Total, single repetition and contraction-phase specific (concentric and eccentric) time-under-tension (TUT) are important exercise-descriptors, as they are linked to the physiological and clinical response in exercise and rehabilitation.To investigate the validity and reliability of total, single repetition, and contraction-phase specific TUT during shoulder abduction exercises, based on data from a stretch-sensor attached to an elastic exercise band.A concurrent validity and interrater reliability study with two raters was conducted. Twelve participants performed five sets of 10 repetitions of shoulder abduction exercises with an elastic exercise band. Exercises were video-recorded to assess concurrent validity between TUT from stretch-sensor data and from video recordings (gold standard). Agreement between methods was calculated using Limits of Agreement (LoA), and the association was assessed by Pearson correlation coefficients. Interrater reliability was calculated using intraclass correlation coefficients (ICC 2.1).Total, single repetition, and contraction-phase specific TUT – determined from video and stretch-sensor data – were highly correlated (r>0.99). Agreement between methods was high, as LoA ranged from 0.0 to 3.1 seconds for total TUT (2.6% of mean TUT), from -0.26 to 0.56 seconds for single repetition TUT (6.9%), and from -0.29 to 0.56 seconds for contraction-phase specific TUT (13.2-21.1%). Interrater reliability for total, single repetition and contraction-phase specific TUT was high (ICC>0.99). Interrater agreement was high, as LoA ranged from -2.11 to 2.56 seconds for total TUT (4.7%), from -0.46 to 0.50 seconds for single repetition TUT (9.7%) and from -0.41 to 0.44 seconds for contraction-phase specific TUT (5.2-14.5%).Data from a stretch-sensor attached to an elastic exercise band is a valid measure of total and single repetition time-under-tension, and the procedure is highly reliable. This method will enable clinicians and researchers to objectively quantify if home-based exercises are performed as prescribed, with respect to time-under-tension. In the rehabilitation of patients, home-based exercises using elastic exercise bands are often prescribed after a thorough initial instruction by the prescribing physician or physiotherapist –3. ElastWe recently showed that automatically stored data from a stretch-sensor attached to a standard elastic exercise band could be used to accurately identify training adherence and quality of shoulder-abduction strength training . In the Total TUT refers to the total time of all concentric, quasi-isometric and eccentric contraction-phases in a single training set . TogetheThe objective of the study was therefore to investigate the validity and reliability of total, single repetition and contraction-phase specific TUT during shoulder abduction exercises, based on data from a simple set-up that included a stretch-sensor attached to a standard elastic exercise band.All participants were provided with verbal and written information about the procedures of the study, and written informed consent was obtained in accordance with the Declaration of Helsinki. The Ethics Committee in the North Denmark Region and the Danish Data Agency approved the study 2012-2410). The participant in 012-2410.The study was a concurrent validity and interrater reliability study. The study investigated if it were possible to determine total TUT, single TUT and contraction-phase specific TUT during shoulder abduction exercises, based on data recordings from a stretch-sensor attached to an elastic exercise band. Video recordings were used as a gold standard, and recorded concordantly, so each rater could determine, from both video recordings and stretch sensor data, the total TUT, single repetition TUT and contraction-phase specific TUT. The reporting of the study follows the Guidelines for Reporting Reliability and Agreement Studies (GRRAS) .A convenience sample of 12 healthy volunteers from the local hospital staff was recruited. They were between 21 and 29 years of age and free of upper extremity symptoms.Two raters with different clinical and technological experience using elastic band exercise were recruited. Hence, we considered the two raters representative of a larger population of raters who, in a future setting, would use the stretch-sensor equipment being investigated. Rater 1 was a male research physiotherapist with three years of research experience and 10 hours of prior experience of rating stretch-sensor data. Rater 2 was a female physiotherapist with three years of clinical experience and no previous experience in rating stretch-sensor data. Rater 1 already had experience in rating video recordings and stretch-sensor data and received no additional practice. Rater 2 was given 30 minutes of practice in rating stretch-sensor data from participants who were not part of the current study sample.The elastic stretch-sensor was based on technology designed by Danfoss PolyPower  and custom-made for the application. It acts as an elastic capacitive material that is stretchable in one direction. To measure the change in capacitance of the stretch-sensor, an electrical circuit was built with a timer. The circuit was built in such a way that a change in capacitance of the stretch-sensor corresponded to a change in the timer frequency, which allowed us to measure how much the sensor was being stretched . The seParticipants were instructed in performing shoulder abduction exercises from 0 to 90 degrees abduction. They were told to perform 10 repetitions at a self-determined speed, meaning there was no restriction to perform either of the contraction-phases at a predetermined speed or at a relative loading (repetition maximum). However, we secured that no slack was present in the exercise band at 0 degrees abduction meaning that the tension in the elastic band increased throughout the shoulder abduction with a peak at the end of the abduction due to the elastic tension. This approach was chosen to ensure a wide range of different speeds of exercises. All participants performed five sets of 10 repetitions with a 2-minute break between each set. The exercises were performed in front of a white wall, and recorded with a 50mm lens on a Canon 5D Mark II mounted on a tripod, Rating of the stretch sensor data was done by analysing data from the stretch-sensor with Matlab 2011a . A custom-written Matlab programme transformed the stretch-sensor data into an image showing the stretch of the sensor as a function of time (see example in The free software programme V1 Home Basic (www.v1golfacademy.com) was used for playback of the video recordings. The software programme allows for frame-by-frame playback of the video recordings and thereby a precise determination of contraction-phase specific time points. The starting point of the concentric phase was defined as the first frame where the hand holding the elastic exercise band moved in the direction of abduction. The end of the concentric phase (the start of the quasi-isometric phase) was defined as the first frame where the hand no longer moved. The end of the quasi-isometric phase (the start of the eccentric phase) was defined as the first frame where the hand moved in the direction of adduction. The end of the eccentric phase was defined as the first frame where the hand no longer moved.The following variables were determined from both video and stretch sensor data: (I) Total TUT, defined as the sum of the concentric, quasi-isometric and eccentric TUT during each set (10 repetitions) , (II) siThis analysis was performed to determine if total TUT, single repetition TUT and contraction-phase specific TUT could be determined validly from data recorded by the stretch-sensor. TUT determined from the video recordings was used as the gold standard. One rater (Rater 1) determined the total, single-repetition and contraction-phase specific TUT from all five exercise sets from three randomly chosen participants, from both video recordings and stretch-sensor data. This corresponded to 15 exercise sets consisting of 150 contraction-specific phases per method for both concentric and eccentric phases. The rater was blinded as to which subject he was rating and which of the five exercise sets he was rating. Firstly, all rating of video recordings was done, followed by rating of the stretch-sensor data. During rating of the video recordings and stretch-sensor data, the rater was randomly presented with one of the five exercise sets from the three different participants, until all 15 exercise sets had been rated.For the statistical analyses of validity, paired t-tests were used to test for systematic bias between the two methods. Systematic bias is reported as the mean difference between methods and graphically presented as Bland-Altman plot in This analysis was performed to determine the interrater reliability and agreement between independent raters, rating the stretch-sensor data. Raters 1 and 2 rated all 60 exercise sets from all 12 participants and determined TUT for each of the 600 single repetitions.For the statistical analysis, a two-way random effects model (2.1), single measures, absolute agreement, and intraclass correlation coefficients (ICC) were used to express interrater reliability, while LoA were used to express agreement between raters . LoAs weWe used a non-inferiority sample size calculation to determine the sample-size required for the validity analysis. This approach was chosen, as the primary goal of the validity analysis was to show that there were no clinically relevant differences between total TUT determined from video recordings and stretch-sensor data. The data used for the sample-size calculation were collected in a pilot study. Using a non-inferiority limit of 2 seconds of total TUT  and a standard deviation of 1.5 seconds at 5% significance and 80% power, it was necessary to rate 14 exercise sets. We chose to increase the number of exercise sets to 15, as this enabled us to use all exercise sets from three randomly selected participants. The sample-size for the reliability analysis included 60 data points for total TUT, 60 data points for total concentric and eccentric contraction-phase and 600 data points for single repetition TUT and contraction specific TUT .Total TUT, single repetition TUT and contraction-phase specific TUT-determined from video recordings and stretch sensor data – were highly correlated (r>0.99). Moreover, the agreement between the two methods was high, as the LoA ranged from 0.0 to 3.1 seconds for total TUT (2.6% of mean TUT), from -0.26 to 0.56 seconds for single repetition TUT (6.9% of mean TUT), and from -0.29 to 0.56 seconds for contraction-phase specific TUT (13.2-21.1% of mean TUT), The ICC for interrater reliability for total, single repetition and contraction-phase specific TUT was above 0.99, Both contraction-phase specific TUT and total TUT from video recordings took significantly longer time to calculate compared to calculating TUT when using stretch-sensor data (p<0.0001). Rating the video recordings of 10 repetitions of contraction-phase specific TUT took, on average, 17: 35 minutes. In comparison, it took, on average, 55 seconds to determine total TUT for 10 repetitions from stretch-sensor recordings and 3: 10 minutes to rate contraction-phase specific TUT for 10 repetitions.Currently, there are no tools available to determine time-under-tension during home-based exercises, performed using elastic exercise bands. Such a tool is especially relevant, as patients most often perform home-based