Making use of sandwich assays on top of the nanorods, we obtain a limit of detection of 110 pM and 470 pM in 10-fold diluted spiked saliva and serum samples, respectively. In conclusion, our results open up numerous applications in direct protein biomarker quantification, specifically in point-of-care settings where resources are limited and ease-of-use is of essence.Detecting biomarkers from complex sample solutions is the key objective of molecular diagnostics. Being able to do so in a simple approach that does not require laborious sample preparation, sophisticated equipment and trained staff is vital for point-of-care applications. Here, we report on the specific detection of the breast cancer biomarker sHER2 directly from serum and saliva samples by a nanorod-based homogeneous biosensing approach, which is easy to operate as it only requires mixing of the samples with the nanorod probes. By careful nanorod surface engineering and homogeneous assay design, we demonstrate that the formation of a protein corona around the nanoparticles does not limit the applicability of our detection method, but on the contrary enables us to conduct  Within the range of methods employed for biomarker detection, homogenous measurement techniques are of special relevance for point-of-care (PoC) testing settings, as they allow for omitting complex sample preparation steps. Thus, the time for sample analysis may be reduced, whilst ensuring at the same time maximal ease-of-use3. Here, magnetic nanoparticles play a major role due to their added capability of magnetic manipulation, which can be exploited, for example, to accelerate binding processes or to enhance the signal to noise ratio4. Alternative non-magnetic nanoparticle-based bio sensing techniques include surface-enhanced Raman spectroscopy6 or methods relying on fluorescent nanoparticle properties7. It has been shown that the combination of nanoparticle labels and surface-enhanced Raman spectroscopy allows to detect various biomarkers in complex sample solutions9.Molecular diagnostics employed for the diagnosis and prognostics of a wide range of diseases is based on the detection of biomarkers in complex sample solutions and is of enormous scientific and clinical interestin-situ referencing.In the present article, we show the applicability of our previously introduced magnetic nanoparticle-based homogeneous measurement principle to molecular diagnostics in complex samples, i.e. serum and saliva samples. The method relies on changes of the hydrodynamic nanoparticle volume upon analyte molecule binding. To that end, antibody-functionalized magnetic nanorods (‘nanoprobes’) are excited in solution by a rotating magnetic field (RMF), which results in a rotational nanoprobe motion. The hydrodynamic volume of the nanoprobes induces a rotational drag torque with the result that the nanoprobes lag behind the RMF by a characteristic phase lag. Binding of the antigen causes an increase of the hydrodynamic nanoprobe volume, which can be observed directly via a change of the phase lag. This effect can be further enhanced by also adding secondary antibodies to form a sandwich-type immunoassay on top of the nanoprobe surface. The phase lag is determined optically by measurements of the actual nanoprobe alignment. This is made possible by the elongated nanoparticle geometry that causes anisotropic absorption and scattering. When applying linearly polarized incident light, this effect allows for deducing the actual nanoprobe orientation in the sample solution via transmission measurements. By correlating the measured actual nanoprobe orientation with the momentary vector of the applied RMF, the phase lag angle can be determined, and our signal is defined by the change in phase lag angle (Δα) between the sample and a suitable reference 13. The extracellular sHER2 protein is shed into the blood stream so that it can be found in serum as well as in saliva samples, currently being mainly of interest for the diagnosis as well as for the prognosis of breast cancer15. Currently, the clinical cut-off value for sHER2 in serum is 170 pM14, while the clinical cut-off value for saliva is one order of magnitude below the serum value15.As model protein we have chosen the soluble domain of the human epidermal growth factor receptor 2 - sHER2, which is the extracellular domain of HER2, a receptor-like tyrosine kinase that is reported to be involved in several types of human carcinomasIn the following sections, we describe the measures that are taken to detect the sHER2 analyte protein in complex samples of serum and saliva, which were spiked with sHER2. These measures include the right choice of type and concentration of secondary antibodies as well as the dilution factor of the complex sample solutions. Finally, we conclude by summarizing the major results and by giving an outlook on how to further apply and improve the measurement method.16, which screens the measurement effect of bound analyte molecules. Hence, additional measures