on the two types of scanners. Results from a study of 20 patients with LBP and VESC, conducted at the Back Research Centre subsequent to data collection for the cohort study, showed that there are differences in the way that VESC is displayed on high- and low-field scanners. When coThe major strength of this study is that the study sample was population-based, representative of the Danish general population, and that all individuals were of the same age. FurtherThe results from this study indicate that the distribution of VESC, its association with disc degeneration and its natural course are dependent on VESC size.The authors declare that they have no competing interests.TSJ took part in all elements of the study and drafted the manuscript. TB contributed to the design of the study, data interpretation and revision of the manuscript. JSS contributed to the study design, data interpretation, and revision of the manuscript. LK contributed to the study design, participated in the data interpretation and supervised the statistical analyses. CM contributed to the design of the study and data interpretation. PK contributed to the design of the study, data interpretation, the statistical analyses, and revision of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Characteristics of VESC at age 40. Characteristics of VESC in relation to vertebral levels in 344 persons from the Danish general population at the age of 40. No type 3 changes were observed at age 40.Click here for fileCharacteristics of VESC at age 44. Characteristics of VESC in relation to vertebral levels in 344 persons from the Danish general population at the age of 44.Click here for file


Few data are available on the incidence of nosocomial Rotavirus infections (NRVI) in pediatric hospitals and on their economic impact. The goals of this study were: to evaluate the incidence of NRVI in various Italian pediatric wards during the course of two peak RV seasons; to investigate possible risk factors for NRVI; to estimate the costs caused by NRVI.prospective cohort study. Population: all the children under 30 months of age who were admitted without any symptom or diagnosis of gastroenteritis in the pediatric hospitals of Florence, Naples, Brescia and Ancona, Italy, during the winter-spring periods 2006-2007 and 2007-2008. Serial RV rapid tests and clinical monitoring were carried out on the cohort. Telephone interviews were performed from 3 to 5 days after discharge.520 out of 608 children completed the study (85.6%). The overall incidence of NRVI was 5.3% (CI95% 3.6-7.5), . The average duration of hospital stay was significantly longer for children who had NRVI  than for non-infected children . The risk of contracting NRVI increased significantly if the child stayed in hospital more than 5 days, RR = 2.8 (CI95% 1.3-6), p = 0.006. In Italy the costs caused by NRVI can be estimated at 8,019,155.44 Euro per year. 2.7% of the children hospitalized with no gastroenteritis symptoms tested positive for RV.Our study showed a relevant incidence of NRVI, which can increase the length of the children's stay in hospital. Limiting the number of nosocomial RV infections is important to improve patients' safety as well as to avoid additional health costs. Rotavirus (RV) is the most frequent cause of viral gastroenteritis in children under 5 years of age. The virus can cause severe diarrhea and dehydration, especially in children aged 6 to 24 months. In developing countries, acute gastroenteritis due to RV infection (RV AGE) causes the death of approximately 440,000 children every year ,2. In thReoviridae family is very diverse, as it consists of different groups (A-G) and of different types based on the characteristics of the surface proteins VP7 (G = glycoprotein) and VP4 (P = protease-sensitive protein). To date, at least 23 G types and 31 P types of group A RVs, the group which most commonly infects humans, have been differentiated  genotype is more common . Other genotypes found during the winter season were G9 P[8]  and G3 P[8] .The distribution of the genotypes responsible for the contraction of NRVI in spring is instead more varied: G1 P[8] , G9 P[8] , G2 P[4] . In one case, it was not possible to type the isolate.At present, several retrospective studies providing estimates of the incidence of NRVI in children are available. Compared to them, prospective cohort studies can provide more reliable and less biased data. However, to date only few researches on the incidence of NRVI have been conducted using a prospective cohort study design and the existing ones were mainly carried out on small populations -26.Our results give an updated and representative picture of the epidemiology of nosocomial RV infections, which affect more than 5% of the children under 30 months of age hospitalized in winter and in spring. This also confirms the clinical and social relevance of this type of infection. The total incidence of NRVI detected in our study deserves attention and it is consistent with the results of other studies having the same design -26. A 20A limit of our study could be the fact that the observed cohort had a loss to follow-up of 14.4%, a statistically acceptable percentage which should not to be overlooked, but which does not alter the reliability of results.Another possible limit of our study may be the fact that no information is available about the percentage of