The aim of this study was to investigate the effects of 10 weeks of strength training with different number of sets and their influence on flexibility of young men. Sixty men were divided into three groups as follows: group that trained 1 set per exercise (G1S), group that trained 3 sets per exercise (G3S) and control group (CG). The training lasted 10 weeks, totaling 30 training sessions. The training groups performed 8 to 12 repetitions per set for each exercise. The flexibility at Sit and Reach Test was evaluated pre and post-training. Both trained groups showed significant increase in flexibility when compared to pre-training and the G3S showed significant difference when compared to CG post-training. According to this study, the strength training carried out without flexibility training promotes flexibility gains regardless the number of sets. According to the American College of Sports Medicine  the physical fitness is related to health through five basic components: body composition, aerobic capacity, strength, endurance and flexibility. Among these components, strength and flexibility are important variables of physical fitness, and their appropriate levels are required not only for the health promotion and maintenance, and functional autonomy but also for safe and effective participation in sports .Regular practice of strength training can be aimed to increase flexibility. Several studies found that isolated strength training promotes improvement in flexibility . FatouroIn sum, the literature reports the positive effects of strength training on flexibility and the available studies used different types of training methods and different combinations of exercises . HoweverSixty men, intentionally chosen, were divided into three groups: 1 set per exercise (G1S) (n=20), 3 sets per exercise (G3S) (n=20) and control group (CG) (n=20). To be included in the study, all participants must have the following characteristics: a) be recreationally trained in strength training and flexibility at least one year before the study began; b) not perform any type of regular physical activity other then the prescribed; c) not present any functional limitations to strength training or for the performance of the tests involved in the study; d) not present any medical limitations that could influence the training program, and; e) not use any nutritional supplementation . All participants signed an informed consent form that explains the testing and training procedures conducted during the study. The study protocol was approved by the “Research Ethics Committee of Rio de Janeiro Federal University (Brazil)”.Data collection pre and post-training (10 weeks) was performed in four days. On the first visit, between 7:00 and 8:00 am, anthropometric and flexibility measures were made. On the second day (24 hours after), all the tests were repeated to determine the test retest reliability. On the third day (24 hours after), the 5 repetitions maximum test (5RM) was applied. On the fourth day, 48 hours later, the 5RM test was repeated.The subjects were evaluated in two non-consecutive days in both pre and post training. The 5RM test was conducted for the exercises: bench press (BP) and leg press (LP). All exercises were performed on machines Rotech Fitness® (Goiânia - Brazil). To minimize the error during the 5RM tests, the following strategies were adopted : (a) staFlexibility was measured before and after 10 weeks using a Sit and Reach Test . The subject sat with their feet firmly against the testing box, keeping their knees extended and hands placed one over the other; reached forward, sliding the hands along the measuring ruler. The considered score was the greatest distance recorded in the three attempts with a 10-second interval between them . The same procedure was performed after training. All flexibility tests were conducted in the same period of the day. Data collected during the first evaluation were not available to the examiner to prevent information bias during the measurements taken after training.Before the flexibility test, a warm-up of 4 stretching exercises was performed for the muscle groups involved in the evaluation. Two sets of static stretching were used for the warm-up protocol, holding the position for 10 seconds in each set, until a point of slight discomfort was reached. A 10-second interval was provided between the warm-up stretching sets. The warm-up exercises followed the protocol of the Sit and Reach Test of American College of Sports Medicine (2000).The exercise order for all groups (G1S and G3S) was: BP, LP, lat pull-down, leg extension, shoulder press, seated leg curl, biceps curl, abdominal crunch and triceps extension. The CG not participated in the strength training program. All subjects performed the sets with moderate intensity (8 to 12 repetitions) in each exercise. During the sessions, the subjects were verbally encouraged to perform all sets to concentric failure and the same definitions of a complete range of motion were used to define completion of a successful repetition. There was no attempt to control the velocity of the repetitions performed. The training load was increased when the individual could perform more than the prescribed number of repetitions (12 repetitions). Frequency of the training program was 3 sessions per week with at least 48 to 96 hours between the sessions. A total of 30 sessions was performed during the training period. Prior to each training session, the subjects performed a specific warm-up, consisting of 10 repetitions with approximately 50% of the load used in the first and second exercises of the training session. Adherence to the strength training was 100% for all participants. All training sessions were monitored by an experient physical education professional and the subjects were not allowed to perform aerobic or flexibility exercises during the training period.All data were expressed as mean ± standard deviation. Statistical analysis was initially performed by Kolmogorov-Smirnov normality test and for the homocedasticity test (Bartlett criterion). All variables showed normal distribution and homocedasticity. An ANOVA one-way was used and, when the differences were significant, the Tukey post hoc test was applied for comparisons. An alpha level of p<0.05 was considered statistically significant for all comparisons. Statistica® 6.1 statistical software was used for all statistical analysis.The trained groups showed a significant increase in flexibility in relation to pre-training and the G3S showed significant difference when compared to CG in the post-training .The purpose of this study was to examine, in young recreationally trained men, if the isolated strength training in different training volumes (1 or 3 sets per exercise) could influence in flexibility gains. The main finding of this research was that the strength training performed without the flexibility training promotes flexibility gains regardless of the number of sets. In fact, both trained groups (G1S and G3S), promoted significant gains post-training when compared to pre-training, but only G3S showed significant gains when compared to CG post-training. Regard to strength gains in 5RM, it was observed that G3S showed greater gains in both the analyzed exercises.Actually, only two studies  investigDespite of methodological differences between our study and those conducted by Concerning the flexibility gains through the isolated strength training, our results corroborate with previously published studies in this area . HoweverIn relation to strength gains in 5RM for bench press and leg press, it was evident that multiple sets promoted greater strength gains. Although it was not the main focus of our experiment, we found that strength gains are dependent of training volume and it is important to observe that both, the flexibility and the strength gains seem to be dependent on the training volume. Our findings regarding strength gains corroborate with the previously showed in the literature, that is, there is a dose dependency relationship in strength gain related to the number of sets .When think in health gains or physical performance, it seems to perform isolated strength training, with a greater volume of training, is sufficient to promote flexibility gains. Health professionals can use this training strategy to increase flexibility when the session time is reduced. However, it is important to emphasize that only the isolated strength training might not be enough to increase flexibility in a specific way.