had to be taken to gain specificity. This can be achieved by employing secondary antibodies (2nd Abs) in a sandwich-type immunoassay format .In an actual assay recipe, a fixed concentration of added 2Furthermore, the assay can be optimized by carefully choosing the best suited antibody pairs. To that end, we tested all combinations of Ab-a and Ab-b as primary and secondary antibodies. Corresponding measurement results are shown in the Supplementary Table 17. In addition, the nanoprobe rotation is also influenced by the concentration of non-bound proteins in solution, thereby affecting the drag torque and measured phase lag18. Consequently, by adjusting the dilution factor, the measurement signal can be maximized. To that end, different saliva dilutions were tested. Here, we have chosen a high initial concentration of sHER2 of 10 nM spiked into pure saliva to ensure that the concentration after dilution remains high enough to be easily detected. On the other hand, the initial concentration was chosen low enough to guarantee less than 5 nM even in low sample dilutions for optimal signal enhancement by the 2nd Abs as shown above. The obtained measurement signals at different dilutions of saliva are shown in Supplementary Table The next aspect to be addressed in order to optimize the assay for complex sample solutions like saliva and serum is the determination of the optimum dilution factor. Dilution is an important aspect of our measurement approach as the structure and the composition of the protein corona depends on the types and the concentrations of the proteins present in the sample solution and, therefore, on the sample dilutionBased on the information gained by the results presented above, we conducted phase lag difference measurements in dependence of the analyte concentration in solutions of pure buffer, serum, and saliva. For all these measurement series, we determined the relevant limits of detection.nd Abs were also added. In this case, the signal saturated at about 5 nM sHER2 concentration, and decreased for higher concentrations. This decrease is due to the now non-optimal ratio of 2nd Abs to analyte molecules (see above). The achieved limits of detection were about 400 pM for the assay without 2nd Ab addition and about 170 pM after addition of 2nd Abs.Figure nd Abs. Generally, the results compared well with the ones obtained with 2nd Ab addition in buffer solution. Specifically, the signals again rose up to a saturation level of about 5 nM sHER2 concentration. While in diluted serum, we deduced a limit of detection of about 470 pM, in diluted saliva samples sHER2 could be detected down to about 110 pM. The reason for the better measurement sensitivity in saliva compared to serum we attribute to different protein composition of serum and saliva samples and the difference in total protein content19. Specifically, the lower reference phase lag in saliva allowed for a larger relative phase lag increase due to analyte and 2nd Ab binding12. We ascribe the slightly better detection limit in diluted saliva samples compared to buffer solution to adsorption of tertiary proteins to the bound 2nd Abs, which further increases the signal.Finally, we conducted assay measurements in 10-fold diluted solutions of spiked serum and saliva with addition of 2in-situ reference measurements by simply not adding 2nd Abs, which is an important feature for PoC testing environments.In this work, we demonstrated that our previously introduced nanoprobe-based homogeneous biosensing approach is also feasible to specifically detect analyte proteins in complex sample solutions . While in buffer solution, direct analyte quantification is possible, the formation of a protein corona in complex samples screens the direct measurement signal. Consequent, the increase in hydrodynamic diameter due to antibody-mediated binding of target protein cannot be distinguished from non-specific adsorption (i.e. the formation of a protein corona). However, upon addition of secondary antibodies to the sample solution, there again is a specific further increase in hydrodynamic nanoprobe size, which results in a detection signal. Thus, what at first glance seems to be a disadvantage, can in fact be exploited to conduct 14, and, due to the substantially lower concentration, about two orders of magnitude above the clinical cut-off value for saliva15. However, our results demonstrate that by advancing the nanoprobe assay conditions, the detection limit can still be improved substantially. For example, in buffer solution we observed a significant enhancement of the signal and detection limit by introducing 2nd Abs, which can be expected to further improve by applying labelled 2nd Abs to bind large molecular weight biopolymers. In addition, while we tested some antibody combinations and observed a substantial effect on our measurement signal, there is a wide variety of different antibodies available to determine the best-suited pair.We systematically elaborated measurement conditions to