recruited children immunized against RV and about the percentage of breast-fed babies among them. This information could have given important indications on how to evaluate the incidence of NRVI resulting from our study. Breastfeeding is indeed associated with a reduced incidence of gastroenteritis . Our datWith respect to the possible risk factors for NRVI, the mean age of the children affected by NRVI did not differ from the one of children who were not. No significant difference in the incidence of NRVI in the two genders was found.In line with the data of other studies , the perOur study also showed that NRVI brings about a statistically significant and clinically relevant increase in hospitalizations, thus causing higher, non-negligible costs for the healthcare and social systems, also in relation to the total healthcare costs of a country.Our data do not seem to suggest a possible decrease in the direct costs of hospitalization following RV vaccination. However, our study population is limited and additional research is necessary to evaluate the possible impact of RV vaccination on the additional costs highlighted in our study.With respect to typing isolates responsible for NRVI, recent global epidemiology studies, used to monitor the emergence of new strains, highlighted that 88% of the strains which can infect human beings have G1, G2, G3 and G4 genotype in combination with P[8] or P[4] elements . The resIt is worth noting that the genotypes of the isolates found in our study are all covered by the two existing Rotavirus vaccines Since the transmission of RV occurs primarily by contact, the results of our study confirm the importance of insisting on handwashing, on the implementation of appropriate hygiene standards and on prevention, as recommended also in the existing guidelines -56 for rIt is noteworthy that 2.7% of the recruitable subjects who were hospitalized for reasons other than gastroenteritis, and who had no gastroenteritis symptoms, tested positive for RV. These children can become a source of outbreaks of NRVI within the ward where they are admitted. Using the RV rapid test upon hospital admission during the period of higher risk of NRVI for children under 30 months of age might be a way to reduce the impact of NRVI in pediatric hospitals. However, additional studies are needed to confirm this hypothesis.Nosocomial Rotavirus infection is an important problem from a clinical, organizational and economic point of view. Although RV is a marginal cause of death in Western countries, limiting the number of nosocomial RV infections is important both to improve patients' safety and to avoid additional health care costs.This research was entirely funded by Sanofi Pasteur.The author declares that no other financial or non financial competing interest exists.All authors read and approved the final manuscript. FF conceived the study, participated in its design and coordinated it, carried out the analysis and interpretation of data and prepared the manuscript, PC coordinated the acquisition of data and performed the RV rapid tests, DM coordinated the recruitment and acquisition of samples in center 2, BT coordinated the recruitment and acquisition of samples in center 3, MC coordinated the recruitment and acquisition of samples in center 4, DC participated in the design and analysis of data, KPB coordinated the recruitment and acquisition of samples in center 1, RS participated in the interpretation of data, FMR Carried out the genotyping of RV, FMDB participated in the recruitment and acquisition of samples in center 4, AP participated in the recruitment and acquisition of samples in center 3, AG participated in the recruitment and acquisition of samples in center 2, MDM Participated in the design of the study and interpretation of data.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/235/prepub


The Arthritis Impact Measurement Scales 2 (AIMS2) has not been translated and validated for Persian-speaking patients with osteoarthritis of the knee. This was to provide a validated instrument to measure functional disability and health-related quality of life in patients with osteoarthritis of the knee in Iran. The aim of this study was to culturally adapt and validate the AIMS2 for Persian-speaking patients with osteoarthritis of the knee in Iran.A consecutive sample of patients with knee osteoarthritis were asked to complete the AIMS2, the Short Form Health Survey (SF-36) and four visual analog scales for pain, joint stiffness, patient's and physician's global assessment. Internal consistency and convergent validity were applied to examine psychometric properties of the AIMS2. In addition, 30 randomly selected patients were asked to complete the questionnaire two days later for the second time for test-retest reliability. Finally factor structure of the Persian AIMS2 was performed using the principal component factor analysis.In all 230 patients were entered into the study. The mean (SD) age of the participants was 56.9 (8.7) years and the mean (SD) duration of disease was 7.2 (3.5) years. Cronbach's alpha coefficient and intraclass correlation coefficient (ICC) for the Persian AIMS2 scales ranged from 0.74 to 0.92 and 0.85 to 0.96, respectively. The correlation between most of the Persian AIMS2 scales and the physical and mental summary scores of the SF-36 and the visual analogue scales for pain, joint