Six patients from 4 families with ADOA underwent detailed ophthalmic examinations including spectral domain optical coherence tomography (SD-OCT). Mutational screening of all coding and flanking intron sequences of the OPA1 gene was performed by DNA sequencing. SD-OCT showed a severe reduction in the retinal nerve fiber layer (RNFL) thickness in all patients. A new splicing defect and two new frameshift mutations with premature termination of the Opa1 protein were identified in three families. A reported nonsense mutation was identified in one family. SD-OCT of one patient showed MME in the inner nuclear layer (INL) of the retina. AO images showed microcysts in the en face images of the INL. Our data indicate that AO is a useful method to identify MME in neurodegenerative diseases and may also help determine the mechanisms underlying the degeneration of the inner retinal cells and RNFL.The purpose of this study was to investigate the characteristics of microcystic macular edema (MME) determined from the  Autosomal dominant optic atrophy , also known as Kjer's disease –4. ADOA OPA1 (MIM no. 605290), located on chromosome 3q28-q29, are responsible for about 60–80% of the cases of ADOA [ADOA has considerable intra- and interfamilial clinical variability with incomplete penetrance estimated to be about 90% in the familial forms of the disease [ of ADOA –12.OPA1 encodes a mitochondrial dynamin-related GTPase, which is anchored to the mitochondrial inner membrane [OPA1 gene result in a loss of function in most ADOA patients indicating that haploinsufficiency is involved in the pathomechanism of the disease [OPA1 gene in the degeneration of RGCs in ADOA.membrane , 14. Altmembrane . The Opamembrane , 14. Themembrane , 16, 17,membrane , 19, orgmembrane . Mutatio disease . HoweverRecently, Barboni et al. detected “microcystic macular edema (MME)” in the inner nuclear layer (INL) of patients with Leber's hereditary optic neuropathy (LHON) and ADOA . The INLμm compared to commercial OCT systems with a resolution of approximately 15 μm. This higher resolution should help in detecting and evaluating en face images of MME.Adaptive optics (AO) technology has enabled clinicians to view the retina with high microscopic lateral resolution , 27. Thien face images obtained by an AO fundus camera in patients with ADOA and also to try to determine the mechanisms underlying the degeneration of the inner retinal cells and RNFL by AO. To accomplish this, 6 patients from 4 families with the OPA1 gene were studied.Thus, the purpose of this study was to investigate the characteristics of MME determined from the The protocol of this study conformed to the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board of the Nippon Medical School. Six consecutive cases of ADOA patients from 4 families who visited Nippon Medical School Chiba Hokusoh Hospital from December 2010 through April 2013 were studied. A written informed consent was obtained from the six patients after an explanation of the nature and possible complications of the experimental protocol.μm, and transverse resolution was 15 μm. The images presented are 6-mm-long scans except for en face OCT images. With this protocol, 128 cross-sectional B-scan images were obtained, each composed of 512 A-scans. In all patients, fixation was centered on the macula. Full-field scotopic and photopic ERGs were recorded using an extended testing protocol incorporating the International Society for Clinical Electrophysiology of Vision standards [The ophthalmological examinations included measurements of the best-corrected visual acuity (BCVA), determination of the refractive error , slit-lamp biomicroscopy, ophthalmoscopy, fundus photography, fluorescein angiography (FA), perimetry, SD-OCT, infrared imaging, and full-field electroretinography (ERG). The visual fields were obtained by Goldman perimetry and Humphrey Visual Field Analyzer . The Swedish interactive threshold algorithm standard strategy was used with program 30-2 of the Humphrey Visual Field Analyzer. Color vision was evaluated with the Farnsworth Panel D-15. SD-OCT (Carl Zeiss Meditec) images were obtained from all of the patients. The B-scan retinal images were composed of 27,000/s consecutive A-scans acquired through the center of the macula horizontally for Figures tandards .OPA1 gene. Coding regions and flanking introns of the OPA1 gene were amplified by polymerase chain reaction (PCR) with published primers [Blood samples were collected from the patients, and genomic DNA was isolated from peripheral white blood cells with a blood DNA isolation kit . The DNA was used as a template to amplify the  primers . The PCROPA1. The mRNAs were obtained from peripheral white blood cells with the TRIzol reagent , and template cDNAs were generated with random hexamer primers. We designed exon-spanning primer pairs and used them to amplify exon 18 to exon 20 of the OPA1 cDNA. They are forward primer (5′-GTTGAACAACAGGCTGATAG-3′) and reverse primer (5′-GCTTGATATCCACTGTGGTG-3′). The recovered DNAs were subcloned into the StrataClone PCR cloning vector . Plasmid DNAs from 20 positive clones were purified with the Qiagen Plasmid Purification Kit  and sequenced with an automated sequencer .RT-PCR was used to amplify the cDNAs of en face images of the retina with a transverse optical resolution of 250 line pairs/mm. Successive AO images were taken at adjacent retinal locations with an angular spacing of 2 degrees in the horizontal and vertical directions. This procedure allowed for a horizontal and vertical overlap of at least 2 degree between successive images. Prior to each acquisition, the focusing depth was adjusted to the inner nuclear layer. The resulting images were stitched together by superimposing retinal vessel landmarks with an image editing software . The size of each pixel was typically 0.8 μm when calculated at the retinal plane, and the values were adjusted for variations in the axial length of the eye [Fundus images were obtained with an infrared AO retinal camera  . This sy the eye . We alsoWe studied 6 patients from 4 families with ADOA,  and the We performed a vertical SD-OCT scan at about 1 mm from the edge of optic disc. The results in the ADOA patients showed that the temporal RNFL was very thin in all of the patients . FA did The clinical findings of a representative case are shown in We identified one already reported pathogenic mutation and three new mutations in the four families . PatientOPA1 transcripts expressed in the white blood cells from this patient. Two distinct RT-PCR products were obtained from the patient (data not shown). To separate the mutant transcripts from wild-type transcripts, the RT-PCR products were cloned into a cloning vector. Twenty clones from the patient were sequenced to verify the inserts, and 5 of them showed truncated inserts with a skipping of exon 19 . This would yield a truncated protein with a premature termination codon due to a frameshift (p.I633MfsX12). We could not test the genetic changes in other family members because they were not willing to have them tested. Although this mutation is a novel mutation, two small deletion mutations within the same exon  have been reported as pathogenic mutations for ADOA , 41.Patient 3-III-1 was found to have a reported nonsense mutation. A heterozygous C to T mutation at position 1096 (c.1096C>T) directly changed an arginine at amino acid position 366 to a stop codon (R366X). This mutation was confirmed to be pathogenic by several studies , 43.Patients 4-II-1, 4-III-1, and 4-III-2 from the same family were found to have a new heterozygous single base-pair deletion at amino acid position 1102 (c.1102delT) that would yield a truncated protein with a premature termination codon due to a frameshift (p.R368GfsX4).All three new mutations identified caused a frameshift with premature termination codon. These mutations would likely be pathogenic by the mechanism of haploinsufficiency as reported .en face OCT imaging. En face OCT imaging of Patient 1-II-1 revealed that cysts were located in the superior, nasal, and inferior macular quadrants in both eyes . Approximately one-half of the OPA1 mutations lead to premature termination codons from nonsense mutations or frameshifts from small insertions, deletions, or splice site mutations [Over 200 mutations in the utations . These tutations , 46, 47.Gelfand et al. reported that MME was associated with lower visual acuity and a thinner RNFL in patients with MS . In our Gelfand et al. hypothesized that the presence of MME was associated with a breakdown of the blood-retinal barrier . HoweverMME has been detected in the INL of the retina with chiasmal glioma . It is hen face AO images of our ADOA patients are areas of degenerated horizontal, bipolar, and amacrine cells in the INL caused by retrograde transsynaptic degeneration.It is well established that retrograde transsynaptic degeneration can occur in the human central nervous system , 49. Vanen face MME structures detected by AO were also found in another ADOA patient who did not show MME clearly in the en face and cross-sectional OCT images. These observations indicate that AO might be useful in identifying MME in other neurodegenerative diseases and may also be helpful in determining the mechanisms underlying RGC and INL degeneration.The en face MME in patients with ADOA; however, there are several other diseases that have MME in their SD-OCT images, for example, MS, recurrent optic neuritis, neuromyelitis optica, LHON, and chiasmal glioma. We need to investigate the en face MME structures in patients with such diseases to identify whether they also show the en face MME in their INL and to compare their features to those of en face MME seen in our patient with ADOA. It will probably be helpful in clarifying the pathomechanisms of the degeneration of inner retinal cell degeneration to investigate several diseases with different etiology.Our study has a number of limitations. We identified the en face AO images, we need to determine why patients with advanced glaucoma did not show en face MME despite the RGC loss. We cannot explain why we did not find MME in the other 4 ADOA patients in this study. It may be related to the disease severity; however, patient 2-II-1 showed comparable peripheral RNFL thickness and better BCVA compared to the other ADOA patients without MME. Some other factors may be needed for MME to develop.We have found MME in the INL of the ADOA patients, but it is important to note that only in two patients. The cross-sectional nature of our study did not allow us to draw conclusions regarding the evolution of MME in ADOA and the other diseases. To address these issues, systematic longitudinal studies incorporating detailed ophthalmologic assessments in large cohort are needed and may help determine the mechanisms involved in the development of MME. Although the controls in our study including those with advanced glaucoma did not show MME in their In conclusion, our findings showed that genetically identified ADOA patients without any sign of inflammation can have MME in the INL of the retina. Our data indicate that the disease severity may be associated with the presence of MME in the INL as reported, because we found the clearest MME in the patients with poorest BCVA, although some other factors may be needed for MME to develop other than disease severity. Our findings indicate that there is a possibility that retrograde trans synaptic degeneration could cause severe damages in horizontal, bipolar, and amacrine cells in the INL after the optic nerve atrophy. Further studies are needed, and these findings will probably be helpful in clarifying the pathology of the degeneration of inner retinal cells by retrograde transsynaptic degeneration in patients with optic nerve atrophies and in developing new therapies.