determine sHER2 analyte protein directly in spiked complex sample solutions by establishing a homogeneous sandwich assay directly at the surface of our dispersed nanoprobes. Recalculated to undiluted samples, our sHER2 limit of detection currently is about 4.7 nM in serum and 1.1 nM in saliva, which is about one order of magnitude above the clinical cut-off value for serumIn summary, for our simple mix-and-measure type homogeneous biosensor we succeeded to overcome a major hurdle that nanoparticle biosensors often suffer from, which concerns signal screening by protein corona formation. This opens up numerous applications in protein biomarker detection, specifically in PoC settings where resources are limited and ease-of-use is of essence.20. Afterwards, the NRs were transferred to aqueous solutions by over-coating them with an amphiphilic polymer as described earlier21. The functionalization of the NRs with antibodies (Abs) was achieved by carboxy-amine linker chemistry. To that end, we employed EDC (N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride) and S-NHS  at ratios of 1 × 104 EDC molecules and 3 × 104 S-NHS molecules per NR. The NR concentration was determined by inductively coupled plasma mass spectrometry and by the geometric NR dimensions determined by transmission electron microscopy as described elsewhere10. We employed a NR concentration of 730 pM and 2-(N-morpholino)-ethanesulfonic acid (MES) buffer solution at a concentration of 50 mM and at a pH of 5.5 throughout the whole functionalization procedure. In a first functionalization step, the NRs were incubated with EDC and S-NHS for 15 min at room temperature (RT) and then dialyzed against MES for 30 min at RT to reduce the concentration of EDC and S-NHS. The dialysis was performed with a Float-A-Lyzer G2 dialysis device with an approximate molecular weight cut-off value of 1000 kDa. Afterwards, the Abs diluted in MES were added at a volume comparable to the NR/EDC/S-NHS solution at a ratio of 200 Abs per NR, and the whole solution was incubated for 100 min at RT. Abs applied here were either trastuzumab in the form of the Herceptin therapeutic drug (Ab-a) or commercial HER2 antibodies (Ab-b) purchased from R&D Systems under catalogue number AF1129. Here, Ab-a is a monoclonal antibody, while Ab-b is of polyclonal nature. In a next step, 100 bovine serum albumin molecules per NR were added, followed by another incubation step overnight at 4 °C to block any remaining binding sites. Finally, to remove all unbound reagents, the functionalized NRs were dialyzed against MES at a pH of 5.5 for 48 h at 4 °C with a change of the dialysis solution after 20 h.The nanoprobe preparation comprises the synthesis of bare Co nanorods (NRs), which were covered by a noble metal shell of Au and Pt according to already published procedures22. All methods were performed in accordance with the relevant guidelines and regulations. Saliva was filtered by a Whatman glass fibre syringe filter with a nominal pore size of 0.45 μm and stored in the fridge until usage. Filtering of the saliva was necessary to reduce the viscosity by removing glycoproteins and larger molecules without losing analyte molecules23. This procedure allows for preparing samples of a viscosity level comparable to serum by a very simple way suitable for PoC analysis.The employed serum  was obtained commercially from Sigma-Aldrich (product number H4522), while unstimulated whole saliva was collected in the morning from a single healthy male individual who refrained from eating before sample taking. The non-invasive saliva sample has been donated by a staff scientist of AIT following informed consent and approval by the Local Ethics Committee of the City of Viennand Abs) were added, and the whole sample was incubated for another 60 min at RT. The overall sample volume amounted to 240 µl, and the nanoprobe concentration was 10 pM. Measurements were conducted at a RMF amplitude of 10 mT and a rotational frequency of 1 kHz. These parameters are chosen for optimal measurement signals to detect target proteins by our specific nanoprobes24. Phase lag differences were recorded with respect to a suitable reference value. For the measurements executed in buffer solution only, we employed reference samples comprising all reagents except of the analyte sHER2 molecules. In complex sample solutions of serum and saliva, the reference phase lag value was determined with samples containing no secondary antibodies. Addition of the secondary antibodies only without analyte molecules did not result in a measurable signal. The errors of each measurement were determined by the standard deviations of the reference and the respective analyte-spiked samples and the error propagation law.The measurement buffer (MB) at a pH value of 7.4 was composed of 10 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) sodium salt, 150 mM NaCl and 0.05% v/v Tween 20. Samples spiked with sHER2 were prepared by mixing the MB or dilutions of serum/saliva (diluted by the MB) with the sHER2 antigen at a chosen concentration. Next, the nanoprobes were added to the sample solution. After an incubation time of 30 min at RT, the secondary antibodies diluted in MB  to the reference phase lag. Next, the assay results of the measured phase lag differences were fitted by a 4-parameter logistic fit model, and the limit of detection was obtained by using the threshold value in the fitting curve equation.Supplementary Information