exercises alone, without being under the surveillance of the prescribing physician or therapist. Therefore, we investigated if data from a new stretch-sensor attached to a standard elastic exercise band could be used to validly and reliably calculate total TUT, single TUT and contraction-phase specific TUT. The current data show that total and single repetition TUT can be validly and reliably determined from stretch-sensor data. We previously validated the ability of this tool to identify specific exercise scenarios with different ranges of motion and contraction speeds when subjects performed dynamic shoulder abductions at a relative intensity of 12 repetition maximum (RM). Combining TUT with range of motion, and relative intensity determined by a test of repetitions maximum allows for a detailed description of the specific strength training stimulus.For illustration purposes, imagine two subjects, both enrolled in a research study investigating the effectiveness of a home-based strength-training intervention. Previous work indicates that total TUT in addition to concentric and eccentric contractions are important for the intervention to be able to induce a clinical effect ,29. The Both patients adhere to the prescribed programme and relative load, but one patient does perform the exercises too fast; two seconds of concentric phase, no quasi-isometric phase at 90 degrees, and then a one second eccentric phase. After 12 weeks of the programme, both patients will have performed an identical number of exercise sets and repetitions and the patient who adhered to the prescribed TUT will have performed exercises corresponding to a TUT of 8640 seconds (144 minutes). However, the patient who did not adhere to the prescribed TUT, will have performed exercises corresponding to only 37.5% of the total training stimulus and 33.3% of the eccentric stimulus of the patient who adhered. It seems plausible that such variations in training volume (TUT) and training specificity (contraction-phase specific TUT) may lead to differences in the physiological and clinical response even though the same number of repetitions were performed by the two patients ,28–30. IIf instead the patients had trained using the elastic exercise band and stretch-sensor investigated in the current study, the treating physician or physiotherapist could have measured TUT for sets, single repetitions and specific contraction-phases. Afterwards, it would have been possible to calculate how close the measured TUT corresponded to e.g. the prescribed eccentric TUT and thereby quantify adherence as a percentage of the prescribed total eccentric TUT during the programme.Another way of measuring TUT during home-based exercises is to use webcams connected to a computer. This approach has been used in the field of tele-rehabilitation of patients with stroke ,32. HoweAll ratings were done manually, using the definition of concentric, quasi-isometric and eccentric phases, stated in “Methods”, and visualised in A possible solution to increase reliability of the TUT of the eccentric phase could be to apply an automatic algorithm to detect the time spent in each of the three contraction-phases. One could also argue that the achieved degree of reliability and agreement using the manual rating procedure leaves little room for improvement, other than using the same rater for multiple ratings from the same patient. The reason for this is that intra-tester ratings are generally reported to be more reliable than inter-tester rating . HoweverAs a clinical gold standard for measuring TUT, we used video recordings with a sampling rate of 24 Hz. This is lower than the 200 Hz, used by the stretch sensor. It may have introduced some random error in the determination of the start and stopping points of the contraction phases, as the contraction may initiate between two image frames. The position transducers, used by Tran et al.  to measuThe example of the two patients above appearing to demonstrate equal adherence to the exercise programme, even though they display a large difference in total and eccentric TUT during an intervention period, suggests that future research should aim to describe adherence to exercise in more detail. Based on the research conducted in healthy subjects, showing that greater TUT induces a larger physiological stimulus, may hold true for patients as well. Using a stretch-sensor attached to an elastic exercise band would allow for similar studies to be conducted in various patient groups.Data from a stretch-sensor attached to an elastic exercise band can be used to calculate total and single repetition times under tension. The procedure is valid and highly reliable between raters. The current method will enable clinicians and researchers to objectively quantify if home-based exercises are performed correctly with respect to prescribed contraction phases and time-under-tension.Appendix S1(DOCX)Click here for additional data file.


In the past twenty years, codeine-containing cough syrups (CCS) was recognized as a new type of addictive drugs. However, the exact neurobiologic mechanisms underlying CCS-dependence are still ill-defined. The aims of this study are to identify CCS-related modulations of neural activity during the resting-state in CCS-dependent individuals and to investigate whether these changes of neural activity can be related to duration of CCS use, the first age of CCS use and impulse control deficits in CCS-dependent individuals. We also want to observe the impact of gray matter deficits on these functional results.p<0.05, corrected for multiple comparisons). The Barratt Impulsiveness Scale 11 (BIS.11) was surveyed to assess participants' impulsivity trait soon after MR examination. Abnormal clusters revealed by group comparison were extracted and correlated with impulsivity, duration of CCS use, and age of first CCS use. ReHo was diminished in the bilateral medial orbitofrontal cortex (mOFC) and left dorsal striatum in CCS-dependent individuals. There were negative correlations between mean ReHo in the bilateral medial OFC, left dorsal striatum and duration of CCS use, BIS.11 total scores, and the subscale of attentional impulsivity in CCS-dependent individuals. There was a significantly positive correlation between mean ReHo in the left dorsal striatum and age of first CCS use in CCS-dependent individuals. Importantly, these results still remain significant after statistically controlling for the regional gray matter deficits.Thirty CCS-dependent individuals and 30 control subjects participated. Resting-state functional MRI was performed by using gradient-echo echo-planar imaging sequence. Regional homogeneity (ReHo) was calculated by using REST software. Voxel-based analysis of the ReHo maps between controls and CCS-dependent groups was performed using two-sample t tests (Resting-state abnormalities in CCS-dependent individuals revealed in the present study may further improve our understanding about the neural substrates of impulse control dysfunction in CCS-dependent individuals. In the past twenty years, codeine-containing cough syrups (CCS) was recognized as a new type of addictive drugs. Long-term and sustained taking CCS would lead to physical and psychological dependence Previous studies had revealed the behavior, personality and mental were abnormal in CCS-dependent individuals Functional magnetic resonance imaging (fMRI) provided a primary method of mechanism detection of illicit substance abuse in the past two decades. And large body of fMRI studies, administered during the performance of diverse tasks, had suggested the disorganization of the reward, impulse control and memory circuits in illicit substance abuse More recently, the regional homogeneity (ReHo) method Impulsivity is a personality trait that presents in healthy individuals, yet the substance-dependent individuals would show higher impulsivity The first purposes of present study are to identify CCS-related modulations of neural activity in the resting-state by using rs-fMRI and ReHo method, and to investigate their correlations with the duration of CCS use, age of first CCS use and impulsivity traits in CCS-dependent individuals.In addition to the functional deficits, previous studies also indicated illicit substances dependent individuals had lower gray matter volume/density than matched controls This prospective study was approved by the Research Ethics Review Board of the Institute of Mental Health at the Guangdong No. 2 Provincial People's Hospital. Written informed consent was obtained from all subjects. Sixty subjects, including 30 (50%) control subjects and 30 (50%) CCS-dependent individuals participated in this study. The CCS-dependent individuals were randomly selected from the patients seeking treatment at Addiction Medicine Division of Guangdong No. 2 Provincial People's Hospital. Patients were screened based on the DSM-IV criteria from the medical history, along with a urine test and an interview. They regularly used cigarettes and denied any use of psychotropic agents in the month prior to the rs-fMRI scan being performed. Inclusion criteria for the control subjects were lack of diagnosis of substance abuse or dependence. Exclusion criteria for all participants included neurological illness, schizophrenia or bipolar disorder, prior significant head trauma, positive HIV status, diabetes, Hepatitis C, other major medical illness and left-handedness.Impulsivity was assessed using the BIS.11, which is one of the oldest and most widely used self-report measures of impulsive personality traits. This 30-item self-rated scale has three oblique factors: attentional/cognitive, which measures toleration for cognitive complexity and persistence; motor, which measures the tendency to act on the spur of the moment; and nonplanning impulsivity, which measures the lack of sense of the future. Items are rated from 1 (rarely/never) to 4 . To determine overall impulsiveness scores, all items were summed, with higher scores indicating greater impulsivity 2, slice thickness = 4.5 mm and slice gap = 0 mm. Each brain volume comprised 22 axial slices and each functional run contained 240 volumes (8 minutes) , during resting state fMRI scanning, subjects were instructed to close their eyes and keep still as much as possible, and not to think of anything systematically or fall asleep. Sagital structural images  were acquired using a fast field echo (FFE) three-dimensional T1 weighted sequence.MRI data were obtained on a Philips Achieva 1.5T Nova dual MR scanner using a 16 channel NV coil in the Department of Medical Imaging, Guangdong No. 2 Provincial People's Hospital. None of the subjects were taking any medications at the time of the scans. Tight but comfortable foam padding was used to minimize head motion, and ear plugs were used to reduce scanner noise. Rs-fMRI scans were performed by an echo planar imaging (EPI) sequence with scan parameters of repetition time (TR)  = 2000 ms, echo time (TE)  = 50 ms, flip angle  = 90°, matrix = 64×64, field of view (FOV)  = 230×230 mmAfter MR examination, all the participants were asked some questions to verify the degree of their cooperation.http://www.fil.ion.ucl.ac.uk/spm). For each participant, the first ten time points were discarded to avoid transient signal changes before magnetization reached steady-state and to allow subjects to get used to the fMRI scanning noise. The raw data were corrected for temporal shifts between slices, corrected for head motion . Subjects with head motion exceeding 1.0 mm of maximal translation  or 1.0° of