Chronic cerebrospinal venous insufficiency (CCSVI) has been associated to multiple sclerosis (MS).To evaluate the possible association between CCSVI and MS, using a population-based control design.A random cohort of 148 incident MS patients were enrolled in the study. We have also studied 20 patients with clinically isolated syndrome (CIS), 40 patients with other neurological diseases (OND), and 172 healthy controls. Transcranial (TCC) and Echo Color Doppler (ECD) were carried out in 380 subjects. A subject was considered CCSVI positive if ≥2 venous hemodynamic criteria were fulfilled.CCSVI was present in 28 (18.9%) of the MS patients, in 2 (10%) of CIS patients, in 11 (6.4%) of the controls, and in 2 (5%) of the OND patients. A significant association between MS and CCSVI was found with an odds ratio of 3.41 . CCSVI was significantly more frequent among MS subjects with a disease duration longer than 144 months  and among patients with secondary progressive (SP) and primary progressive (PP) forms  than in patients with relapsing remitting (RR) MS (14.3%). A stronger association was found considering SP and PP forms ; the association was weaker with the RR patients  or not significant in CIS group .A higher frequency of CCSVI has been found in MS patients; it was more evident in patients with advanced MS, suggesting that CCSVI could be related to MS disability. Multiple sclerosis (MS) was firstly described by Charcot in 1868; since that it was known that plaques in MS are venocentric In order to produce stronger evidence, we carried out a population case-control study to assess the possible association between CCSVI and MS. The presence of CCSVI was also evaluated in patients suffering either from clinically isolated syndrome (CIS), suggestive of demyelinating diseases, or from other neurological diseases (OND).The study was approved by the two different local ethical committees  and patients and controls were enrolled only after they signed the informed consent.The survey was carried out in Catania, Sicily. Its official population in 2001, date of the last official census, was 313,110 inhabitants . Since immigrants from other countries represent only 1% of the entire province of Catania (census 2001), our population can be considered ethnically stable.Epidemiological surveys to determine the prevalence and temporal trend of MS in the city of Catania from 1975 to 2004 have been previously carried out At least one healthy control subject per each case, group matched by age and sex, were enrolled from the general population of Catania using a multistage sampling methods. In particular from the ten municipalities of the city of Catania we selected one or more General Practitioners (GPs). Control subjects, not affected by neurological disorders, were randomly selected from every GPs’ cabinet list. In order to exclude the presence of neurological disorders among controls we used a validated screening instrument already adopted in the Sicilian neuroepidemiologic surveys Exclusion criteria for both patients and controls were preexisting medical conditions known to be associated with brain pathology , history of cerebral congenital malformations, thrombosis of jugular veins, deep venous thrombosis, central venous catheter, head and neck surgery, transient global amnesia, vasculitis and pregnancy. Patients with MS were excluded according to the presence of relapses and steroid treatment in the 30 days preceding study entry.Other 20 patients, also resident in the city of Catania, who had had the onset after 31 December 2004, and who fulfilled the McDonald’s criteria Sample size was calculated considering a frequency of the exposure in the general population of 8.8%, average of the frequency of CCSVI among healthy subjects reported in published studies http://www.neurostatus.net); demographic and clinical data have been also recorded. For each patient the Expanded Disability Status Scale (EDSS) All enrolled patients underwent a complete neurological and physical examination including blood pressure measurement, detailed medical history of vascular risks with particular emphasis on venous disease. All neurological examinations were performed by trained and certified examining neurologist , equipped with a 8L-RS (4–12 MHz) linear array transducer was employed for the study of internal jugular vein (IJV) and vertebral veins (VVs) while a 3S-RS Sector Array Probe (1.5–3.6 MHZ) was used for the study of the deep cerebral veins (DCVs). Furthermore a special C-RS Microconvex Ultrasound Probe was used to study internal jugular veins (IJV) under clavear points.Following the Zamboni’s procedures The following five parameters were evaluated for each case and control subjectsReflux in the IJV and/or VVs in sitting and supine posture;Reflux in the DCVs;High-resolution B-mode evidence of IJV stenosis;Flow not Doppler detectable in the IJVs and/or VVs;Reverted postural control of the main cerebral venous outflow pathways.In agreement with literature data, presence of CCSVI was defined as the presence of at least two out of the five parameters Each ECD examination was recorded on a DVD and the presence of CCSVI was evaluated by an adjudication panel composed by two expert evaluators (P.F.V. and D.P.L), both unaware of the subjects’ status. Only in case of disagreement between the two experts the sonographer joined the panel to obtain a consensus on CCSVI status.The panel’s members were blinded to subjects’ status (cases or controls) and unaware of the number of patients with MS, OND, or CIS and controls enrolled in the study. All the members of the adjudication panel attended the course on CCSVI and received a specific training at the University of Ferrara before the study.To avoid the possible lack of blinding we adopted the following strategies:instructing subjects not to reveal their disease status during Doppler examination;all the enrolled subjects were always placed on the tilt table before the arrival of Doppler evaluator.Data were analyzed using STATA 10.0 software packages Quantitative variables were described using mean and standard deviation (m ± SD). The difference between means and the difference between proportions were evaluated by the t-test and the Chi-square test respectively. In case of not a normal distribution appropriate non-parametric tests were performed. Unconditional logistic regression analysis was performed and for each study variable, we calculated OR, 95% confidence interval (CI), and p-value . Multivariate analysis was performed to investigate the independent effect of a risk or protective factor after adjustment for one or several other factors or to adjust for confounding variables. Parameters associated with the outcome at the univariate analysis with a threshold of p = 0.10 were included in the model. The model was manually constructed using the likelihood ratio test (LRT) to compare the log-likelihood of the model with and without a specific variable.Whenever variables were dichotomized or polychotomized, the cut-offs were derived from the pooled distribution of cases and control subjects .The possible interaction was also evaluated by the LRT . For quantitative exposure the test for linear trend was performed to evaluate the linear or trend effect. Stratified analyses were performed for MS form and disease duration.Thirty ECD and TCC ultrasonographies have been repeated within one week and Kappa coefficient has been estimated in order to assess intra-rater agreement.According to the sample size calculation from the incident cohort of 367 MS patients, we randomly selected 200 MS patients (54%). Out of the 200 randomly selected patients, 40 were not traced, 10 refused, one was bedbound and two were already dead. Finally 148 (74%) MS patients were effectively enrolled in the study. Among the enrolled patients, 105 had relapsing-remitting (RR) MS, 26 had secondary progressive (SP) MS and 17 had primary progressive (PP) MS. No significant differences were found between baseline characteristics  of MS patients enrolled in the study and the MS patients selected from the incident cohort but not enrolled (n = 52) in the study, except for a longer disease duration recorded among the enrolled patients .At the end of the study 177 controls have been selected of whom 23 were positive at the screening questionnaire for neurological diseases and underwent a complete neurological examination. Five of them were excluded because presented abnormal neurological examination Finally 172 controls were enrolled. No significant differences were found for age and gender distribution between MS patients and controls. Baseline characteristics of patients affected by MS, CIS, OND and controls are reported in Intra-rater agreement of TCC-ECD evaluation was good with a kappa value of 0.79 (p<0.001).CCSVI, defined as the presence of at least two positive venous hemodynamic criteria Considering only the 148 defined MS patients selected from the incidence-cohort and the 172 population-based controls a significant association between MS and CCSVI was found with an OR of 3.41 .Disease duration was dichotomized on the basis of the median value of 144 months; CCSVI was significantly more frequent (p = 0.03) among MS patients with a longer disease duration  respect to those with a shorter disease duration . CCSVI was also more frequent among SP and PP patients (30.8% and 29.4% respectively) than among RR MS 14.3%;  and, as When multivariate analysis was stratified considering only the SP and PP forms, a stronger association was found with CCSVI , while significant but weaker association was found when the analysis was restricted to the RR patients . We also found a positive, but not significant, association of CCSVI and CIS patients with an age adjusted OR of 2.04 .As shown in At the time of the TCC-ECD evaluation, out of the 148 CDMS, 24 (16.2%) did not receive any treatment, 91 (64.5%) were taking Disease-Modifying Drugs (DMD) and 33 (22.3%) Immunosuppressive agents (ISA). In particular among the MS patients with CCSVI, 11 (39.3%) were taking DMD and 12 (42.8%) ISA; on the other hand out of the 120 MS patients without CCSVI, 80 (66.7%) were under DMD treatment and only 21 (17.5%) were under ISA (p = 0.009). However when analysis was stratified according to the MS form not significant differences concerning the type of treatment were found as shown in In our study we found a significant association between CCSVI and MS. Differences were evident among the groups studied: MS patients showed the highest CCSVI prevalence compared to normal subjects and OND patients. Furthermore CCSVI was more frequent in patients with progressive MS than in patients with RRMS or CIS and in patients with longer disease duration.During the last few years, several studies have been carried out to evaluate the possible association between MS and CCSVI and most of them have reported a higher frequency of CCSVI among MS patients, suggesting a possible pathogenetic role To the best of our knowledge, this study represents the first population-based case control-study carried out to evaluate possible association between CCSVI and MS. Frequency of CCSVI, in fact, has been estimated in a randomly selected sample of a population-based incident cohort of MS patients, thus reducing the risk of a possible selection bias among cases and allowing also the generalizability of the obtained results. Nevertheless the choice of appropriate control subjects is probably the most challenging aspect in case–control studies, and, as it is well known, several strategies should be adopted for selection in order to obtain a representative control population avoiding selection bias. To this reason, to obtain a representative sample of the general population, controls were randomly selected from the GPs roster and the GPs involved in the study were selected from the 10 municipalities of the town. Furthermore, in order to avoid a possible observer bias, great attention has been made to guarantee the blinding procedure.In agreement with literature data, we observed an higher frequency of CCSVI among MS patients respect to the control population. However in our population-based study, CCSVI was significantly more frequent among MS patients with a longer disease duration respect to those with a shorter disease duration. This observation seems to be supported by the higher frequency of CCSVI among the SP patients, and consequently with a longer disease duration, respect to the RR MS Nevertheless the prevalence of CCSVI found in our sample is far from that initially reported by Zamboni et colleagues Another important point to take into account is related to the retrospective nature of the study that did not allow us to establish the exact sequence of the events. To this reason we are not able to be certain that CCSVI exposure occurred before or after the disease onset. Our results indicate that only 18.9% of patients with MS, and 10% with CIS presented with CCSVI. We further found CCSVI to be significantly more frequent among MS patients with longer disease duration, and in patients with the progressive forms of MS. According to these observations, CCSVI could be subsequent to the disease onset, making impossible to exclude a possible “reverse causality”. Even if a “causative relationship” between CCSVI and MS has been suggested, it should be underlined that association not necessary imply causation and that observational study alone does not allow us to establish a cause-effect relationship. To the best of our knowledge all the data available in literature concerning the relationship between CCSVI and MS come from retrospective case-control studies that are unable to determine whether the exposure precede the outcome , the only absolutely essential or necessary criterion of causality Proximal IJV stenosis followed by reflux in deep cerebral veins, reverted postural control of the main cerebral venous outflow pathways and reflux in IJVs and/or VVs in both supine and upright position were more frequently found in MS patients than in CIS and OND patients and in controls. Similar findings with different percentages were found by other groups Whether or not venous abnormalities can contribute to the severity of the disease is questionable. However, our results, in agreement with literature data In conclusion, even if the suggested causal-relationship could be supported by the positive associations reported in different studies, partially fulfilling the consistency criterion Moreover it has to be kept in mind that DMD and ISA treatment may also have an influence on the venous system and the results of the ultrasound investigation which should be addressed in further studies.From our point of view, at the state of the art, results from observational studies must be interpreted with caution and longitudinal multicentre studies on large cohort of CIS should be performed to better understand the correlation between CCSVI and progression.