stiffness, patient's and physician's global assessment were statistically significant indicating a good convergent validity (p < 0.05). The results obtained from factor analysis indicated three latent factors that jointly accounted for 67.5% of the total variance.The results showed that the Persian AIMS2 had reasonably good internal consistency, test-retest reliability, and convergent validity in patients with osteoarthritis of the knee. It is simple and easy to use and now can be applied in the future studies in Iran. However, its sensitivity to change needs still to be studied. Osteoarthritis (OA) is a major medical, social, and economic problem in both developed and developing countries -3. It ofThe Arthritis Impact Measurement Scales 2 (AIMS2) is a shorter version of the AIMS and has been developed for the purpose of assessing the impact of arthritis on patient's quality of life ,13. ThisThere is an increasing need for internationally standardized instruments to measure health status in patients with OA of the knee in a manner that allows comparison across cultures and countries ,22. OsteForward-backward procedure was applied to translate the English version of the AIMS2 into Persian (the Iranian language). Two independent professional translators produced two forward translations. Both translators were instructed to aim for conceptual rather than literal translation. Together with one of the authors, the translators compared their translations and produced a single provisional version of the questionnaire. Totally blind to the original version, two other professional translators translated the provisional Persian questionnaire back into the English language. The two translators were not aware of the questionnaire. Finally, an expert committee consisted of the translators, the researchers, two rheumatologists, one orthopedics surgeon and one specialist in psychometric reviewed all the translation and cultural adaptation processes. They also evaluated the final English backward version with the original questionnaire. Consensus in terms of semantic, idiomatic, experiential, and conceptual equivalence was reached and a pre-final version of the questionnaire (the Persian AIMS2) was provided. In general the original scales were remained unchanged except some minor modifications in items 7, 9 and 33. In items 7 and 9, 'walking several blocks' and 'walking one block' have been changed to 'walking several alleys' and 'walking one alley', respectively to refer to a similar distance in Persian language. In item 33, 'church' was changed to 'religious meetings' to imply comparable meaning.The AIMS2 consists of 78 items and the first 57 items are broken to 12 scales and measuring: mobility level (5 items), walking and bending (5 items), hand and finger function (5 items), arm function (5 items), self care (4 items), household tasks (4 items), social activities (5 items), support from family and friends (4 items), arthritis pain (5 items), work (5 items), level of tension (5 items) and mood (5 items). Scores on each scale range from 0 to 10 with lower scores indicating better quality of life [However, the remaining 21 items of the questionnaire are related to satisfaction with each area of health (1 item), arthritis impact on each area of health (1 item), three areas most like to see improvement (1 item), current and future health (5 items), overall arthritis impact (3), medication (1 item), co-morbidity (3 item), socio-economic status (6 items).The SF-36 is a general health questionnaire that consists of 36 items, summarized into eight subscales. The eight subscales are summarized into physical component scores (PCS) and mental component score (MCS). The PCS and MCS provide greater precision, and reduce the number of statistical comparisons needed . The psyThe patient's perception of pain, joint stiffness, and global severity assessment level was measured on a 100 mm visual analogue scale (VAS) ranging from zero  to 100 '. Similarly, the physician's global assessment of disease severity was recorded using a 100 mm VAS anchored by word descriptors at each end .A consecutive sample of 230 patients with symptomatic OA of the knee for at least one year duration who were referred to 6 rheumatology and physical therapy clinics in Tehran, Iran, were entered into the study over a period of 11 months. All patients fulfilled clinical and radiological criteria for OA of the knee according to the American College of Rheumatology (ACR) . A rheumKolmogorov-Smirnov test was used to assess distribution of the Persian AIMS2, MCS, PCS, and the VAS scores. The interval measurements were normally distributed, and therefore several parametric tests were employed to analyze data. The critical values for significance were set at p < 0.05.Reliability of measurement refers to the extent, to which the measured variance in a score reflects the true score, rather than random error; that is, the extent to which measures give consistent or accurate results . Two comConvergent validity examines the extent to which a particular measure relates to other measures that is believed to be assessing the same construct . In the Finally factor analysis was applied to determine the dimensionality of the items of a questionnaire that is, to determine whether they formed only one overall dimension or more than one . Factor The Ethics Committee of the Tehran University of Medical