maximal rotation through the resting-state run were discarded from further analysis. Moreover, we also performed a two-sample t test on the mean absolute estimated movement parameters (shift and rotation) on all three axes to examine between-group differences in the degree of head motion. Following the motion correction, all data were spatial normalized to the Montreal Neurological Institute (MNI) template (resampling voxel size  = 3×3×3 mm3). Then all images spatially normalized to the MNI template were transformed to Talairach and Tournoux coordinates.The imaging data were mainly preprocessed with SPM8 (http://resting-fmri.sourceforge.net) Further data preprocessing and ReHo analysis were performed with REST software (i is the sum rank of the ith time point (Ri = ij where rij is the rank of the ith time point in the jth voxel); i; k is the number of time series within a measured cluster ; and n is the number of ranks . The KCC value was calculated to this voxel, and an individual KCC map was obtained for each subject. To reduce the influence of individual variations in the KCC value, normalization of regional homogeneity maps was preformed through dividing the KCC among each voxel by averaged KCC of the whole brain. The resulting data were then spatially smoothed with an 8-mm full-width at half-maximum (FWHM) Gaussian kernel to reduce noise and residual differences in gyral anatomy.3) 3D anatomical images of each subject within the brain mask were automatically segmented into gray matter, white matter and cerebrospinal fluid (CSF) by using a DARTEL method In order to investigate the impact of regional brain gray matter deficits on the functional results, we calculated a regional gray matter index of each region showing significant between-group ReHo difference as follows. First, the Talairach-normalized and re-sampled (1 mmp<0.05 (two-sided) was deemed significant.Two-sample t tests were performed to assess the differences in age, duration of education, cigarette smoking, head motion and regional gray matter index between CCS-dependent individuals and control groups, Pearson Chi-Square test was performed to assess the differences in gender between the two groups using SPSS (version 13.0), and a p<0.05 (corrected for multiple comparisons) was considered to show significant differences between the two groups. Threshold correction was done by using the AlphaSim program in AFNI, which applies Monte Carlo simulation to calculate the probability of false positive detection by taking into consideration both the individual voxel probability thresholding and cluster size p<0.05 was obtained by clusters with a minimum volume of 2106 mm3 at an uncorrected individual voxel height threshold of p<0.01. . Moreover, to identify the association between the alteration of ReHo, the duration of cough syrup usage, and age of first cough syrups use. The average ReHo values of all voxels in the reduced areas revealed by voxel-based analysis were extracted separately using the extract time series in REST and were input into SPSS. Then, bivariate correlation was introduced to indicate the relationships between the ReHo values of the significant differences in the CCS-dependent individuals with the duration of cough syrup usage, age of first cough syrups use, and BIS.11 scores, the significance levels were set at p<0.05 .To explore the ReHo differences between the CCS-dependent individuals and controls, a second-level random-effect two-sample t-test was performed on the individual normalized ReHo maps in a voxel-by-voxel manner with head motion as covariate To determine whether the functional results were influenced by the structural deficits, we further examined the between-group differences in mean ReHo using a two-sample t test by taking individual regional gray matter indices as covariates. Additionally, the correlation of the mean ReHo and the duration of cough syrup usage, age of first cough syrups use, and BIS.11 scores in the CCS-dependent individuals were also examined by a bivariate analysis with individual regional gray matter indices as covariate. Finally, the statistical significances were compared with the functional results obtained from the same processes but without accounting for the regional gray matter deficits.p<0.05, corrected; There were no significant differences in age, gender, education, number of cigarette smoked per day, and head motion between the CCS-dependent individuals and controls . Comparep<0.05).Comparison of average BIS.11 scores in the CCS-dependent group and control group was showed in p<0.05/2, Bonferroni correction; p<0.05/2, Bonferroni correction; Significantly negative correlation was observed between the mean ReHo values in dorsal striatum which showed group differences and the duration of CCS use in CCS-dependent individuals and significantly positive correlation was observed between the mean ReHo values in dorsal striatum which showed group differences and the age of first CCS use in CCS-dependent individuals  . There wBilateral medial OFC demonstrated both significant ReHo decreases and gray matter deficits. After controlling for the regional gray matter deficits by statistical analysis , significant bilateral medial OFC ReHo differences  between the two groups and significant correlation of bilateral medial OFC ReHo against the BIS.11 total scores  in the patients group was still observed. However the statistical significance was reduced compared to the results without accounting for the regional gray matter deficits with  for the ReHo differences, and  for the correlation analysis.Given the detection of signal differences between groups in the bilateral medial OFC, a region prone to fMRI signal loss To our best knowledge, this is the first study to investigate the ReHo of brain spontaneous activity in CCS-dependent individuals. Compared with the controls, reduced ReHo was found in prefrontal-striatal system, including bilateral medial OFC  and left dorsal striatum . No increased ReHo was found in CCS-dependent individuals. We further found that the mean ReHo value in bilateral mOFC and left dorsal striatum revealed by group comparison was associated with total BIS.scores and the subscale of attentional impulsivity in CCS-dependent individuals. The mean ReHo value in left dorsal striatum was related to the duration of CCS use, age of first cough use in CCS-dependent individuals, i.e. the longer (younger) the CCS-dependent individuals have been using (start to use) CCS, the lower of the mean ReHo in this region. Most importantly, these results still remain significant after statistically controlling for the regional gray matter deficits. Thus, the present investigation expands our understanding of CCS abuse on the brain function in human.The medial OFC (BA10) is a part of the limbic system which involves ongoing monitoring of the reward value of a reinforcer 2 (D2R) availability in striatum that persisted months after protracted detoxification Dorsal striatum forms a continuous and large mass Taken together, we demonstrate that CCS abuse can destroy the brain function of the addicts as other illicit drugs do, especially for prefrontal-striatal system, including the bilateral mOFC and left dorsal striatum. This may raise community awareness of CCS abuse, and help rectify the false beliefs that the cough syrups are non-addictive and less harmful Consistent with previous studies about illicit drug addiction, the CCS-dependent individuals scored significantly higher on the impulsivity questionnaires when compared with healthy volunteers. It is known that high scores on BIS.11 imply on increased tendency to shift attention quickly causing inappropriately rapid decision making, acting without thinking and failure to consider the future implication of behavior Interestingly, the mean ReHo value in bilateral mOFC showing group differences was negatively correlated with BIS scores in both CCS-dependent individuals and controls, which was consistent to previous studies declaring the mOFC was functional for impulse control. In a structural MRI study, Matsuo et al. demonstrated the lower OFC volume was associated with the higher impulsivity in healthy people We also observed a negative correlation between the mean ReHo in left dorsal striatum  and the subscale of attentional impulsivity in CCS-dependent individuals, which means the lower mean ReHo in left striatal, the more rapid shifts in attention and more impatient with complexity in patients. This finding declared the higher attentional impulsivity directly related with dorsal striatum dysfunction. This phenomenon was also observed in attention deficit hyperactivity disorder (ADHD) patients, who are characterized primarily by the co-existence of attentional problems and hyperactivity. In an rs-fMRI study, Cao et al. also found boys with ADHD showed decreased ReHo in the frontal- striatal-cerebellar circuits A negative correlation was observed between duration of cough syrup use and mean ReHo value in the left dorsal striatum in CCS-dependent individuals. In other words, greater duration of CCS use was associated with relatively greater destruction of local synchronization of spontaneous low-frequency blood oxygenation in this structure. Previous neuroimaging studies related to illicit drug addiction also demonstrated this effect. For example, Yuan et al found that the gray matter density of the prefrontal, temporal and insular was negatively correlated with the duration of heroin use in heroin dependent individuals We also found a modest, but significantly positive correlation between the age of first CCS use and the mean ReHo value in the left dorsal striatum that showed group differences. This meant that the earlier use of CCS, the greater the impact on brain function. This phenomenon was also proved in previous studies in illicit drug addiction, Wison et al revealed subjects who started using marijuana before age 17 had smaller whole brain and percent cortical gray matter than those who started later, and male who started using marijuana before age 17 had significantly higher CBF than others It is important to note here that we took into account the impact of regional gray matter deficits on the functional results (ReHo) shown in the bilateral medial OFC. Numerous studies have demonstrated that addicts have lower gray matter volume/density in the bilateral medial OFC than normal controls Some limitations in our study need to be addressed. Firstly, because the present study is a cross-sectional one, the cause-effect relationships among the variables cannot be rigorously assessed. Secondly, the results of BIS.11, as a self-report scale, may not fully reflect the impulsivity of all the subjects for someone may not answer the questions honestly. Thirdly, the CCS-dependent individuals changed dose of CCS abuse over time, so we cannot assess the relationship between the total amounts and the clinical data directly. In the end, methodological issues concerning the use of ReHo should be considered when interpreting these results.In conclusion, compared with controls, the CCS-dependent individuals exhibit decreased ReHo in the bilateral frontostriatal brain system progressively, which relate to the first age of CCS use and higher impulsivity trait in CCS-dependent individuals. Importantly, these results still remain significant after statistically controlling for the regional gray matter deficits. Thus this resting-state fMRI study suggests that the decreased ReHo of these regions may implicate the underlying pathophysiology in CCS-dependent individuals.