The aim of this study was to evaluate the association between experienced physical violence and health-related quality of life (HRQoL) by comparing self-reported health status for individuals with and without experience of physical violence. Our hypothesis was that individuals exposed to violence would experience worse HRQoL than non-exposed individuals. We tested whether men and women and different age groups experience similar reductions in HRQoL, and the extent to which such differences might be associated with social circumstances and lifestyle conditions. Finally, we explored the HRQoL consequences of exposure to violence in a longer time perspective.We used data from self-completed questionnaires in two Danish nationally representative, cross-sectional health interview surveys. Exposure to violence was indicated through specific survey questions  enquiring about different types of violence during the last 12 months. Health status of respondents was elicited by the EQ-5D and SF-36 questionnaires. The health status profiles were converted to health score indexes using the Danish algorithm for EQ-5D and the revised Brazier algorithm for SF-6D. Differences in score indexes between the exposed and non-exposed individuals were explored separately for men and women using ordinary least square regression with four age categories as explanatory variables.In the 2000 and 2005 surveys, respectively, 4.9% and 5.7% of respondents indicated that they had been exposed to physical violence within the last 12 months. Exposure to violence was more prevalent in the younger age groups and more prevalent for men than women. Respondents exposed to violence had lower score indexes on both the EQ-5D and the SF-6D compared with the non-exposed. Respondents who reported exposure to violence in both 2000 and 2005 reported lower HRQoL than individuals who only reported exposure in one of the surveys.The results of this study provide evidence for an association between exposure to physical violence and reduction in health-related quality of life. The World Health Organisation recognizes violence as a public health problem, and violence against women is considered a priority health issue . ViolencConsequences on health-related quality of life (HRQoL) of violence may be measured using condition-specific or generic HRQoL instruments. Generic instruments are advantageous in that they allow comparison of the impact of violence on HRQoL with the impact of other conditions or illnesses. The Short Form–36 (SF-36) is an example of such a generic instrument . Some geWe searched the research literature to identify studies that have assessed the consequences of violence on generic HRQoL. We identified relatively few studies that analyzed the consequences of exposure to violence on HRQoL using generic measures and no studies that used preference-based generic measures. These studies differ in terms of target group , type and definitions of violence  and study design.The SF-36 was used in several studies. In a small Norwegian study where 87 women were interviewed about violence and HRQoL -11. Men The Danish National Health Interview Surveys are large population-based representative surveys that are undertaken every five to six years . The surThe aim of the current study was to evaluate the association between exposure to physical violence and health-related quality of life (HRQoL) by comparing self-reported generic preference-based HRQoL index scores for individuals with and without experience of violence.Our hypothesis was that individuals exposed to violence would experience worse HRQoL than non-exposed individuals. We wanted to investigate whether there was a gender difference in the health impact of violence, and the extent to which the reported health impact might be associated with social and other lifestyle conditions. Finally, we aimed to explore HRQoL consequences of exposure to physical violence over a longer time perspective.This study used data from the 2000 and 2005 National Health Interview Surveys (NHIS) . The 200The 2005 survey comprised two sub-samples: i) all those invited to the follow-up sample in 2000 if they were still alive and living in Denmark in 2005, and ii) a supplementary sample to ensure 3000 completed interviews in each of the five Danish regions. The total sample size was 21,832 individuals, of whom 14,566 (67%) participated in a face-to-face interview where half were asked to self-complete the SF-36 and a set of questions on physical and sexual violence. A total of 5,202 individuals (72% of those invited to complete the questionnaire) completed these supplementary questionnaires on violence and 93% also completed the SF-36 , each with three levels  ,20. ThesThe SF-6D is derived from responses to 11 questions in the SF-36 questionnaire. It covers six dimensions , each with 4–6 levels. A single index score was assigned to each health state using the revised Brazier SF-6D valuation algorithm ,23, thatLevel of schooling and highest completed professional education  were combined into a variable of education duration: less than 10 years, 10 years completed, 11–12 years completed, 13–14 years completed, 15+ years completed and ‘other’ including still in education, foreign education and missing data  (AppendiData from the two samples (2000 and 2005) were analyzed separately and stratified by gender. Age was categorized in broad groups to maintain statistical power . Chi-squared tests were used to compare the distribution of categorical data, and unpaired t-tests were used to test mean differences in the EQ-5D and SF-6D score indexes.Model 1, the explanatory variables were the four age categories (reference = 16–29 year-olds) and a dummy variable for exposure to violence (=1). The estimated parameters for age categories represent the average change in HRQoL from the reference group; negative estimates were expected as HRQoL typically deteriorates with increasing age. The estimate for violence exposure represents the average difference in score index for those who are exposed to violence; a negative estimate was expected as individuals exposed to physical violence were assumed to have reduced HRQoL.Ordinary least squares regression models were used to test the hypotheses concerning HRQoL differences. Separate models were estimated for each HRQoL instrument, where the HRQoL score index was used as the dependent variable. In Model 2 we investigated whether exposure to physical violence had a greater effect on older age groups and introduced a set of interaction variables between exposure and age category. The parameters of these interaction variables were interpreted as the marginal change in HRQoL for individuals in the particular age group, who had been exposed to violence.In Model 3 social and lifestyle characteristics were introduced as explanatory variables. As some of the HRQoL differences between persons exposed and non-exposed to violence might be related to social factors, we expected the HRQoL loss associated with violence exposure to reduce but remain negative.In Respondents who had responded to the questions on physical violence in both the 2000 and the 2005 survey could be identified by their unique person identification number. These respondents were categorized as: a) reporting exposure to violence in either 2000 or 2005, b) reporting exposure to violence only in 2000 or c) only in 2005, or d) reporting exposure to violence in both 2000 and 2005. The change in SF-6D index score between the 2000 and 2005 surveys was calculated. To explore the consequences of long term physical violence exposure, a random effect regression model was fitted for both genders , with the index score as the dependent variable and the categories of violence exposure (three binary variables), gender and age as explanatory variables. All other things being equal, we expected the HRQoL of non-exposed individuals to stay the same or be slightly reduced due to age-related health deterioration over the five years. Individuals exposed to violence in 2000 but not exposed in 2005 were expected to show an improvement in HRQoL, while those with new exposure in 2005 were expected to show deterioration in HRQoL. Individuals exposed to violence in both years were expected to have deterioration of HRQoL over time. As a supplementary analysis, the score index in 2000 was included as an explanatory variable in order to improve the fit of the model.All statistical analyses were undertaken using Stata MP – parallel edition, version 11 at Statistics Denmark’s research server. P-values < 0.05 were considered significant.In the 2000 and 2005 survey, respectively, 520 of 10,563 respondents (4.9%) and 296 of 5,202 (5.7%) respondents indicated that they had been exposed to physical violence  are