Sciences approved the study. All patients gave informed consent.Table Table Table Moderate to high correlations were observed between mobility level, walking and bending, work, and level of tension scales and VAS measures for pain, joint stiffness, patient's and physician's global assessment. The highest correlations were found between arthritis pain scale and the VAS for pain and joint stiffness. The scores for hand and finger function, arm function, household tasks and mood scales were moderately correlated with most measures. The social activities and support from family and friends scales were not significantly correlated with clinical measures.A factor analysis with varimax rotation was performed and 3 latent factors extracted with eigenvalues greater than 1 that jointly accounted for 67.5% of the total variance. The lower-limb  and upper-limb  functioning scales together with the self care, household tasks, arthritis pain and work scales formed the first factor, which explained 47% of the total variance measured. The psychological scales (level of tension and mood) were loaded on the second factor, which explained 11.1% of the variance. The third factor (9.4% of the variance) was determined by the social dimension, consisting of the social activities and support from family and friends scales , Turkish (0.72–0.90), and German (0.73–0.88) versions of the AIMS2 ,19,20. IThe Persian AIMS2 showed significant good convergent validity, as assessed with the physical and mental component scores of the Persian SF-36 and clinical measures. Satisfactory significant correlations were found between the PCS score and most of the Persian AIMS2 scales, especially mobility level, walking and bending, and arthritis pain. Correlation between support from family and friends and the PCS was not significant. Fair to good correlations were observed between MCS and most of the AIMS2 scales. The highest correlation was found between the MCS and mood, social activities and level of tension. No significant correlation was found between the MCS and arm function and self care. In a Chinese validation study the World Health Organization Quality of Life questionnaire (WHOQOL-BREF) was used to test convergent validity of the AIMS2. In their study significant correlation were reported between physical and psychological dimensions of the WHOQOL-BREF and the Chinese AIMS2 . HoweverFair to good correlations were observed between most of the Persian AIMS2 scales and the subjective clinical measures of pain, joint stiffness and global assessment of disease severity. All or some of these clinical measures were used in previous studies to test convergent validity of the Italian , ChineseA three-factor solution was extracted from the factor analysis of the Persian AIMS2 that jointly accounted for the 67.5% of the total variance. In fact the result was satisfactory and consistent with the theoretical basis for the original conceptual model of the instrument. A German study also confirmed the postulated three-factor structure for the AIMS2 with a physical, physiological and social dimension, explaining 48.5%, 13.9% and 6.8% of the variation, respectively . SimilarThis study has several limitations. Perhaps the main concern is that this study did not provide evidence for responsiveness to change or other psychometric tests . Secondly, the statistical analysis was limited. For instance, to indicate the factor structure of the instrument it would be interesting to carry out confirmatory factor analysis instead of exploratory factor analysis. The future studies could focus on other psychometric properties of the questionnaire and also on different applications of the AIMS2 among Iranian patients.The present study presents initial step in evaluating psychometric properties of a well-known instrument to measure the impact of arthritis on Persian-speaking patients with osteoarthritis of the knee in Iran. Since health related quality of life was rarely assessed as primary end-point in studies of osteoarthritis of the knee in Iran, indeed the Persian AIMS2 might possibly provide both clinicians and patients with numerous advantages as an important outcome measure in the future studies. However, its sensitivity to change needs still to be studied.The authors declare that they have no competing interests.All authors were involved in data collection, analysis, interpretation of results, and manuscript preparation. In addition, SJM, MP, and AM designed the study and prepared the first draft of the paper. AM provided the final manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The Viral Genetic Diversity Network, established in Brazil to enhance the study of viruses, antiviral drug resistance, and the spread of specific viral genotypes, also increased the net scientific production of virology research, and may provide a viable model for other developing countries to reduce the risks of pandemics and facilitate basic scientific research. Xylella fastidiosa genome sequencing consortium [X. fastidiosa sequencing consortium also had a major qualitative impact on genome sciences in Brazil. Taking inspiration from the success of the genome sequencing effort, the VGDN envisaged a network of laboratories at several research institutions in the state of São Paulo, with the aim of studying key viral diseases within an applied context. Its main goal was to provide competence