To test the hypotheses that community-dwelling veterans with spinal cord injury (SCI) who receive the Wheelchair Skills Training Program (WSTP) in their own environments significantly improve their manual wheelchair-skills capacity, retain those improvements at one year and improve participation in comparison with an Educational Control (EC) group.We carried out a randomized controlled trial, studying 106 veterans with SCI from three Veterans Affairs rehabilitation centers. Each participant received either five one-on-one WSTP or EC sessions 30–45 minutes in duration. The main outcome measures were the total and subtotal percentage capacity scores from the Wheelchair Skills Test 4.1 (WST) and Craig Handicap Assessment and Reporting Technique (CHART) scores.Participants in the WSTP group improved their total and Advanced-level WST scores by 7.1% and 30.1% relative to baseline (p < 0.001) and retained their scores at one year follow-up. The success rates for individual skills were consistent with the total and subtotal WST scores. The CHART Mobility sub-score improved by 3.2% over baseline (p = 0.021).Individualized wheelchair skills training in the home environment substantially improves the advanced and total wheelchair skills capacity of experienced community-dwelling veterans with SCI but has only a small impact on participation. Wheelchairs are among the most important of rehabilitation interventions . WheelchOne way to enhance the benefits and minimize the problems of wheelchair use is better wheelchair provision. The World Health Organization (WHO) guidelines on wheelchair provision  include Despite the well-recognized importance of training, there is a surprisingly low prevalence and/or intensity of wheelchair skills training –20. EarlOne available resource for addressing this problem is the Wheelchair Skills Training Program (WSTP) . The WSTWhat remains under-represented in the literature are RCTs with large sample sizes, RCTs for different training venues , follow-ups of more than three months, active versus standard-care control groups, evidence of a cause-and-effect relationship between training and participation outcomes and RCTs for specific populations .Our primary objective was to test the hypothesis that community-dwelling veterans with SCI who receive the WSTP in their own environments significantly improve their manual wheelchair-skills capacity in comparison with an Educational Control (EC) group. Our secondary objectives were to describe differences in the success rates for individual skills, to test the hypothesis that any improvements would be retained at one year, and to test the hypothesis that such training has an impact on participation.2 = 0.07), tested with two-tailed two-sample t tests of pre- vs post-training change scores and a conservative Bonferroni-adjusted α of 0.0125. Calculations showed that 94 participants would be needed for such an analysis. We oversampled, in anticipation of drop-outs between the baseline and post-training assessments.We studied community-dwelling veterans with SCI who used manual wheelchairs, a sample of convenience. A power analysis was conducted using Cohen’s methods, conventions as implemented in Power Analysis and Sample Size (PASS) software  and assuAn un-blinded RCT design was used for this multi-site study. CONSORT guidelines were followed  see S1 .Gail.Powell-Cope@va.gov). Data will be available upon request to all interested researchers who meet the VA policy guidelines.The Clinical Trial Registration Number is NCT00434018. The original protocol can be viewed in The three sites were the James A. Haley Veterans’ Hospital in Tampa, Florida, the Charlie Norwood Veterans Administration (VA) Medical Center in Augusta, Georgia and the West Roxbury campus of the VA Boston Healthcare System in Boston, Massachusetts.Ethical approvals for this study were obtained from the Institutional Review Boards (IRBs) of each of the three participating centers—the Institutional Review Board of the James A. Haley Veterans’ Hospital in Tampa, FL (IRB #006372), the Office of Human Research Protection, Human Assurance Committee (HAC), Medical College of Georgia, Augusta GA (HAC #0705290), and the Veterans Affairs Boston Health Care System IRB—Human Studies Subcommittee (IRB # 2030). Each participant provided his/her written informed consent prior to any study participation.Participants were recruited by recruitment flyers, word of mouth and review of health records for individuals who met eligibility criteria. Potential participants who met initial