shown in Table The results of the gender-stratified OLS regressions with score index as dependent variable and exposure to physical violence and age categories as explanatory variables . The deteriorations in HRQoL for older age categories remained statistically significant (data not shown).The results for Model 3). The model fit improved as expected, with R-squared ranging from 0.11 to 0.16. The estimated differences in HRQoL were reduced but remained statistically significant in all models.Table Inclusion of gender into the regression model showed an expected difference in score index between men and women of 0.03 on both EQ-5D and SF-6D (2000 and 2005). However, the interactions between gender and age categories were insignificant in all three samples, suggesting an age-independent difference in HRQoL for men and women. The inclusion of interaction between violence and gender provided small and insignificant parameter estimates, suggesting that the difference in HRQoL between those exposed and unexposed to physical violence is independent of gender (data not shown).Analysis of long-term exposure to physical violence was undertaken using only the SF-6D index score, as EQ-5D data were not collected in the 2005 survey. Of the 1972 respondents (47% men) who participated in both the 2000 and the 2005 survey, 63 reported exposures to violence in 2000 but not in 2005; 78 reported exposure to violence in 2005 but not in 2000 and 20 reported exposure to violence in both 2000 and 2005. The remaining 1811 respondents (92%) did not report exposure to violence in either survey.The non-exposed respondents had a small, insignificant fall in SF-6D index score (p = 0.46). The health index score reduced by −0.029 (p = 0.02) for those exposed to physical violence in 2000 but not in 2005. Respondents who had been exposed to violence in 2005 but not in 2000 showed no difference in HRQoL (p = 0.94), whereas respondents who reported exposure to violence in both 2000 and 2005 showed an insignificant reduction in HRQoL of 0.041 (p = 0.10). The model that included variables for social factors provided similar results, with a slightly larger difference for individuals exposed for violence in 2000 but not in 2005 and a smaller difference for respondents exposed for violence in both 2000 and 2005.The main objective of this study was to assess the association between exposure to physical violence within the past 12 months and self-reported health status. The analysis was based on data from two nationally representative health interview surveys undertaken in 2000 and 2005.The study results suggest that 5-6% of Danish adults have experienced physical violence during the last year. The numbers are slightly higher for 2005 than 2000, but are within the bounds of statistical uncertainty. More men than women reported physical violence: 6.2% and 6.4% of men in 2000 and 2005, respectively, compared to 3.8% and 5.0% of women. Young people were more likely than others to be exposed to violence: nearly one out of five men and one out of ten women aged 16–29 years had experienced violence during the last year, although experience of violence was also reported by older age groups. Exposure to violence was also more likely for individuals with less education, single people and those with a high weekly alcohol intake. These findings are similar to those found in .Individuals who had experienced physical violence had lower HRQoL according to the EQ-5D and SF-6D instruments. After adjustment for age category and stratification for gender, the index score differences ranged from 0.032 to 0.058 depending on the instrument used and the survey year. Research into minimally important differences suggests that health states with less than 0.03 units difference on a generic utility-based index cannot be considered as different from one another . The minWith these minimally important differences in mind, there appeared to be only a small difference between men and women in the HRQOL consequences of violence, although women exposed to violence had a larger reduction in HRQOL than men. When interpreting such gender differences it is important also to consider that there is a gender difference in the general population in HRQoL where men score higher than women ,30. The Although there was some evidence of older individuals experiencing more severe consequences in terms of reduced HRQOL than younger individuals, this finding was not statistically significant. Lower HRQoL can also be related to social and lifestyle factors . When thIndividuals who were exposed to physical violence in both 2000 and 2005 showed a reduction in HRQoL score over time of 0.04 (p = 0.10), though this result was not statistically significant – possibly due to the low statistical power as only 20 individuals (1.0%) reported exposure to violence in both surveys.While the main finding of this study is a consistent association between exposure to violence and lower HRQoL, the causality of this relationship is not clear. A number of the risk factors for violence exposure, such as educational level and marital status, have themselves an impact on HRQoL. We found no strong confirmation that continued exposure to violence reduces HRQoL even further, although there was a non-significant trend towards this.These results using generic health measures fit well with results from other studies reporting effects on physical and psychological health measured using non preference-based instruments. Thus, several studies report significant associations between current intimate partner violence and physical and/or psychological symptoms after adjustment for lifestyle factors ,33. An aGeneric preference-based HRQOL measures are increasingly used to describe population health status and to assess outcome from health care interventions. The major advantage of such measures is the opportunity to compare scores across groups with very different health problems. Preference-based measures also allow interventions to be compared in terms of their social value.The main strength of the study is its basis in a large nationally representative population sample using paper-based questionnaires to assess exposure to violence. This approach may be less susceptible to underreporting of violence due to embarrassment and social stigma than for example data collection through personal interviews .Our definition of exposure to physical violence was fairly general being based on yes/no responses to questions about specific types of violence included in national health surveys. We explored regression models that included the different subtypes of violence reported in the surveys and found that more severe types of violence tended to have greater impact on HRQoL, but these analyses relied on relatively few observations (data not reported).Respondents in the 2000 survey also reported frequency of exposure to physical violence within the last year. However, inclusion of a frequency variable did not improve the fit of the regression models and the estimated parameter was not significant. It was unexpected that the application of more detailed description of violence exposure (type and frequency of violence) did not improve the regression models, as severity and frequency of violence would be expected to influence HRQoL. This finding may be due to a weak linear association between frequency of violence exposure and HRQoL deterioration.Although the study population was sampled to be representative of the general population, the low response rate for parts of the data collection may be a concern. As the prevalence of exposure to violence was relatively low (5-6%) in the study population as a whole, the sample of exposed individuals was limited. The statistical power for subgroup analysis was especially weak in the longitudinal analysis, where only few of the participants reported violence exposure at both survey points.As in previous studies -37 the EThis study found that respondents exposed to physical violence had lower score indexes on both the EQ-5D and the SF-6D compared with the non-exposed. Respondents who reported exposure to violence in both 2000 and 2005 reported lower HRQoL than individuals who only reported exposure in one of the surveys. The results of this study provide evidence for an association between exposure to physical violence and reduction in health-related quality of life.The authors declare that they have no competing interest.This study was developed as part of a long-term collaboration between KHL, HBH, MK and JS. The first draft of the manuscript and the statistical analyses were devised by JS and discussed with KHL, HBH and MK. All authors contributed to the contents. CG revised the manuscript. All authors read and approved the final manuscript.