in genetic studies of viruses and to acquire primary data of immediate use . The VGDN proposal was evaluated by a panel of top international scientists and eventually approved for funding by FAPESP.In 1997, in an effort to improve the pursuit of virology in the state of São Paulo, Brazil, a group of scientists proposed the establishment of a Viral Genetic Diversity Network (VGDN) to São Paulo's major scientific funding agency, Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). The major impetus for the project was the earlier establishment of the Under the concept of a “virtual institute” fostered by FAPESP , the proA core set of VGDN laboratories started operations in 2001, conducting a pilot study on HIV-1 together with state-run sexually transmitted disease/AIDS reference centres at a total cost of US$2 million. Over the next few years, FAPESP invested a total of US$7.5 million in the VGDN. Crucially, the infrastructure, technical capabilities, and some research time provided under the VGDN were also utilised by each participating member as seed for the establishment of research projects beyond the four coordinated tasks. The VGDN had 41 researchers, the direct involvement of 115 graduate and 29 under-graduate students, and 69 technicians. Almost all of the institutions provided funds in addition to the resources included under the programme, and in three campuses across the state, new virology units (including new buildings) were established. A total of 20 laboratories (seven of which are biosafety level 3) were either newly built, renovated, or re-equipped.The large amount of basic virological data resulting from each coordinated task has already been passed on to the public health system, and is being prepared for publication. The HIV-1 coordinated task has generated 20% of the viral genome, coupled with 495 pieces of socio-economic and clinical data from each of the 1,279 georeferenced patients from eight cities within the state of São Paulo sampled between 2001 and 2004. These data are currently being used for an intensive study of the patterns of dispersal of subtypes and circulating recombinants of HIV-1, as well as of resistance patterns relating to treatment outcome. As the Brazilian government spends US$731 million annually on free drug treatment and clinical follow-up for HIV-1 carriers, these unique data allow an in-depth assessment of the success of this important health programme, particularly in the face of considerable friction among pharmaceutical companies and the government on issues of drug prices and patents.Similarly, the hepatitis C virus coordinated task involves the study of 1,000 patients undergoing antiviral therapy in three cities. The data produced as part of this project have provided vital information on the role of blood donation policies in the dispersal of viral genotypes over the last 20 years. Since 2005, a total of 2,000 samples have been collected for the HRSV coordinated task, which, combined with retrospectively sampled sequence data, has provided a unique insight into the intricate fluctuating dynamics of HRSV-type genotypes A and B. Finally, hantavirus pulmonary syndrome is an increasing health problem in Brazil (884 cases were reported in 2007, with a 39% case fatality rate [In addition to all of these direct benefits accrued from the operation of the VGDN, the indirect impact on the net scientific output in the state of São Paulo has been extensive. This is evident from PubMed searches for publications by virologists based in São Paulo state. A remarkable change in the annual rate of publishing was observed from 2001 onwards . As a coIt is clear that the VGDN has been pivotal in establishing a more efficient infrastructure for the surveillance and monitoring of emerging and reemerging viral diseases in São Paulo state, using modern techniques and facilities and bringing public health experts and academic researchers together under the same virtual roof. We believe that this unique initiative may constitute a viable model for other developing countries, not only to help alleviate the risk of pandemics, but also to facilitate and support basic scientific research.


A comparison has been made between the carcinogenic activities of benzo(a)pyrene and the proposed proximate carcinogen, benzo(a)pyrene 7,8-dihydrodiol, in the adult C57BL mouse submandibular salivary gland. In preliminary studies using a range of doses, the dihydrodiol was slightly less active than the parent hydrocarbon in this system. There was a difference in the type of tumour induced by the 2 compounds. Benzo(a)pyrene induced tumours of the salivary glands at the site of injection, whereas the dihydrodiol induced malignant lymphosarcomas, particularly of the thymus, which were often metastatic to other orgnas. Possible reasons for the different sites of action of the 2 compounds are discussed.