Exaggeration of the long-latency stretch reflex (LLSR) is a characteristic neurophysiologic feature of Parkinson's disease (PD) that contributes to parkinsonian rigidity. To explore one frequently-hypothesized mechanism, we studied the effects of fast muscle stretches on neuronal activity in the macaque primary motor cortex (M1) before and after the induction of parkinsonism by unilateral administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We compared results from the general population of M1 neurons and two antidromically-identified subpopulations: distant-projecting pyramidal-tract type neurons (PTNs) and intra-telecenphalic-type corticostriatal neurons (CSNs). Rapid rotations of elbow or wrist joints evoked short-latency responses in 62% of arm-related M1 neurons. As in PD, the late electromyographic responses that constitute the LLSR were enhanced following MPTP. This was accompanied by a shortening of M1 neuronal response latencies and a degradation of directional selectivity, but surprisingly, no increase in single unit response magnitudes. The results suggest that parkinsonism alters the timing and specificity of M1 responses to muscle stretch. Observation of an exaggerated LLSR with no change in the magnitude of proprioceptive responses in M1 is consistent with the idea that the increase in LLSR gain that contributes to parkinsonian rigidity is localized to the spinal cord. Rigidity, one of the cardinal signs of Parkinson's disease (PD), is defined clinically as a sustained increase in resistance to passive movement of a joint throughout its range   were used for these experiments (monkeys V and L). All aspects of animal care were in accord with the Guide for the Care and Use of Laboratory Animals , and all procedures were approved by the institutional animal care and use committee.Two female monkeys , single flexing or extending torque impulses (0.1 Nm–50 ms duration) were applied to the manipulandum by a DC brushless torque motor  at an unpredictable time beginning 1–2 s (uniform randomized distribution) after initial capture of the center target. Each square-wave torque impulse induced an angular displacement of the joint (mean = 10-deg) causing a sudden stretch of arm extensor or flexor muscles. The animals were not trained to produce a specific response to these unpredictable perturbations, but the animals naturally adopted a strategy that returned the joint to its initial pre-impulse position.Later aspects of the behavioral trial, which evaluated instruction-related neuronal activity, are irrelevant to the current study.The animals were prepared surgically using aseptic techniques under Isoflurane inhalation anesthesia  electrodes was verified post-surgically. Following surgery, animals were given prophylactic antibiotics and analgesic medication.PTNs and CSNs were identified by antidromic activation from electrodes implanted in the cerebral peduncles and posterolateral striatum. Sites for implantation were identified using standard electrophysiological mapping techniques . A cortical region was targeted for data collection if neurons responded to active and/or passive movement of the arm and microstimulation at low currents evoked contraction of forelimb muscles . Microelectrode penetrations were performed throughout the targeted cortical area using sequential stimulation of each putamen and peduncle stimulating site as search stimuli . Neurons were selected for data collection if they were activated antidromically or if they were located in close proximity (<0.5-mm) to an antidromically-activated neuron. Standard tests for antidromic identification were used: constant antidromic latency (<0.2-ms jitter), reliable following of a high-frequency train of stimuli (three or four shocks at 200-Hz), and collision of antidromic spikes with spontaneously occurring spikes . The action potentials of single neurons (sampled at 60-kHz) were discriminated on-line using template-based spike sorting . The timing of detected spikes and of relevant task events was sampled digitally at 1 kHz and saved to disk for offline analysis. EMG signals were differentially amplified (gain = 10-K), band-pass filtered (20-Hz to 5-kHz), rectified and then low-pass filtered (100-Hz). EMG data were collected during only a subset of data recording sessions.  Analog data reflecting angular position of the manipulandum , the torque produced by the motor, and EMG were digitized at either 200-Hz (monkey L) or 500-Hz (monkey V).Neuronal activity was collected while the animal performed the step-tracking task. The microelectrode signal was amplified ×10After an adequate number of neurons were sampled from the neurologically-normal state, a hemiparkinsonian syndrome was induced by injection of MPTP into the left internal carotid artery . A neuron was judged to be torque-related if it generated a significant post-torque response for at least one movement direction. Response onset times, defined as the time at which the SDF first crossed the p = 0.002 threshold, were determined separately for each torque direction. For comparisons between neuronal populations, a cell's earliest response onset across directions was used as that cell's latency. We only analyzed cortical responses that began at relatively short latencies (between 20–60-ms), so as to exclude responses related to volitional compensatory movements ] were generated by convolving a spike train's delta function (1-ms resolution) with an asymmetric Gamma function kernel . Second, we calculated the maximum change of firing rate away from baseline between times of response onset and offset. And third, we calculated the area under the curve in the SDF between times of response onset and offset. The temporal dispersion of a response was measured as its full-width half-maximum (FWHM).P)|, where NP equals the maximum change infiring rate from baseline in the non-preferred direction and P equals the maximum change in firing rate in the preferred direction during the post-torque period (200-ms).The preferred direction is defined as the direction that elicits the largest change from baseline firing rate (either positive or negative).By this convention, DSI = 0 meant that the response to torque perturbation was equal for both directions and therefore was not directionally selective . DSI ≈1 meant that the cell response (either increase or decrease in firing) was present for only one movement direction . A cell's torque response was considered directionally selective if the DSI was >0.50 . Responses with DSIs between 0.5 and 1 were defined as bidirectional whereas those with DSIs >1 were considered reciprocal (changes of opposite sign for opposing directions of movement).The directional selectivity of torque-evoked responses was parameterized using a directional selectivity index (DSI)  = 0.0125]. The second analysis tested for relationships within individual recording sessions. We tested for correlations between a single neuron's trial-to-trial response latency and an animal's trial-to-trial response to torque perturbations (peak velocity and EMG). Spearman's rank correlations were performed for individual cells and results were compared between populations recorded pre- and post-MPTP.To study MPTP-related changes for different subsets of cells, we corrected for multiple comparisons using the Bonferroni–Holm method Holm, . Then, tSingle unit recordings were obtained from the arm-related areas of the left M1 of two monkeys. A total of 227 neurons were studied in the neurologically normal state. Of these, 66 were activated antidromically from peduncle stimulation . This result is consistent with previous descriptions of the marked differences in resting firing rates between intra-telencephalic-like CSNs and PTNs . MPTP had markedly different effects on the two identified neuronal populations. The pre-torque firing rate of PTNs was reduced by 27% following MPTP treatment . The antidromic latencies of PTNs remained unchanged between pre- and post-MPTP periods .Also consistent with previous reports . More specifically, the mean peak velocity and the amplitude of torque-induced displacements averaged across recording sessions were reduced by 16.2% and 6.6%, respectively, following MPTP administration, consistent with an increase of rigidity in the parkinsonian condition. The latencies of torque-induced displacements remained unmodified post-MPTP . Because of this, data were analyzed separately for flexion and extension torque directions.Induction of parkinsonism also led to a slowing of torque-induced displacements of the arm  was poorly formed and generally much smaller than the long-latency response . Following the administration of MPTP, the m2 component was markedly larger , primarily due to the appearance of an m3 component. The m1 short-latency response remained weak in all the case, and latencies of the earliest EMG responses showed no significant change (p > 0.05).EMG responses to sudden, unpredictable muscle stretches were analyzed in their separate latency components Figure . The sho2 = 32.3, p < 0.001; Table A large fraction of the general population of M1 cells  responded to torque perturbations with a phasic response at short latency , induction of parkinsonism did not increase the prevalence of torque responses in the general population of M1 cells (62% for both states), in CSNs (26%), or in PTNs , we selected subsets of recording sessions from pre- and post-MPTP sessions in which velocities were equivalent .Induction of parkinsonism also did not alter the balance of torque-elicited increases and decreases in firing. For PTNs, most torque-evoked changes in activity consisted of an increase in firing . M1 response latencies were reduced by similar degrees for flexion and extension directions .The latencies of torque-evoked responses, however, were significantly shorter following MPTP administration. This observation held true for the general population of M1 neurons and specifically for PTNs, and for responses to flexions and extensions, all considered separately .In addition to the shift in latencies, torque-evoked increases in PTN activity were more tightly synchronized or time-locked to the torque perturbation following MPTP Figure . For theMethods), it became clear that highly directional  response types became less common following MPTP administration (10% reduction in incidence), and bi-directional or non-directional response types became more common  in the incidence of highly directional response patterns were observed in CSN and PTN sub-populations. This general decrease in directional selectivity could not be attributed directly to MPTP-induced changes in resting firing rates because pre-torque mean firing rates did not differ between the different directional categories . In addition, EMG responses to torque perturbations did not show alterations in directional selectivity or sign of co-contraction between antagonist muscles . This analysis was restricted to the relatively homogenous population of PTNs because their activity was known to be affected by MPTP administration. No significant correlation was found for either analysis . A second approach searched for correlations trial-by-trial between torque-evoked neuronal responses and measures of the LLSR (Velmax or m2max). Measures of PTN response latencies did not correlate with any of the measures of LLSR . Similar negative results were obtained from correlation analyses of the general population of M1 cells (results not shown).Although torque-evoked cortical responses, movement velocity, and the The present study addresses a long-standing but untested hypothesis for why the long-latency component of the stretch reflex is exaggerated in Parkinson's disease. Despite the fact that exaggeration of the LLSR is a significant contributor to parkinsonian rigidity .