The aim of the study was to find a simple intravenous glucose tolerance test (IVGTT) that can be used to estimate insulin sensitivity.bw (glucose uptake) obtained during a hyperinsulinemic euglycemic glucose clamp. Plasma glucose was used to calculate the volume of distribution (Vd) and the clearance (CL) of the injected glucose bolus. The plasma insulin response was quantified by the area under the curve (AUCins). Uptake of glucose during the clamp was corrected for body weight (Mbw).In 20 healthy volunteers aged between 18 and 51 years  comparisons were made between kinetic parameters derived from a 12-sample, 75-min IVGTT and the Mbw. Algorithms based on the slope of the glucose-elimination curve (CL/Vd) in combination with AUCins obtained during the IVGTT showed statistically significant correlations with Mbw, the linearity being r2 = 0.63-0.83. The best algorithms were associated with a 25-75th prediction error ranging from -10% to +10%. Sampling could be shortened to 30-40 min without loss of linearity or precision.There was a 7-fold variation in MSimple measures of glucose and insulin kinetics during an IVGTT can predict between 2/3 and 4/5 of the insulin sensitivity. The best established methods of measuring insulin resistance are the hyperinsulinemic euglycemic glucose clamp and the intravenous glucose tolerance test (IVGTT), of which former is the "gold standard" -3. TheseThe aim of this project is to evaluate a simplified IVGTT test that lasts for 30, 40 or 75 min. This test is less labour-intensive than both the glucose clamp and the conventional IVGTT. Analysis of the data is based on a comparison between the "strength" of the insulin response and the elimination kinetics of glucose. A commonly used expression for the "strength" of a physiological factor is the area under the curve (AUC), which was applied here on insulin, while the slope of the elimination curve for glucose served to quantify the "effect".The hypothesis was that the test could predict insulin resistance with the same or higher precision than the "minimal model" (MINMOD) which is typically based on a longer IVGTT and quite demanding mathematically ,7. We asTwenty non-obese healthy volunteers, 8 females and 12 males, aged between 18 and 51  years and with a body weight of 49-88  kg, were studied. None of them had any disease requiring medication, and routine blood chemistry confirmed the absence of metabolic disease . In the opposite arm an intravenous catheter was inserted into the left antecubital vein for insulin and glucose infusion.-2 · min-1  was infused along with 20% dextrose . Baseline blood samples were drawn and the euglycemic hyperinsulinemic clamp was initiated by infusion of a bolus dose of insulin for 4 minutes followed by a step-wise increase in glucose for 10 min. The glucose infusion rate was adjusted to keep the subjects' blood glucose level constant at 5 mmol/L on the basis of arterialized samples withdrawn every 5 min from the dorsal hand vein catheter . The change in prediction error obtained by restricting the analysis period from 75 to 40 and 30 min was tested by Friedman's test. All reported correlations were statistically significant by P < 0.05.The results were presented as mean and standard deviation (SD) and, when there was a skewed distribution, as the median .Consistently weaker correlations were obtained on correcting Mbw with a linearity of only r2 = 0.41  and the G/I ratio (r2 = 0.39) [This key algorithm has the same construction as "Quicki" which uses only the baseline values of plasma glucose and insulin. The original "Quicki" equation correlated with M1 Figure  which wabw with the insulin sensitivity as obtained by "minimal model analysis" (MINMOD) of the IVGTT data  obtained by MINMOD indicated that the insulin concentration at the effect site was highest at 18 min (13-33) min.Weaker correlations were also obtained when comparing Met al. [bw with a linearity of r2 = 0.54 for the period 0-40 min. Logarithm-transformation of Tura's surrogate measure for insulin sensitivity increased r2 to 0.65.The recently published equation by Tura et al.  correlatAnother equation applied the parameters of the glucose kinetics directly and might therefore be easier to handle Table .2 = 0.83 for the relationship between the IVGTT and Mbw .The overall linear correlation between the AUC for C-peptide and insulin was rbw of the glucose clamp (control) with minor mathematical variations of two key algorithms based on data derived from a short IVGTT. Any of them may be used as substitutes for a glucose clamp in healthy volunteers, although some offer stronger linearity and a smaller prediction error than others.The present study searched for an approach to estimate insulin sensitivity that requires only minimum of resources. The results are presented as a number of regression equations that compare MThe first of the key algorithms, shown on top of Table CL/Vd, and the insulin "pressure", AUCins, in a multiple regression equation. This approach could explain up to 83% of the inter-individual variability in Mbw  ,3 and thin level .bw for the steady state insulin level in plasma to yield the Mbw/I ratio, although this is often done. The high concentration of insulin at the effect site at the end of a glucose clamp probably changes CL very little for a large increment in plasma insulin. Correcting Mbw for steady state plasma insulin also resulted in poorer correlations vis-à-vis the IVGTT.The saturation kinetics makes it questionable to correct Mins when estimating Mbw from an IVGTT test. Although being a logical and commonly used correction, disregarding the baseline strengthened the correlations in the present study. Inhibition of the endogenous glucose production taking place early during the IVGTT is likely to make the insulin concentration below baseline govern the disposition of both the exogenous and the endogenous glucose later during the test. Differences in the mathematical correlations between the glucose clamp and the IVGTT were fairly small, however, and we therefore conclude that correcting for baseline insulin can be done, but is not essential.Likewise, one may question whether baseline insulin should be subtracted from AUCet al. [The precision by which our 12-sample IVGTT could predict insulin sensitivity stands out favourably in comparison with other and more complex approaches, as presented in a review by Borai 2 = 0.34 found here [CL and Vd, which makes them less sensitive for a reduction of sampling time and/or sampling intensity. With 12 samples, CL and Vd were estimated with the standard errors that averaged less than 10% (data not shown).A previous study of MINMOD based on a series of 25 blood samples showed a linearity to the glucose clamp that was quite similar to the rund here . The newet al. [ins with SI and Mbw in a retrospective analysis of studies comprising both volunteers and diabetic and postoperative patients who had undergone a frequently sampled 50-min IVGTT and a conventional 2-hour glucose clamp. Good correlations between these indices of insulin sensitivity were claimed for all subgroups. The basic equation used is quite similar to the one we propose on the top of Table 10log of AUCins and corrected this area for the group average SI value. They also divided the expression by the sampling time, which we find questionable since plasma insulin but not KG decreases with time. This fact must be handled by using a unique equation for each sampling time, as in Tables Tura et al.  recentlyThe present study suggests two key algorithms, together with various modifications thereof, that may be used to estimate insulin sensitivity based on data derived from a short IVGTT performed in healthy volunteers. In a subsequent study, these algorithms will be validated in the pre- and postoperative settings. Our interest in this topic stems from a wish to study insulin resistance during surgery. Virtually all non-diabetic patients develop transient type 2 diabetes as a part of the stress response to surgery ,5. Too l2 = 0.63-0.83) with the insulin sensitivity as obtained by the glucose clamp technique in healthy volunteers.The ratio of the slope of the glucose elimination curve and the AUC for plasma insulin during a short IVGTT showed a strong linear correlation (rCL: clearance; IVGTT: intravenous glucose tolerance test; MINMOD: minimal model analysis; Vd: volume of distribution; T1/2: half-life.AUC: area under the curve; The authors declare that they have no competing interests.RH provided the study idea, made the calculations, and wrote the manuscript. SL and FL assisted during the experiments. TN wrote the ethics application and arranged for the experiments.