HIV-1 integrase (IN) is an attractive target for the development of drugs to treat AIDS, and inhibitors of this viral enzyme are already in the clinic. Nevertheless, there is a continuing need to devise new approaches to block the activity of this viral protein because of the emergence of resistant strains. To facilitate the biochemical analysis of wild-type IN and its derivatives, and to measure the potency of prospective inhibitory compounds, a rapid, moderate throughput solution assay was developed for IN-catalyzed joining of viral and target DNAs, based on the detection of a fluorescent tag.++- and Mn++-dependent activities of the IN proteins from human immunodeficiency virus type 1 (HIV-1) and the avian sarcoma virus (ASV). The results confirm that ASV IN is considerably more active than HIV-1 IN, but with both enzymes the initial rates of joining, and the product yields, are higher in the presence of Mn++ than Mg++. Although the pH optima for these two enzymes are similar with Mn++, they differ significantly in the presence of Mg++, which is likely due to differences in the molecular environment of the binding region of this physiologically relevant divalent cation. This interpretation is strengthened by the observation that a compound that can inhibit HIV-1 IN in the presence of either metal cofactors is only effective against ASV in the presence of Mn++.A detailed, step-by-step description of the new joining assay is provided. The reactions are run in solution, the products captured on streptavidin beads, and activity is measured by release of a fluorescent tag. The procedure can be scaled up for the analysis of numerous samples, and is substantially more rapid and sensitive than the standard radioactive gel methods. The new assay is validated and its utility demonstrated via a detailed comparison of the MgA simplified, assay for measuring the joining activity of retroviral IN in solution is described, which offers several advantages over previous methods and the standard radioactive gel analyses. Based on comparisons of signal to background ratios, the assay is 10–30 times more sensitive than gel analysis, allows more rapid and accurate biochemical analyses of IN catalytic activity, and moderate throughput screening of inhibitory compounds. The assay is validated, and its utility demonstrated in a comparison of the metal-dependent activities of HIV-1 and ASV IN proteins. Retroviral integrase (IN) catalyzes the insertion of a duplex DNA copy of the viral RNA genome into the DNA of its host cell. This process establishes the retroviral provirus as a permanent component of the host cell genome, and is required for normal viral gene expression via host cell components. IN proteins are members of a superfamily of polynucleotidyl transferases, which include transposases and other recombinases. The HIV-1 IN is of special interest as a target for the development of drugs to treat AIDS . I,3. I2,3]g factor -8. For Hg factor ,10. Theret al. , containing the plasmid pET29 that expresses wildtype HIV-1 IN (NY5 strain), were induced to express IN, harvested from 1 liter of Luria broth culture and stored frozen. The frozen cell pellets were thawed and resuspended in lysis buffer  at 0.13 g of cells/ml. The cells were lysed by passage through a French Pressure cell as above, and the lysate was then sonicated for 30 s. The preparation was subjected twice to centrifugation for 30 min at 12,000 × Viral DNA (donor) oligodeoxynucleotides with a covalently attached 6-carboxyfluorescein (6-FAM) were purchased from Integrated DNA Technologies , and purified by Tris-borate urea denaturing polyacrylamide gel electrophoresis. The efficiency of labeling was quantified by comparison of the absorbance at 260 nm with the peak absorbance of the fluorophore (495 nm for 6-FAM). The labeled oligodeoxynucleotides were annealed with unlabeled complementary oligodeoxynucleotides to obtain viral donor oligodeoxynucleotide duplexes. Complementary strands of the target oligodeoxynucleotide containing biotin at their 3'-ends were synthesized and purified in the Fox Chase DNA Synthesis Facility. These were then annealed to obtain a 27 bp duplex with single nucleotide overhang on each 3'-end to which biotin was attached.Processing activity was measured using fluorescence-anisotropy ,15. The 2  or 10 mM MgCl2,  and ionic strength ≤ 100 mM NaCl equivalents. The reactions were stopped by the addition of 10 μl of 30 mM EDTA. For the comparisons described in Figures The double stranded, 6-FAM-labeled viral oligodeoxynucleotide (donor substrate) was mixed with IN and the metal cofactor, and the mixture was left on ice for 15 min. The biotin-conjugated, double stranded target oligodeoxynucleotide was then added, and the mixture left on ice for an additional 15 min, after which it was transferred to a waterbath at 37°C and incubated for the desired period. The total reaction volume was 20 μl. We determined the optimal ratio of IN:viral oligodeoxynucleotide:target oligodeoxynucleotide, to be 4:1:6, and this ratio was used to test the potency of the inhibitors. These reactions contained 1 μM IN, 0.25 μM 6-FAM-labeled viral oligodeoxynucleotide (26nt/28nt recessed duplex), 1.5 μM biotin-conjugated target oligodeoxynucleotide duplex, 5 mM DTT or 2 mM mercaptoethanol, 10% DMSO, 25 mM Hepes, pH 7.5 (at 37°C) with 10 mM MnClA 96 well filter plate  was prepared for use by adding 50 μl of a 1:1 slurry of streptavidin agarose beads to each well . The assay reactions were transferred to the wells, and incubated at room temperature for 30 min  to allow the biotin-conjugated target and joined products to bind to the beads. The wells were then washed 10 times with 200 μl Wash Buffer  using a vacuum manifold . In some cases, the last wash was also collected by centrifugation into a reader plate and analyzed to confirm that all of the unbound, unjoined FAM-labeled viral oligodeoxynucleotide