criteria were mailed letters informing them of the study and asking any interested individuals to contact the study coordinator for additional information. To ensure the privacy of potential participants, no identifying information was utilized until the participants provided consent to participate in the study.Each participant was a veteran, had a SCI for at least one year, had a level of injury at C6 and below, used a manual wheelchair as the primary means of mobility, was able to self-propel the wheelchair, was between the ages of 18 and 75 years, was living within 241 km (150 miles) of the research site, was able to follow simple instructions and was willing to participate (as manifested by providing informed consent and completing the baseline [T1] assessment). Potential participants were excluded if they had a progressive disease, had a cardiac or respiratory condition that limited physical performance, had any unstable medical conditions or were pregnant.To describe the sample, we collected demographic and clinical data at intake by chart review and interview. We recorded age, sex, SCI injury level, duration of SCI, number of comorbidities , highest level of education completed, employment status, marital status, race/ethnicity and research site.Participants used the wheelchairs that they ordinarily used. No alterations were made by study personnel to optimize fit or function. Wheelchair specifications were recorded at T1.Participants were randomly assigned to either the WSTP or EC groups by using a computer-generated blocked randomization schedule. This was done to ensure that at no time during randomization was the imbalance large and that at certain points the number of participants in each group would be equal. At the end of baseline data collection, each participant was handed a sealed envelope that had the study-group assignment and the schedule for skills training or education.The WSTP Version 4.1 included 32 individual wheelchair skills (listed later) divided into three skill levels  . The WheThe trainers  were trained in WSTP administration. Wheelchair skills trainers familiarized themselves with the WSP website and received in-person practical training from the WSP developers. For any research personnel who joined the study later, the outgoing person and research coordinator at that site trained the incoming person.The initial participant training session provided the therapist with an opportunity to establish training goals based on the baseline evaluation of the participant’s skill level and his/her personal goals for training . ExampleThe EC intervention mirrored the WSTP in intensity, duration and process. The difference was in the content. Participants in the EC group received five home-based sessions about 45 minutes in duration that focused on health promotion for persons with SCI. The EC participants each had discussion with a research assistant  on the topics related to general wellness after SCI, including nutrition, pressure ulcer prevention, prevention of infections, prevention of respiratory complications and the importance of exercise.Education Control trainers received training from the research coordinators at each site. The material covered in the sessions was part of standard care for people with SCI and the research personnel were already well-versed in the content.Using principles of adult learning, each session began with an informal pre-test. The trainer then used a printed “Fact Sheet”, 3–10 pages of information that was discussed with the participant. The same Fact Sheets were used at all sites and with all participants. Examples of the content of such sessions were the importance of maintaining strength and range of motion for health and function and the importance of frequent weight relief from the buttocks as a means of preventing pressure ulcers. The sessions were individualized based on the pre-test and the specific health issues of the participant. During training, whenever possible, a significant other or caregiver was present. The session ended with an informal post-test and the participant received printed materials to keep.The WST has been highly ranked in independent surveys of such tests ,49 and iA number of participation measures are available for people who use wheelchairs ,6,53–55,We recorded any of the participants’ spontaneous comments that were of relevance to the training intervention.The enrollment process consisted of having interested individuals contact the site project manager who verified eligibility criteria, answered any study-related questions and obtained contact information in order for research staff members to schedule an initial visit. During the initial visit, the research staff verified eligibility criteria, obtained informed consent and collected demographic, clinical and wheelchair data. The participants were randomized to the WSTP or EC group. Participants were provided with either wheelchair-skills training or education in their own environments over a 5-week