Protein-protein interactions (PPIs) are frequently mediated by the binding of a modular domain in one protein to a short, linear peptide motif in its partner. The advent of proteomic methods such as peptide and protein arrays has led to the accumulation of a wealth of interaction data for modular interaction domains. Although several computational programs have been developed to predict modular domain-mediated PPI events, they are often restricted to a given domain type. We describe DomPep, a method that can potentially be used to predict PPIs mediated by any modular domains. DomPep combines proteomic data with sequence information to achieve high accuracy and high coverage in PPI prediction. Proteomic binding data were employed to determine a simple yet novel parameter Ligand-Binding Similarity which, in turn, is used to calibrate Domain Sequence Identity and Position-Weighted-Matrix distance, two parameters that are used in constructing prediction models. Moreover, DomPep can be used to predict PPIs for both domains with experimental binding data and those without. Using the PDZ and SH2 domain families as test cases, we show that DomPep can predict PPIs with accuracies superior to existing methods. To evaluate DomPep as a discovery tool, we deployed DomPep to identify interactions mediated by three human PDZ domains. Subsequent in-solution binding assays validated the high accuracy of DomPep in predicting authentic PPIs at the proteome scale. Because DomPep makes use of only interaction data and the primary sequence of a domain, it can be readily expanded to include other types of modular domains. Intrinsic specificity determinants, such as the amino acid sequence of a domain and the characteristics of its ligand-binding site, are context-independent because they are retained even in the isolated domains in silico models to predict novel PPI events Cellular signal transduction is regulated by the formation and dissolution of specific protein complexes. These complexes is often mediated by modular protein-interaction domains such as the Src homology 2 (SH2) or 3 (SH3) and the Post-synaptic density protein, Disc large, Zonula occludens-1 (or PDZ) domain that are capable of binding partner proteins via short peptide motifs in silico prediction of binding partners for that domain or for other members of the same domain family. Scansite et al.et al.Intrinsic specificity information of a domain, obtained from sequence analysis and/or experimental data on domain-peptide binding, may be harnessed for While the above methods make use of specificity and sequence information for PPI prediction, other approaches exploit the binding site characteristics of the relevant domain-peptide complex structures. For instance, a position-specific domain-ligand-contact frequency matrix could be constructed from the contacting residues in the corresponding domain-peptide complex structures To overcome limitations of the existing methods for domain-based PPI prediction, we developed DomPep. Unlike previous methods that group related domains according to a single parameter , DomPep identifies specificity-similar domains by combining two parameters, namely DSI and Position-Weighted-Matrix (PWM) distance. PWM, represented by the sequence logo of the bound peptides Using this new strategy of domain classification, we have built DomPep prediction models for the SH2 and PDZ domains that differ significantly in specificity. The accuracy of prediction in each case was benchmarked against experimental data DomPep employs both the DSI and PWM distance to assess the similarity in specificity for two domains. The PWM of a domain is a matrix  are used to calculate the PWM distance (in vitro and in vivo), the specificity-similar domains to a query domain are identified. These domains are iteratively grouped together with the query and the binding data for each group of the domains are employed to construct prediction models. These models are compared, using leave-one-out cross-validation, with the one constructed using the peptides of the query itself and the model with the best accuracy is retained as the final model for the query. The same procedure is repeated for every domain for model construction. We employed the SVM algorithm to build the DomPep models DomPep comprises two modules – model construction and PPI prediction . The moddistance . Domain The utility of the DomPep was demonstrated on the PDZ and SH2 domain families that have distinct specificities and for which comprehensive binding data are available from large-scale proteomic analysis and conventional experiments d cutoff of 100 µM was applied, 731 positive interactions and 1,361 negative interactions were identified that involved 85 PDZ domains and 181 peptides d cutoff was adopted. The five C-terminal residues of the bound peptides were considered in our analysis as they were shown to be the major specificity-determinants in PDZ binding We analyzed the specificity of PDZ domains based on published data. MacBeath and colleagues recently conducted a study to characterize the specificity of 157 mouse PDZ domains using 217 peptides representing potential physiological PDZ-binding sites We first investigated the relationship between LBS and DSI. To ensure that the calculation for LBS and subsequently, for PWM distance was accurate, PDZ domains with fewer than 10 experimentally verified ligands were omitted . As showTo further explore the relationship between DSI and binding specificity, we calculated the PWM distances see . PWM disTo characterize the relationship between domain sequence identity and ligand-binding specificity, we plotted the percentile distribution of different DSI groups against the PWM distance. As shown in PDZ domains with 10 or more binding peptides identified from the arrays were analyzed in pairs to generate the corresponding DSI, PWM distance and LBS values. We used the corresponding LBS value to calibrate DSI and PWM distances. To this end, we arbitrarily set LBS = 0.7 as the cutoff to assign two PDZ domains similar (when LBS ≥ 0.7) or dissimilar (when LBS<0.7). This cutoff value was used, in turn, to determine the threshold values for DSI and PWM distance . In the We compared DomPep to two methods developed recently by MacBeath and coworkers −4, Wilcoxon signed-rank test; −4). Therefore, we concluded that the DomPep strategy leads to improved prediction accuracy.The training set was previously employed to construct both the MDSM and SPSSM models −3, Ideally a prediction method for domain-ligand interactions should be capable of identifying binding partners not only for domains included in the training set but also for family members not included in training. We therefore compared the performance of DomPep with that of SPSSM, a method that can be used for predicting binding partners of an untrained PDZ domain http://netphorest.info/and used to evaluate the accuracy of DomPep in predicting SH2-peptide ligand interactions. We selected peptide sequences corresponding to residues from positions –2 to +4 with respect to the phosphotyrosine (pY) because these residues are sufficient in conferring specificity to most SH2 domains studies The specificity of SH2 domains was systematically analyzed by probing an array of 6,200 phosphotyrosine-containing peptides respectively with 66 human SH2 domains We further investigated the relationship between PWM distance and LBS for SH2 domain pairs grouped according to DSI . A reverSimilar to PDZ domains, we considered SH2 pairs with LBS≥0.7 as having similar specificity. This threshold was used to determine the cutoffs for DSI and PWM distance, respectively. According to the above analysis , LBS≥0.7We evaluated the performance of SH2 DomPep models by comparing them to Netphorest The training set was based on the published SH2 domain-peptide array data Several differences between the two methods may have contributed to the improved performance of DomPep over NetPhorest for the eight SH2 domains tested. For example, the two methods employ different strategies for the identification of specificity-similar domains. Furthermore, while DomPep randomly selected negative peptides of the training set from the Swiss-Prot protein sequence database, NetPhorest used positive peptides of other domains with small DSIs as negative training peptides for the query domain . We found that the difference in performance originated largely from the different strategies by which the negative peptide training set was selected. DomPep showed comparable performance to NetPhorest when the same negative peptide sets were used as in the latter (data not shown). This is consistent with our observation  that twoin-vitro array binding data that may contain non-specific binding events.Because DomPep compared favorably to MDSM in identifying ligands for a PDZ domain included in the training set and to SPSSM in identifying binding peptides for an untrained PDZ domain, we next tested it whether it could reliably PPIs mediated by a PDZ domain on the genome scale. To this end, we employed three DomPep models to predict interactions between the Scrib PDZ domains and human proteins from the Swiss-Prot protein database. The three prediction models, which were generated for the mouse Scrib PDZ-1, PDZ-2 and PDZ-3 domains, were trained with 6, 5 and 30 positive peptide, respectively . Searchihttp://lilab.uwo.ca/DomPep.html) to predict binding ligands for the PDZ and SH2 domains . LBS is defined as the percentage of peptides commonly selected by a domain pair ij is the number of peptides selected by both domains i and j, BiNBj is the number of peptides that bind to domain i but not to domain j. LBS ranges from 0 to 1, where “1” indicates that both domains have the same set of binding and non-binding peptides, or identical specificity. This formula, suitable for processing the MacBeath dataset on the PDZ domain, needs to be modified to accommodate the SH2 domain-peptide array binding data used in NetPhorest Where BAccording to this formula, the LBS value for the ABL1 and ABL2 SH2 domains increases to 1  program SVMlight with the conventional linear kernel to build prediction models Peptide arrays were synthesized following established protocols Figure S1Phylogenetic tree of the human PDZ Domains. 5 pairs of domains with similar specificities selected as example are shown with different colors. They are the first PDZ domain in protein ZO2 (ZO2/1) and HtrA3 with SI = 15% (pink), DLG3/2 and MAGI3/2 with SI = 17% (grey), SHANK1 and PDZK1/1 with SI = 23% (blue), MAGI1/5 and MAGI3/4 with SI = 24% (green), LRRC7 and SCRIB/2 with SI = 36% (red). The first three pairs are identified by DomPep which have BS>0.7 based on PDZ domain arrays (TIF)Click here for additional data file.Figure S2Respective binding profiles for the SH2 domains from HSH2D (A), GRB7 (B), GADS (C) and for control GST (D) on arrays of phosphotyrosine-containing peptide ligands. These peptides are derived from the PhosphoSite database and were predicted to be highly connected to the four SH2 domains using SMALI program (TIF)Click here for additional data file.Table S1Comparison of DomPep with MSMD on predicting PDZ domain-ligand interactions.(DOC)Click here for additional data file.Table S2Mus musculus.Comparison of DomPep with SPSSM for ability to predict PDZ domain-ligand interactions for PDZ domains in other species than (DOC)Click here for additional data file.Table S3Comparison of DomPep with Netphorest with ANN models on predicting SH2 domain-ligand interactions.(DOC)Click here for additional data file.Table S4Peptides tested for binding the SH2 domains of GADS, GRB7 and HSH2D in the peptide array experiment.(DOC)Click here for additional data file.