had been removed.The viral oligodeoxynucleotide strand that included the 6-FAM probe was dissociated from the bound product by denaturation via addition of 150 μl of freshly prepared 50 mM NaOH to each well. The plate was then left at room temperature for 5 min. The soluble fractions were collected by centrifugation  into a black, round bottom 96 well plate .50 values were determined from non-linear fitting of the triplicate data to a four parameter sigmoidal dose response equation:The wells were read using a Tecan GENois Pro fluorescent microplate reader equipped with Magellan Standard V5.03 software  set to the fluorescence intensity mode. In this instrument the excitation of 6-FAM is at 485 nm and the emission is measured at 535 nm. The data from the plate scanner are expressed as relative fluorescence units (RFUs). The experimental RFU readings, including the data from the background wells and the controls were transferred to the Visual Enzymics  module running with Igor Pro  graphing software. In the experiments described in Figure 50 value, and D is the Hill coefficient. The Hill coefficient, which is proportional to the slope of the sigmoidal curve, reflects the cooperativity and the tightness of binding of the inhibitor to the enzyme. All four parameters are fitted, and the standard error and Chi squared goodness of fit statistics confirm adequate data quality. The data are then plotted as percent joining activity to compare the various enzymes, metals, and inhibitors used.where A is the activity at maximal inhibition, B is the activity in the absence of inhibitor, X is the inhibitor concentration, C is the IC32P-labeled at its 5' end. These strands were then annealed with complementary oligodeoxynucleotides that were labeled with 6-FAM, as described for the fluorescent assay above. The target DNA and reaction conditions followed those described for the fluorescent assay. The products were separated by electrophoresis in a Tris-borate-urea 20% polyacrylamide gel and quantified using a Fuji phosphorimager. The processed products migrated below the substrate bands, and the joined products migrated in a series of bands above the substrates.The same viral donor DNAs were assembled after the strand to be processed was ++ or Mg++) in a suitable buffer on ice. The target oligodeoxynucleotide, which contains biotin at both 3' ends, is then added in molar excess over the donor. In step 2, the mixture is incubated at 37°C for the desired period, after which catalysis is stopped by the addition of an excess of EDTA. In step 3, the reaction is transferred to a well in a 96 well filter plate that contains a slurry of streptavidin agarose beads. This mixture is left at room temperature for 30 min and shaken gently at 5 min intervals. In step 4, the beads are washed thoroughly with suction applied in a multi-well plate vacuum manifold. A solution of 50 mM NaOH is then added to each well to denature the DNA and the mixture left for 5 min at room temperature. In step 5, the solution containing the released FAM-labeled donor single strands is collected by centrifugation into a 96 well plate. The fluorescence of the FAM-labeled donor in each well is recorded in a plate reader.This assay employs a short DNA duplex  comprising the sequence at the end of one or the other viral LTR, hereafter called the donor oligodeoxynucleotide. As illustrated in Figure During optimization studies, we measured the relative activities of ASV and HIV-1 IN in the presence of both cofactors, and observed an increase with increasing divalent metal concentration to a maximum at approximately 15 mM for both proteins. We note that higher metal concentrations promote the non-specific endonuclease of IN proteins and can raise the ionic strength to inhibitory levels. To avoid these problems and to establish uniform conditions for our comparisons we chose the close to optimum concentration of 10 mM to measure the joining activities of these two proteins.++ or Mg++ was also determined. The results from our joining assays indicated that with Mn++ as cofactor, both enzymes exhibit activity maxima in the range of pH 7–7.5; maxima for processing with Mn++ are higher, at pH 8.1 for both enzymes. Rather different results were obtained with Mg++ as cofactor. In this case, optima for ASV IN were in the range of pH 8–8.5 for both processing and joining, whereas the optima for HIV-1 IN were substantially lower, pH 7 for processing and pH 6.5 for joining, although the ranges were fairly broad.The pH-dependence of both the processing and the joining reactions with ASV and HIV-1 IN proteins in the presence of either Mnin vivo is believed to be Mg++ [++ as the cofactor. For comparison of the activities of these proteins, as donor oligodeoxynucleotides we used sequences from the U3 (ASV IN) and U5 (HIV-1 IN) LTRs, because previous studies have shown that the enzymes are most active with these DNA ends [++ as the cofactor was 6.7 times faster than HIV-1 IN, and with Mg++ it was 5.3 times faster , and then product continued to increase at a much reduced rate. A similar response was observed in the presence of Mn++, but the rate of the second phase was higher. In both cases, the initial bursts are likely to represent product from donor-enzyme complexes formed during the preincubation step  label to the 5' end of the donor strand to be joined, and then analyzed the products using