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The method can be integrated with mass spectrometry, allowing for the discovery of novel protein interactions. Here, we describe and characterize the sensitivity and physical features of DiG-Stamping. We demonstrate the biologic utility of DiG-Stamping by (1) identifying the binding partners of a target protein within a cellular lysate and by (2) visualizing the dynamics of proteins with multiple post-translational modifications.Proteins and small molecules are the effectors of physiological action in biological systems and comprehensive methods are needed to analyze their modifications, expression levels and interactions. Systems-scale characterization of the proteome requires thousands of components in high-complexity samples to be isolated and simultaneously probed. While protein microarrays offer a promising approach to probe systems-scale changes in a high-throughput format, they are limited by the need to individually synthesize tens of thousands of proteins. We present an alternative technique, which we call diffusive gel (DiG) stamping, for patterning a microarray using a cellular lysate enabling rapid visualization of dynamic changes in the proteome as well protein interactions. A major advantage of the method described is that it requires no specialized equipment or  Protein patterning on microarrays has been shown to be useful for high-throughput analyses, probing with multiple targets, and for minimizing reagent quantities The precursors to protein microarrays are membrane blots transferred from cellular lysates separated by polyacrylamide gel electrophoresis (SDS-PAGE). Screening of novel binding interactions can be accomplished through the use of Far-western blots de novo via sequencing, enabling novel protein binding partners to be discovered. However, mass spectrometry, which is limited in throughput, must be combined with other techniques in order to determine binding interactions. Often, the proteins of interest for a particular study are those that change in response to specific biologic stimuli or genetic manipulations. In principle, restricting microarray analyses to such proteins would significantly reduce sample complexity in a hypothesis-relevant manner and would allow for a synergistic combination with mass spectrometry.While protein microarrays can characterize samples based on their specific binding to patterned proteins, mass spectrometry identifies proteins Towards this objective, we have developed a novel method, DiG-Stamping, that allows for biologically-motivated complexity reduction in high-content samples and subsequent unbiased discovery of novel protein interactions and proteome dynamics. The basics of DiG-Stamping are straightforward. The protein array is assembled by transferring a cellular lysate (or subset thereof) separated via SDS-PAGE to a chemically functionalized slide by direct contact with the gel . The resTo characterize the transfer efficiency, serial dilutions of Alexa 555 Ovalbumin ranging from 0.3 µg to 19 pg, were run on a 1 mm, 15 well 10% Tris glycine gel, under denaturing conditions  in triplicate. The gels were transferred for 60 minutes to aminosilane-coated glass slides with no applied voltage according to the protocol in The slides were scanned with the Axon 4000B (Gain = 600)  1e, and To investigate the limit of detection for a brief (30 minute) gel transfer assay, we measured the detection efficiency by using biotinylated BSA , probed with streptavidin Cy3. 10% Tris-Glycine, 15 well, 1 mm gels were run in triplicate with serial dilutions of biotinylated BSA ranging from 10 µg to 100 pg and transferred for 1 hour. The slide was probed with 20 nM solution of streptavidin Cy3, imaged and quantified .To compare the DiG-Stamping method to a standard Western blot , gels weWe describe the preparation of HeLa cell lysate in Preparation of T-47D cells lysates with and without Heregulin-β treatment are described in To identify the parameters that modulate protein transfer and detection with DiG-Stamping, we characterized the transfer of a serial dilution of fluorescently-labeled Ovalbumin with respect to concentration, gel thickness, applied electric field  1a–c, anIn order to demonstrate the applicability of this technique to biological assays and compare it to conventional methods, we used DiG-Stamping to identify protein-protein interactions in comparison with Western blotting . To gaugWe next investigated whether DiG-Stamping enables the probing of protein interactions in a format that allows for the identification of residual protein remaining in the gel. In principle, this could be accomplished by identifying the location of a binding event on the slide and then extracting the corresponding location in the gel. To demonstrate the feasibility of this approach, we used the biotin-streptavidin system to detect the binding partner(s) of streptavidin within a high-complexity cellular lysate. To do so, we added biotinylated-BSA (bBSA) and a fluorescent-labeled molecular protein ladder to cellular lysate from the HeLa cell line, transferred the lysate mixture following SDS-PAGE to a glass slide by DiG-Stamping, and then probed with streptavidin Cy3 . The gelThis resulted in the identification of Albumin (ALB Protein), as well as several other proteins  Table 1 in-vitro labeling, and SILAC in-vivo labeling) can be integrated with DiG-Stamping by labeling the lysate prior to patterning.Although the proteome contains tens of thousands of proteins, often only a subset of these are of interest and warrant further study in any given study; these could include proteins that interact with a target, change in expression level, or post-translational modification in response to a particular stimulus. In principle, DiG-Stamping could allow for biologically relevant complexity-reduction in lysates by first identifying dynamic protein changes on patterned microarrays, prior to selective follow-up analysis by mass spectrometry. This approach could help reduce the low-throughput bottleneck of mass spectrometry. Additionally, quantitative mass spectrometry  cells upon activation of the Her2/Neu receptor with Heregulin-β The unprocessed slide images  show traa priori, providing a demonstration that DiG-Stamping can provide novel information that may help direct further analyses.Following this analysis, simultaneous visualization of both the phosphoproteome, as well as phosphoproteins with an ubiquitin post-translational modification, were directly compared across short-term and long-term Heregulin-β stimulation. Significant changes in the phosphoproteome could be readily identified, while levels of proteins having dual phosphorylation and ubiquitinylation modifications did not significantly change over time following Heregulin-β treatment . This biMethods for the analysis of novel proteins and small molecule interactions are essential for the study of biological systems. We have developed and demonstrated a novel technique, DiG-Stamping, to rapidly fabricate protein microarrays by leveraging the use of cellular lysates. The primary advantage of the proposed technique is that it enables rapid visualization of binding interactions (with multiple ligands simultaneously) as well as proteome-level changes, with high sensitivity and minimal reagents. DiG-Stamping does not require any specialized laboratory equipment, making it accessible to all researchers. Furthermore, by utilizing only a small amount of protein for detection, DiG-Stamping enables further assay integration with existing biochemical techniques by preserving the majority of the protein sample in the gel.Until now, the partial transfer of proteins from a gel to membrane-based substrate has been limited by the sensitivity of Western Blots. With the integration of recently developed microarray technology, as demonstrated here with DiG-Stamping, it is feasible to transfer only a portion of the protein sample without compromising protein detection sensitivity. As with all protein assays, the fundamental detection limits are set by the amount of protein sample being probed, combined with the sensitivity of the assay and detector itself. Therefore, the partial transfer of a quantity of protein by passive diffusion – as opposed to the complete transfer of the sample via electrophoresis– will inherently have a lower detection limit when the same method (e.g. Western Blotting) is used for detection. This was illustrated by Kurien et al The primary limitation of DiG-Stamping in comparison to spotted arrays is the preferential transfer of high-abundance and low molecular-weight proteins (LMWPs), and the lateral diffusion of LMWPs diminishing spatial resolution. This makes isolation of individual proteins more difficult, but this is consistent with the limitations of all gel-based techniques. Care must also be taken in correlating the protein position on the slide image to the gel.Gel transfer efficiency and protein detection limits were characterized using a fluorescently-tagged streptavidin as a secondary binding agent. This allowed for accurate comparison across both Western blots and DiG-Stamping, but the robust nature of the biotin-streptavidin bond does not reflect typical protein-protein interactions. To address this, and illustrate the applicability of the current technique to typical protein-protein interactions involving antibody identification of a target, we have also demonstrated simultaneous labeling of the T-47D lysate with fluorescently-tagged antibodies.As illustrated by the analysis of T-47D cancer cell migration, the approach presented here allows for reduction in the complexity of a sample in a hypothesis-driven manner by identifying relevant dynamic changes in subsets of the proteome by DiG-Stamping. These relevant protein components can then, in principle, be identified by mass spectrometry, since the vast majority of protein following DiG-Stamping remains in the gel. This synergistic combination of easily fabricated protein microarrays and mass spectrometry addresses the low-throughput limitations of mass spectrometry.As we have shown here, DiG-Stamping can be applied to screening for protein-protein interactions. In principle, any set of proteins can be patterned, and probed against multiple targets, using a fraction of the sample required in typical Western Blot applications. In the present study, these advantageous features of microarrays were used here to identify multiple post-translational modifications associated with cancer cell migration. Many other applications can be envisioned, e.g. patterning proteins associated with specific signaling pathways and subsequently probing either with other proteins or small-molecules.Figure S1Characterizing Transfer Efficiency. (a) Protein transfer versus concentration. (b) Protein transfer versus electric field. (c) Protein transfer versus gel thickness. (d) Protein transfer versus time. (e) A sample image of serial dilutions transferred to an aminosilane slide.(TIF)Click here for additional data file.Figure S2Gel transfer mechanism.(TIF)Click here for additional data file.Protocol S1Reagents, Equipment and Procedure for Diffuse Gel Stamping; Hela Cell Lysate Preparation; T47D Culture and Lysate Preparation; Western Blot Methods. Detailed figure captions.(PDF)Click here for additional data file.Data S1Table 1: Proteins from Extracted Gel Band. Table 2: Table 2: Proteins from immunoprecipitated Gel band.(PDF)Click here for additional data file.