Plasmodium DNA. However, prior purification of genomic DNA from blood is necessary since PCR inhibitors and quenching of fluorophores from blood prevent efficient amplification and detection of PCR products.Real-time PCR is a sensitive and specific method for the analysis of Plasmodium DNA directly from blood. Whole blood from clinical samples and dried blood spots collected in the field in Colombia were tested.Reagents designed to specifically overcome PCR inhibition and quenching of fluorescence were evaluated for real-time PCR amplification of ® Green dye and 5% blood volume in the PCR reaction. Plasmodium DNA was detected directly from both whole blood and dried blood spots from clinical samples. The sensitivity and specificity ranged from 93-100% compared with PCR performed on purified Plasmodium DNA.Amplification and fluorescence detection by real-time PCR were optimal with 40× SYBRThe methodology described facilitates high-throughput testing of blood samples collected in the field by fluorescence-based real-time PCR. This method can be applied to a broad range of clinical studies with the advantages of immediate sample testing, lower experimental costs and time-savings. Plasmodium DNA using specific reagents and platforms, such as real-time PCR, provide far greater sensitivity, but are not yet usable at the point-of-care. However, these methods have important applications in clinical research studies that involve the analysis of blood samples collected in the field, including genotyping parasite populations and monitoring drug resistance, genetic characterization of vaccine candidates, anti-malarial efficacy trials and surveillance programs [The availability of sensitive diagnostic tools for malaria is critical to ensure appropriate treatment for patients and to preserve the lifespan of effective anti-malarials. In the field, the most common tools for malaria diagnosis are microscopy and rapid detection tests (RDTs), which are performed directly from the blood sample. Molecular methods that amplify and detect programs -3.The performance of molecular tests largely depends on the quality of the parasite DNA. Highly purified DNA requires laborious sample processing and costly reagents, kits or equipment, whereas cruder extraction methods often produce DNA that is insufficiently pure for downstream testing. The presence of PCR inhibitors from the blood, such as haemoglobin, reduces the efficiency of the molecular reaction and compromises sensitivity ,5. HowevTaq mutants in combination with an enhancer cocktail within the PCR master mix for optimal amplification and fluorescence detection [Plasmodium DNA by real-time PCR from raw patient samples and from dried blood spots collected in the field.One of the major advances in molecular diagnostics is the integration of fluorescence-based detection of DNA in real-time PCR. This poses a new challenge to direct PCR from blood as fluorophores are quenched in the presence of haemoglobin. Amplification can be achieved, but the product is not detected. One approach to overcome the quenching effect uses inhibitor-resistant etection ,8. With Plasmodium falciparum 3D7 in vitro culture [Plasmodium falciparum (n = 39), Plasmodium vivax (n = 23), Plasmodium ovale (n = 9), and Plasmodium malariae (n = 2). Blood samples were collected in EDTA or citrate tubes, stored at -20°C and thawed at 4°C prior to testing. Genomic DNA from Plasmodium knowlesi was obtained from MR4.DNA for optimization of the PCR from blood was purified from  culture  using DN culture . Of thes culture . Of the Plasmodium DNA, DNA was extracted using Chelex ® 100 (Sigma) and tested by nested PCR according to the protocol described by Snounou et al [Plasmodium falciparum, 18 were positive for Plasmodium vivax and 10 were negative by microscopy and nested PCR. For real-time PCR, 1.5 mm diameter blood spots were excised from filter papers using a sterilized hole punch. Spots were tested directly or soaked first for 3 minutes in sterile distilled water at 50°C. This study was approved by the Health Research Ethics Board at the University of Alberta and Comité de Bioética, Sede de Investigación Universitaria, Universidad de Antioquia (Acta 07-32-126) in Colombia.Dried blood spots were prepared on 3 MM paper (Whatman) from blood samples collected from patients who attended the malaria clinic at Puerto Libertador, in Cordoba, Colombia between 2008 and 2010. Filter papers were dried at ambient temperature in the field, shipped to Medellín and stored in plastic bags at -20°C. Positive patients were symptomatic for malaria and had infections ranging from 120-39,920 parasites/μL  by microscopy performed on thick smears in the field. To confirm the presence of ® Green , and blood at 5-10% of the total reaction volume. To detect the 18S rRNA gene from all species of Plasmodium, primers reported by Kamau et al [Plasmodium falciparum  with reduced primer-dimer interactions was substituted for the rOVA reverse primer. Primers were used at 400 nM except the Plasmodium malariae set, which was used at 800 nM. Real-time PCR was performed on the ABI 7500 Fast system  with the following conditions: 95°C for 10 min, 40 cycles of 95°C for 30 sec, 58°C for 1 min and 72°C for 1 min. This was followed by the default dissociation cycle for melt curve analysis.PCR was performed as described  in a 20 au et al  , Plasmoprimers; ,15). A nAll samples were tested in triplicate. For whole blood samples, the baseline was set to cycles 3-15 and the threshold was set to 20,000 fluorescence units. Samples were considered negative if the cycle threshold (Ct) value was greater than 35 or if the melt curve did not align with the positive control. For dried blood spots, the baseline was set to 5-15 and the threshold was set to 250,000 to overcome the high background fluorescence from the filter paper itself. The amplification curves generated were sometimes shallow with some variability among replicates. Samples were considered positive when at least two of the three replicates had amplification curves that crossed the threshold before cycle 35 and had the expected melt curve profile. Panel samples were tested in a blind fashion.® Green dye was tested to determine the optimal concentration for detection of Plasmodium DNA directly from blood. PCR master mix with final concentrations of 10×, 20× and 40× SYBR® Green was added to blood spiked with gDNA from Plasmodium falciparum 3D7. Two blood volumes were tested: 5% or 10% of the PCR reaction volume  was negligeable at this concentration. In subsequent experiments, 40× SYBR® Green and 5% blood volume were selected as the optimal reaction conditions.The melt curve profiles were also affected by the concentration of SYBRPlasmodium. Examples of amplification and melt curves for each species, including blood spiked with gDNA from Plasmodium knowlesi, are shown in Figure Plasmodium falciparum (9.2% parasitaemia) was serially diluted and spiked into uninfected blood  and tested under the same reaction conditions used for whole blood with the Plasmodium screening primers. Spots excised from filter paper were either directly added to the PCR master mix or washed briefly in water. Amplification of Plasmodium DNA was detected in both the neat and washed filter papers, although the amplification curves for the neat samples had lower background and tighter replicates compared with the washed samples (Figure An important application of this methodology is the direct amplification of s Figure . The mels Figure . Given tPlasmodium knowlesi spiked into uninfected blood. These samples had been tested previously by real-time PCR performed on purified DNA using the method implemented at the Alberta ProvLab for malaria confirmation. This method uses a TaqMan probe and a different set of primers to screen for Plasmodium [Plasmodium knowlesi sample was positive, giving a total of 74 positive and 26 negative samples in the panel. In the real-time PCR test directly from blood samples, 69 samples were positive and 31 were negative for Plasmodium (Table The sensitivity and specificity of this method for whole blood and blood spots was determined using blind panels of clinical samples Table . For whoasmodium . AccordiMicroscopy results were available for 95/100 samples, 57 of which were positive with parasitaemias ranging from < 0.01% to 9.2%. Of these, 25 samples had an undetermined parasitaemia < 0.1%. Four of the false negative samples in the direct blood panel were from asymptomatic refugees and the fifth was from an acute malaria sample. All five samples were negative by microscopy.et al [For dried blood spots, patient samples collected in the field in Colombia were tested in a blind panel that consisted of 38 positives and 10 negatives (Table et al . The panThis report describes a methodology for DNA amplification directly from blood that is compatible with fluorescence detection by real-time PCR. Although this method cannot be used directly in the field, it facilitates the analysis of blood samples collected in field studies. Clinical trials on antimalarial or vaccine efficacy and prevalence or surveillance studies are just a few examples of applications of this test for high throughput analysis of blood samples. Either blood spots or whole blood samples can be transported to the lab and immediately tested by real-time PCR without the need for DNA extraction.Plasmodium screening reaction [Plasmodium DNA was achieved with these primers with good sensitivity, sporadic non-specific amplification was observed in the negative controls. Use of the primers recently published by Kamau et al. [This method is particularly suited to studies seeking a rapid confirmation of malaria. The sensitivity is similar to real-time PCR performed on purified DNA and specificity is achieved through primer design to reduce non-specific amplification. At the Alberta ProvLab, malaria diagnosis is confirmed by a TaqMan real-time PCR assay that uses a previously published primer set for the reaction . The priu et al.  reduced ® Green required to overcome the quenching effect from the blood results in higher background fluorescence and the threshold was set at 20,000 or greater for these assays. With blood spots, fluorescence from the filter paper itself resulted in even higher background. Therefore, preliminary testing with blank filter papers and spots of uninfected blood is highly recommended to define the Tm and expected melt curve for a positive sample relative to background. Once this is established, the interpretation of results is straight-forward. Another important consideration is that the real-time PCR must be performed in tubes and not capillaries. Preliminary testing in capillaries on the LightCycler instrument from whole blood samples failed to generate amplification curves. It is hypothesized that during the course of the PCR, dried blood accumulates along the walls of the capillaries and blocks the emission of fluorescence.In general, the background fluorescence in this assay is higher than in other real-time PCR assays; the high concentration of SYBRPlasmodium DNA in blood, the reagents for direct PCR can also support the detection of other blood-borne pathogens. These reagents have previously been used to detect herpes simplex virus 2 and other viruses spiked into blood [Streptococcus pneumoniae, Salmonella enterica, Staphylococcus aureus, Escherichia coli and Haemophilus influenzae as the major causes of bloodstream infections in African children [In addition to the detection of to blood . In malachildren . The PCRIn addition to the practical use of this assay for the qualitative analysis of field samples, the ability to perform real-time PCR for malaria directly from blood can be exploited in new technologies to bring molecular diagnostics for malaria to the point-of-care . A numbePlasmodium infections.The methodology described here supports the amplification of gDNA by fluorescence-based real-time PCR directly from blood samples infected with malaria. The advantage of this assay is the elimination of the DNA extraction step, thereby facilitating high-throughput analysis of samples collected in the field using state-of-the-art tools for the detection and characterization of The authors declare that they have no competing interests.BJT and KAM performed the real-time PCR testing of blood samples and contributed equally to this work. AM provided dried blood spots from samples collected in the field and EA and OMA tested these samples by nested PCR. SKY and BJT designed the experiments and SKY wrote the manuscript. All authors read and approved the final manuscript.