gel electrophoresis. The donor and target oligodeoxynucleotides in these reactions were otherwise identical to those used in our standard fluorescence assay  and 205 with Mg++ (30 min).Table 50 values for integrase inhibitors. In this context, Zhao et al [++ or Mg++. Of special interest for our analyses, was a related series of 2,3-dihydroxybenzoic acid hydrazides  than Mg++ (0.6–1.3). This is indicative of a greater cooperativity of inhibitor binding with the Mn++ cofactor, and is consistent with results from previous studies of this class of inhibitors [The concentration dependence for compound hibitors . The Z' hibitors  calculat50 values calculated for all three inhibitors is shown in Figure ++. From these results, it appears that in the presence of this metal cofactor, all three compounds interact with structural elements that are conserved in these two IN proteins, and this interaction inhibits the joining reaction. Results with compound 1 indicate that this inhibitor is able to discriminate between the two proteins in the presence of Mg++.A summary of the ICIn this report we describe a simplified assay measuring the joining activity for retroviral integrases in solution. The assay offers several advantages over the gel analyses used in many laboratories. Limitations of the gel assays include the length of time needed to separate and quantify the products and relatively low sensitivity. The latter problem derives from the fact that the ligated products detected in this assay are of different sizes and therefore spread through a large portion of the gel  the placement of the reporter on the donor strand complementary to that which is actually joined, and its dissociation from the bound product and 2) the attachment of biotin to the 3' ends of both strands of the target DNA. The first feature allows for better detection of the reporter, as its signal is obscured when retained on agarose beads. After developing this protocol we discovered that a similar strategy was employed by Landgraf et al.  in devel++ is likely to be the biologically-relevant cofactor, the initial rates of joining by both isolated enzymes with Mg++ are less than half the rate, with Mn++. Both enzymes also exhibit a similar pH optimum (7–7.5) in the presence of Mn++. However, with ASV IN, the optimum for joining in Mg++ is somewhat higher (pH 8–8.5), and with HIV-1 IN lower (pH 7–6.5) than with Mn++. The reason for these differences is unknown, but these data suggest that the two metals are bound differently by these enzymes, and/or that the microenvironment for binding Mg++ is not the same in the two proteins. Finally, the rate of joining by ASV IN is 6–7 fold faster than HIV-1 IN in the presence of either metal cofactor.A side-by-side detailed comparison of the cofactor-dependent joining activities of purified HIV-1 and ASV IN proteins used to illustrate the utility of this new assay revealed a number of similarities, as well as some notable differences. Although Mg50 values obtained with HIV-1 IN were similar to those previously reported with a gel assay, despite the fact that our assay conditions are quite different [++, although the IC50 values were approximately 2 to 5 times higher with this enzyme. This finding is consistent with the notion that Mn++ is bound in similar ways by these two proteins. Inhibitor 1 was of special interest as it was reported to be equally effective with HIV-1 IN in the presence of either metal cofactor, and those results were also confirmed by our assay. Our finding that this compound was ineffective against ASV IN in the presence of Mg++, further supports the notion that the determinants for binding of Mg++, or a Mg++-inhibitor complex, are different in the two enzymes.We also demonstrated the utility of the joining assay for screening inhibitors, by testing the potency of a related series of compounds known to block HIV-1 IN, on the activities of both IN proteins. The ICifferent . We alsoThe abbreviations used are: IN: retroviral integrase; HIV-1: human immunodeficiency virus; ASV: avian sarcoma virus; 6-FAM: 6-carboxyfluorescein.The authors declare that they have no competing interests.MDA supervised the work and data analysis, and contributed to writing and editing the manuscript. JC designed the assay and performed some of the preliminary experiments. GM conducted all of the optimization studies and performed all of the assays and some of the calculations included in the manuscript. XZZ synthesized and tested the HIV-1 inhibitors under the supervision of TRB, Jr. AMS provided overall direction and had primary responsibility for writing and finalizing the manuscript, which all authors have read and approved.


A kinetic study was made on murine myeloid leukaemia, using the myeloid leukaemia colony in the spleen as a model system. The number of myeloid leukaemia colony-forming units (MLCFU) recoverable from the bone marrow, spleen and liver increased exponentially as a function of time from day 1 up to day 9, after the initial lag period of 12-24 hours. The f-values were estimated to be about 67 per cent in the above 3 organs. After day 10, the number of MLCFU declined in these organs. The myeloid leukaemia was sensitive to cyclophosphamide and chlorambucil, as determined by the slopes of the survival curves obtained for MLCFU. Finally, an entity of the mixed type in the myeloid leukaemia colony was described.