To evaluate the impact of structural changes of the retinal nerve fiber layer (RNFL), and visual field loss, on functional impairment assessed by patient-reported visual functioning in glaucoma.Patients with glaucomatous optic nerve damage were enrolled in this cross-sectional study. Peripapillary RNFL thickness was obtained with spectral-domain optical coherence tomography (SD-OCT). Function was assessed by patient-reported visual functioning using the Rasch-calibrated Glaucoma Activity Limitation 9 (GAL-9) questionnaire and standard automated perimetry. The impact of peripapillary RNFL loss on functional impairment was analyzed with correlation and linear regression analyses.2=0.334), while structural parameters could not enhance the prediction of GAL-9 scores. A total of 176 eyes from 88 glaucoma patients were included. The SD-OCT assessed temporal-superior and temporal-inferior RNFL sector of the worse eye revealed significant correlation with the GAL-9 scores . In mutivariate regression analysis, the best predictors for patient-reported visual functioning were visual acuity of the better eye and mean defect of the worse eye (RSelf-reported visual functioning of patients with glaucoma is better predicted by visual performance data than structural parameters. However, some structural changes of the worse eye are significantly correlated with patient-reported visual functioning.  Glaucoma is one of the leading causes of blindness with more than 70 million people affected worldwide. HoweverPrecise measurements of the RNFL thickness are feasible with optical coherence tomography (OCT), particuThis study was performed to evaluate the structure/function/patient-reported visual functioning relationships in patients with glaucoma. Structural peripapillary RNFL thickness was obtained using the SD-OCT, function was assessed with standard automated perimetry and visual acuity (VA), and patient-reported visual functioning was measured with the GAL-9. A secondary aim was to look for differences in the predictive value of structural and functional parameters depending on weather the better or the worse eye is regarded. This was a cross-sectional investigation conducted in a German university-affiliated glaucoma center: the glaucoma unit of the Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. Potential study participants, patients with glaucoma attending for a regular review, were approached on a consecutive basis. All study participants underwent a full ophthalmic examination, including objective and subjective refraction, slit-lamp biomicroscopy, intraocular pressure (IOP) measurement with Goldmann applanation tonometry, gonioscopy, dilated fundus examination by indirect ophthalmoscopy and central corneal thickness (CCT) measurement. Inclusion criteria were age >18 years and a spherical refraction between -7.0 and +5.0 diopters. Patients with all stages of primary and secondary glaucomatous optic nerve damage undergoing all types of treatment or surgery were included. However, cases with secondary glaucoma associated with systemic disorders were excluded to minimize a potential confounding influence of comorbidity. Patients with a visual acuity of less than 20/50 in the better eye were excluded, as the items of the GAL-9 require at least minimal visual function for utilizable results. Further exclusion criteria included diabetes or history of other ocular or neurologic diseases. Approximately every tenth consecutive patient could not be enrolled due to unreliable standard automated perimetry or not meeting the 15 dB threshold for image quality in SD-OCT. Glaucomatous eyes were defined as those with reliable abnormal standard automated perimetry  and optic nerve damage . Ethical approval of the study was obtained from the Institutional Review Board of the University Eye Hospital Munich in Germany and all the patients who agreed to participate signed a consent form. The study adheres to the tenets of the Declaration of Helsinki. There have been two recent reviews of PRO measures for glaucoma patients,24 that SD-OCT-based assessment of the RNFL provides a technological advance in the diagnosis of glaucoma. The difStandard automated perimetry is currently the gold standard for visual field testing. Visual field was assessed using the Humphrey Field Analyzer  and the SITA 30-2 standard algorithm; whereby the parameters mean deviation (MD) and the pattern standard deviation (PSD) were recorded. A reliable visual field test was defined as having a <25% rate of fixation losses, and <20% false positive and false negative values. The MD was used to determine, which eye of each patient was the better or worse eye; the eye with the lower MD was considered to be the worse eye (more glaucomatous damage). Glaucoma staging was determined following a simplified Hodapp-Anderson-Parrish modification ,32 as ea<-12dB) glaucoma. We performed Rasch analysis on the German GAL-9 using Winsteps software (version 3.68), Chicago, Illinois, USA. The Andrich rating scale model was used and four rating scales were applied  values. Descriptive statistical analyses were performed to characterize the patients’ clinical, functional and structural data. Difference testing between eyes was done using the Student t-test. For correlation analysis, the Pearson correlation coefficient was calculated for normally distributed values (according to the Kolmogorov-Smirnov test), otherwise the Spearman’s rank coefficient was used. Relationships were also tested with multiple linear regression; t-based 95% confidence intervals (CIs) for the regression coefficients were used. All statistical analyses were performed with the SPSS statistical software .A total of 88 patients were included (176 eyes); fifty-one (58.0%) patients were female. Perimetry, GAL-9 testing and SD-OCT of the peripapillary RNFL were obtained from all patients. The best-corrected VA of the MD-guided better eye was 20/28 (logMAR 0.14) and of the worse eye 20/33 . The global RNFL of the worse eye was highly correlated with the MD of the worse eye . Regarding functional parameters and patient-reported visual functioning, there was a trend to a higher correlation of GAL-9 scores with the MD of the better eye than GAL-9 score with the MD of the worse eye .Linear regression analyses were performed to analyze the impact of RNFL loss on functional parameters and patient-reported visual functioning. SD-OCT-assessed RNFL thickness of each sector of the MD-guided better or worse eye entered linear regression to predict GAL-9 person estimates . A multivariate linear regression analysis was performed to predict GAL-9 person estimates entering significant structural and functional parameters. The best model to predict GAL-9 person estimates includes the VA of the worse eye and the MD of the better explaining 33.4% of the variance in patient-reported visual functioning . This reTo the best of the authors´ knowledge, this is the first study that compares glaucomatous RNFL thickness and patient-reported visual functioning. It could be hypothesized that structural change should lead to visual change, which in turn should impact person-reported measurements. However, a major finding of our study is that the RNFL is not well correlated or highly predictive for the GAL-9. There seems to be a degree of separation between structural and patient-reported changes. The functional parameters visual acuity and MD predict GAL-9 scores much better than the SD-OCT assessed structural parameter RNFL. 2=0.334). The finding that the VA of the worse eye is a better predictor for functional impairment is noteworthy, as numerous previous publications suggested that patient-reported visual functioning is at least partially driven by the eye with the better visual acuity  How How4] HoFor patients with good binocular visual acuity, the worse eye may play a significant role in their performance, whereas for patients with poor binocular visual acuity (e.g. 20/100 to 20/200), the better eye may be more important. In this study, there is more variance in the worse eye data which improves the probability of a strong correlation. However, most patients with a diagnosis of glaucoma have good central visual acuity, and therefore, the results appear to be representative of a randomly selected glaucoma population. A strength of this study is the use of Rasch-scaled person estimates of the GAL-9. The GAL-9 was shown to have excellent psychometric characteristics except for targeting of item difficulty to person ability. Poor taThe ultimate gain of glaucoma therapy is to prevent loss of patients´ quality of life. High quality measurement of structural, visual and patient-reported outcomes and an understanding of their inter-relationships is critical to achieving this goal. It would be of interest to investigate longitudinally, whether localized RNFL thinning or other structural change in glaucoma can predict future deficits in patient-reported visual functioning. New measurements of structural changes, e.g. the optic nerve head using the minimum rim width by SD-OCT , might rStructural measurements of the peripapillary RNFL thickness by SD-OCT are weak predictive factors of patient-reported visual functioning as assessed by the GAL-9. The best predictors of patient-reported visual functioning are the visual acuity of the worse eye and the MD of the better eye.


MicroRNAs (miRNAs) are potential regulators that contribute to the pathogenesis of cancer. Microarray technologies have been widely used to characterize aberrant miRNA expression patterns in cancer. Nevertheless, the miRNAs expression signatures identified for a same cancer differs among laboratories due to the cancer heterogeneity. In addition, how the deregulated miRNAs coordinately contribute to the tumourigenic process of prostate cancer remains elusive.We evaluated five outlier detection algorithms that take into account the heterogeneity of cancer samples. ORT was selected as the best method and applied to four prostate cancer associated microRNA expression datasets. After microRNA target prediction and pathway enrichment mapping, 38 Gene Ontology terms, 16 KEGG pathways and 99 GeneGO pathways are found putative prostate cancer associated. Comparison with our previous studies, we identified two putative novel pathways important in prostate cancer. The two novel pathways are 1) ligand-independent activation of ESR1 and ESR2 and 2) membrane-bound ESR1: interaction with growth factors signalling.We proved that expression signatures of at the pathway level well address the cancer heterogeneity and are more consistent than at the miRNA/gene levels. Based on this observation, we identified putative novel microRNA regulatory pathways which will help us to elucidate the cooperative function of different microRNAs in prostate cancer. MicroRNAs (miRNAs) are small non-coding RNAs of approximately 22-nucleotides. They play important roles in gene regulation at post-transcriptional level. They are able to repress the activity of complementary mRNAs by targeting the 3'-untranslated regions . ReleaseMany efforts have been made in search of common molecular signatures, however without obvious success. This is partly due to the highly heterogeneous nature of cancer. Tumour samples often comprise of subpopulations with different genomic alterations. However, the most popular outlier detection algorithm, t-test or its analogues, simply removes heterogeneity between subtypes, and fail to identify the subgroup-specific gene alterations -8. RecenIn this study, we applied these outlier detection methods to analyze our collection of four miRNA expression microarray datasets to identify differentially expressed miRNAs (DE-miRNAs). The DE-miRNAs were then compared among the four data sets at both gene and gene set  levels for comparison. By considering the cancer heterogeneity, we applied different statistical methods to identify the consistent prostate cancer (PCa) associated pathways that are coordinately targeted by miRNAs.Most of the previous expression data studies used fold-change, t-test and other statistics alike to detect cancer-related genes. Recently, it has been recognized that many oncogenes show altered expression in only a small proportion of cancer samples . Such feTo address this complexity, a series of new heterogeneous detection algorithms have been proposed in recent years. Among these methods are Least Sum of Ordered Subset Squared (LSOSS) , Cancer The performance of the above algorithms and the traditional t-test were compared on the detection of the outliers in our collection of prostate cancer (PCa) associated microRNA expression data. The outliers here refer to the deferentially expressed microRNAs (DE-miRNAs). For all these methods applied to the different data sets with different numbers of samples, we set the quantile of outliers to 0.05 (5%). Those DE-miRNAs detected by at least three methods were considered to be putative PCa associated outliers, and then the percentages of the putative outliers in the original result of each method were calculated to determine the method's accuracy . Additional file The collection of the four different datasets are from different platforms, the overlapping of miRNA probes between these data are about 40~60% while the detected differently expressed miRNA profiles only have 3% overlapping . We aim p < 0.001), and 99 GeneGO pathways (FDR < 0.001) that are enriched with target genes of the PCa associated DE-miRNAs.Figure Among the 99 enriched GeneGO pathways, 67 (67.7%) pathways were also significantly enriched in our previous study in which we processed 10 mRNA microarray datasets . In the i.e., 1) ligand-independent activation of ESR1 and ESR2, this is the most significant pathway we mentioned in the last section, and 2) membrane-bound ESR1: interaction with growth factors signalling.To identify potential microRNA regulatory pathways in prostate cancer, the 15 most significantly enriched  pathways were chosen for the analysis. Of those, four had previously been reported to be related to prostate cancer in PubMed citations. We verified the other 11 pathways indirectly by analysis of the component genes in PubMed citations although the wet-lab experiments can direct validate them , which is a public functional genomics data repository supporting MIAME-compliant data submissions. The datasets were downloaded in single-matrix file format, and named according to the name of the first authors of the references  for each pathway and adjust it using the FDR value as the threshold. In MetaCore databases, p value means the probability of a random intersection of two gene sets, with low p values indicating a high potential of non-randomness of the finding.To study the function of the PCa DE-miRNAs, we mapped their target genes to GO, KEGG and GeneGO databases. To decrease the number of the false positives pathways, we first identified target genes shared by at least three PCa DE-miRNAs datasets, which were then mapped to GO, KEGG pathway database by DAVID, and GeneGO pathway database by MetaCore . Both DAVID and MetaCore use hypergeometric distribution to calculate the significance level Click here for fileEnriched KEGG pathways with P-value. Gene list was generated by extraction of targeting genes from at least 3 datasets. Click here for fileEnriched GeneGO pathways with P-value. Gene list was generated by extraction of targeting genes from at least 3 datasets. (FDR<0.001)Click here for fileCitation counts of corresponding genes of each potential GeneGO pathway. Citation count was calculated by searching PubMed in the fields of title and abstract, this may change with the update of PubMed.Click here for file


Longitudinal studies of HIV-1-infected individuals or those at risk of infection are subject to missed study visits that may have negative consequences on the care of participants and can jeopardize study validity due to bias and loss of statistical power. Distance between participant residence and study clinic, as well as other socioeconomic and demographic factors, may contribute to interruptions in patient follow-up.HIV-1-serodiscordant couples were enrolled between May 2007 and October 2009 and followed for two years in Nairobi, Kenya. At baseline, demographic and home location information was collected and linear distance from each participant’s home to the study clinic was determined. Participants were asked to return to the study clinic for quarterly visits, with follow-up interruptions (FUI) defined as missing two consecutive visits. Cox proportional hazards regression was used to assess crude and adjusted associations between FUI and home-to-clinic distance, and other baseline characteristics.10 increase; 95% CI: 1.01, 1.51).Of 469 enrolled couples, 64% had a female HIV-1-infected partner. Overall incidence of FUI was 13.4 per 100 person-years (PY), with lower incidence of FUI in HIV-1-infected (10.8 per 100 PY) versus -uninfected individuals (16.1 per 100 PY) . Among HIV-1-infected participants, those living between 5 and 10 kilometers (km) from the study clinic had a two-fold increased rate of FUI compared to those living <5 km away . Other factors associated with FUI included paying higher rent , having at least primary school education , and increased HIV-1 viral load (HR = 1.23 per logHome-to-clinic distance, indicators of socioeconomic status, and markers of disease progression may affect compliance with study follow-up schedules. Retention strategies should focus on participants at greatest risk of FUI to ensure study validity. Missing data resulting from participant attrition, or incomplete study follow-up, threaten the integrity of a study, with consequences ranging from a reduction in statistical power to significantly biased results and diminished validity. While numerous techniques have been developed to address the problem, none are simple, most require manipulation of the data, and the precision of results can be difficult to determine Predicting attrition is challenging and has been the subject of a number of studies in the U.S., Western Europe and sub-Saharan Africa. Reviews of published studies have listed up to 18 different definitions for patients who were considered lost to follow-up In sub-Saharan Africa and other resource-limited settings, reasons for not completing a study are not fully understood. Despite the paucity of data on the subject, some factors have been associated with attrition in antiretroviral treatment (ART) programs in sub-Saharan Africa, including younger age Distance-decay is used to explain how health care use is negatively correlated with the distance patients must travel to access care Written informed consent was obtained from all participants. This study received ethical approval from the institutional review board of the University of Washington and the Ethics and Research Committee of the University of Nairobi and was conducted according to the guidelines set forth by the United States Department of Health and Human Services.HIV-1-serodiscordant heterosexual couples were enrolled in a prospective cohort study between May 2007 and October 2009. Couples were eligible if one member of the couple was HIV-1-infected and the other HIV-1 susceptible, both partners reported having sexual intercourse at least 3 times in the previous 3 months, and planned to stay together for at least two years. Participants who were pregnant or using antiretroviral therapy at enrollment were excluded.Couples were referred to the study from HIV voluntary counseling and testing (VCT) sites throughout the greater Nairobi region and from a clinical trial site at Kenyatta National Hospital that had identified discordant couples. Couples provided written informed consent at the screening and enrollment visits as well as contact and locator information for retention-tracking purposes. All couples received at least one home visit from a peer counselor to verify the location of their residence. Study clinicians collected baseline sociodemographic, sexual/reproductive and medical history data from both members of the couple, and blood and genital samples were collected at screening, enrollment and subsequent visits.Couples were asked to return to the study clinic quarterly to provide blood and genital specimens, complete questionnaires on recent sexual behavior and current health status, and be tested for HIV-1 seroconversion. HIV-1-infected partners were supplied with trimethoprim-sulfamethoxazole to be taken daily as prophylaxis for opportunistic infections. Couples continued to be followed in the study even if they separated, the female partner became pregnant, or one partner left the study.Support groups for those with and without HIV infection were available throughout the study. Drinks were supplied free of charge during clinic visits, and participants were reimbursed 500 Kenyan Shillings (KSh), (approximately 3.50 US dollars [USD]) per visit for transportation expenses to and from the clinic.At enrollment and quarterly visits, HIV status of the uninfected partner was determined by two rapid tests conducted in parallel using the Determine HIV-1/2 rapid test  and the Bioline HIV 1/2 rapid test , with confirmation by the Vironostika HIV Uni-form II Ag/Ab ELISA kit  for positives or indeterminates. Urine pregnancy tests were also performed every three months . CD4 cell counts  and HIV-1 RNA viral load  were measured in HIV-1-infected participants at enrollment and every six months.The locations of participants’ residences were mapped using locator information provided by each participant. Based on this information, a member of the study retention team mapped each residence using a web-based mapping application built on the Google Maps engine . The geographic coordinates for all VCT sites and the study clinic were determined using a handheld global positioning system (GPS) unit . The resulting coordinates were used to estimate linear (as the crow flies) distances between each residence and the referring VCT and between each residence and the study clinic using a geospatial algorithm .a priori that HIV status would modify the associations between FUI and distance variables, and therefore all analyses were conducted separately for HIV-1-infected and HIV-1-uninfected individuals. For regression models, distance was considered both continuously and categorically. We assumed that distances beyond 5 kilometers (km) were likely to necessitate transport other than walking, and therefore categorized linear distance into 5 km intervals . Potential confounders were included stepwise in a multivariable model and remained in the model if the adjusted association differed from the unadjusted hazard ratio by more than 10% in either the HIV-1-infected or -uninfected model. Robust standard errors were used to relax the assumption of normally distributed standard errors. All statistical analyses were conducted in Stata 10.1  with statistical significance criteria set at α<0.05.The outcome in this analysis was time until study follow-up interruption (FUI), defined as missing two consecutive quarterly study visits. We used Cox proportional hazards regression to evaluate the time from enrollment until the first FUI, calculated as the length of time between the date of the enrollment visit and the scheduled date of the first missed visit (three months after the last completed clinic visit). Participants were censored at the date for their last scheduled follow-up visit, or at the date of death for participants who died during follow-up. If the exact date of death was unknown, the censoring date was defined as three months after the last completed visit. The crude associations between FUI and factors of interest were estimated by hazard ratios (HR) and 95% confidence intervals (CI). We hypothesized 10 copies/ml . Among all enrolled, the median age at enrollment was 35 years  for men and 29 years  for women  were enrolled into the study. The male member of the couple was HIV-1-infected in 168 (36%) of enrolled couples, and the female was the infected partner in 301 (64%) of the couples. Median CD4 cell count at baseline in the HIV-1-infected was 415 cells/µl  and median HIV-1 viral load was 4.6 logor women .A total of 186 (20%) participants had less than a primary school education: 70 (15%) men and 116 (25%) women. Fifty-five (12%) of the couples enrolled reported no household income at study entry, 36% reported earning less than the median of 8,000 KSh per month, and 197 (52%) earned more than 8,000 KSh per month, equivalent to approximately 130 USD at the time of the study. Eighty (9%) participants reported owning a home at study entry with the remaining 858 (91%) renting their current residence.Of those enrolled, 197 individuals missed two consecutive study visits and were categorized as having an FUI during 1471 person-years (PY) of follow-up, for a rate of 13.4 FUI per 100 PY. Among HIV-1-infected partners 46 women and 35 men experienced FUI and among HIV-1-uninfected partners, 46 women and 70 men experienced FUI . The ratFor each couple we ascertained a linear distance between the residence and the study clinic . PatientAmong HIV-1-infected participants, living between 5 and 10 km from the study clinic was significantly associated with a greater than two-fold increased likelihood of FUI when compared to those living <5 km from the clinic  . SimilarTo further investigate the finding that a residence 5–10 km from the study clinic was the highest risk group among HIV-1-infected participants, we conducted a series of post-hoc analyses. We found no significant difference in risk of FUI between HIV-1-infected participants living ≥10 km from the clinic compared to those living 5–10 km , and when we collapsed these categories, there was an attenuated association between distance from study clinic and FUI .10 HIV-1 viral load was associated with increased FUI rates , and having less than a primary school education was associated with lower FUI rates . Also among HIV- infected individuals, paying greater than or equal to the median monthly rent  compared to rent less than the median rent was associated with a 67% increase in the rate of FUI . We found no significant associations between baseline characteristics and FUI among HIV-1-uninfected participants. A multivariable model fit separately for HIV-1-infected and -uninfected patients including primary school education, monthly rent and log10 HIV-1 viral load (for the HIV-1 infected group only) found no independent associations with FUI (data not shown).We evaluated other sociodemographic factors as potential correlates of FUI using Cox regression with separate models for HIV-1-infected and -uninfected participants. Among HIV-1-infected participants, higher baseline logTo determine whether gender modified the association between linear distance and FUI, we stratified the analysis by both gender and HIV-1 status. Among the HIV-1-positive women, living between 5 and 10 km from the study clinic was associated with a nearly three-fold increase in the FUI rate compared to those living <5 km from the clinic  . DistancIn a longitudinal cohort of HIV-1-discordant couples followed quarterly, we found that rates of FUI were high  and that distance between home and the study clinic was significantly associated with FUI, particularly among HIV-1-infected women. While HIV-1-infected female partners were the least likely to miss two consecutive quarterly visits, they were approximately three times more likely to experience FUI if they resided greater than walking distance (>5 km) from the clinic.We found a complicated relationship between distance from participants’ residences and the study clinic and their likelihood of an FUI. The association between home-to-clinic distance and FUI was restricted to HIV-1-infected participants, and the strongest association was among those living 5–10 km from the study clinic with a weaker relationship among those living >10 km from the clinic. This pattern was stronger among HIV-1-infected women compared to infected men. Transportation challenges related to traveling more than 5 km to reach the clinic may pose a barrier to attending study visits. For example, those living within 5–10 km from the centrally located study clinic resided within city limits, potentially resulting in higher transportation costs (approximately 200 KSh per study visit) if they could not walk the distance to clinic. Participants living <5 km from the study clinic may have walked to study visits, or if not, were unlikely to have spent a large proportion of the compensation on direct transportation costs (likely less than 60 Ksh). Therefore, the 500 KSh per visit incentive, an amount equal to a quarter of the median monthly rent , may pose a greater incentive for those residing closer to the clinic compared to those living at or near the city limits. We did not find a significant association between longer home-to-clinic distances  and the risk of FUI. However, the point estimates for this category were slightly lower than in the 5–10 km category, and the lack of statistical significance may simply represent a loss of statistical power. We hypothesize that those who lived a greater distance from the study clinic and chose to enroll may have a greater level of motivation to continue participation, possibly due to a desire to obtain specialized health care not available near their home or to avoid stigma related to HIV infection. Additionally, living farther from the center of town may be indicative of lower socioeconomic status and increased reliance on the financial reimbursement provided by the study, and thus decreased rates of FUI.In this study, we found that women experienced fewer FUI than men, which is consistent with other studies that have found greater adherence to clinic visits and less program attrition among HIV-1-infected women compared to -infected men We also found that paying higher rent was associated with a 67% increase in the FUI rate compared to paying lower rent. If a higher rent is an indicator of greater employment obligations, then the need to take time off from work to attend study visits may explain this association. However, since we saw no statistically significant association between household income and FUI, the association with rent may be influenced by other factors, such as the distance between the place of employment and study clinic. Having less than a primary education was also significantly associated with nearly half the rate of FUI compared to those with at least a primary education. Lower levels of education could limit a person’s ability to find stable employment, which could increase the need for the financial benefits of participating in a research study.This study had several limitations. We were able to obtain measurements of the distance between participants’ residences and the clinic; however, we were unable to determine the time or financial cost required, or methods of transportation available. In one study, travel cost was found to be significantly associated with FUI, while the distance was not Despite its limitations, our study provides important information for further investigation. We found that living further from the study clinic was not significantly associated with an increased risk of FUI, with the exception of HIV-1-infected women between 5 and 10 km, indicating that enrollment of participants living far from the clinic may not be inherently problematic. We also found that HIV-1-uninfected men and women had higher rates of interruption of study follow-up and are an important group on which to concentrate retention efforts in clinical trials and prospective studies that depend on retaining both HIV-1-infected and -uninfected participants. In this study, higher rates of FUI among HIV-1-uninfected partners may have been due to the lack of direct benefits from attending study visits. Unlike their infected partners who received CD4 cell count testing and drugs to prevent opportunistic infections at the study clinic, the uninfected partners may have experienced limited direct benefits from study participation, and consequently may have been less motivated to attend visits.There has been limited research on retention of HIV-1-discordant couples in clinical research studies in sub-Saharan Africa. Yet these couples bear one of the greatest burdens of HIV transmission, and are already the focus of several large HIV prevention studies. Understanding and addressing transportation challenges in settings without easily accessible and inexpensive modes transportation could improve retention, both in research studies as well as in routine care and disease management. Studies combining quantitative and qualitative methods to address the barriers and incentives to study retention are needed to improve study retention and more importantly, to improve utilization and access to health care in sub-Saharan Africa settings.