period. Data were collected at three time points: baseline (T1), early post-intervention  and 12 months post-intervention (T3). Scheduled phone calls every two months between T2 and T3 were used as a strategy to increase subject retention.Teleforms  were used for data entry, then data were verified for accuracy. Demographic, clinical and wheelchair data were reported descriptively for the T1 time point, comparing the two groups to assess comparability using Chi square for categorical data and two-sample t tests for continuous data. Two-sample t tests were used to compare the T1-T2 and T2-T3 latencies (in days) of the two groups. To assess whether there was a training effect due to the WSTP intervention, we used two-tailed, two-sample t tests to compare the groups’ change scores (T2-T1), initially using only data from the participants who completed the study. For these analyses, we looked at total and subtotal  WST scores and total and subscale CHART scores. Additionally, we used repeated measurement Analysis of Variance (ANOVA) to assess the interaction between group and time and multivariate models that included the baseline demographic and clinical variables.We assessed the effect of drop-outs by comparing the demographic and clinical characteristics of drop-outs with those who completed the study  and by conducting Intention to Treat (ITT) analyses on the WST outcome variables. For the two ITT analyses, we replaced missing values with either the previous value or the mean value for that group at that time point.To assess whether there was retention, we used paired t tests to evaluate the WSTP group with respect to the total and subtotal WST change scores (T3-T2). For each of the individual skills, we calculated the n (%) of participants in each group who were successful at each time point. We used a criterion of ≥20% difference between time points for our definition of a clinically significant difference  , 30. We All data analyses were completed in Statistical Analysis Software version 9.4 . A Type 1 error rate (α level) of <0.05 was used to define statistical significance. We elected not to Bonferroni-adjust the α level because the primary objectives were independent a priori; rather we reported the actual p values .The CONSORT  flow diaThe wheelchair specifications at T1 are shown in The total and subtotal WST scores for the participants who completed the study are shown in As shown in The same pattern of significant differences between the groups was seen when using the multivariate models for T2 vs T1 that included the baseline demographic and clinical characteristics except that the magnitudes of the differences were slightly higher—the mean (SD) total WST T2-T1 change scores were 5.9% (1.3) and 1.5% (1.2) for the WSTP and EC groups (p < 0.001); the Advanced-level subtotal WST T2-T1 change scores were 15.6% (3.1) and 4.4% (3.0) (p < 0.001). Although not statistically significant (p = 0.11), the Community-level change scores were also higher for the WSTP than the EC group, 3.0% (1.8) and 0.3% (1.7). There was no difference in this pattern for either of the ITT analyses.Regarding retention of training benefits, in addition to the T3-T2 data shown in ascends 15 cm curb and performs 30 s stationary wheelie) in the WSTP group only.Individual skill success rates for the two groups are shown in The total and subscale CHART scores for the participants who completed the study are shown in The participants in both the WSTP and EC groups generally reported their experiences as being beneficial. The WSTP group participants’ comments included that they appreciated being able to personalize their goals, that they would have never attempted trying some of the skills if they had not had someone to work with one-on-one and that they were able to participate in the comfort of their own environments. A selection of representative quotes were: “I’ve conquered my fear”, “I feel so empowered”, “I am not afraid anymore to be alone” and “I don’t have to rely on others to help me”. Transfer from floor to wheelchair was indicated by our participants as one of the most important skills for them to learn. For the EC group, there was educational content that some of the participants said they had never heard before.There were no adverse incidents affecting the participants during assessment or training activities. One trainer reported injuring his back while moving study equipment in and out of the van for a home visit. Reporting of the incident followed policy and the trainer was placed on light duty responsibilities during his recovery period.The mean total WST scores were high, but roughly similar to those reported for non-veteran manual wheelchair users with SCI –25. Of tThe hypothesis that training would increase WST capacity scores was corroborated for the total and Advanced-level WST scores. For the total WST scores, the mean absolute magnitudes of T2-T1 improvement (from the multivariate models) were 5.9% and 1.5% for the WSTP and EC groups respectively, corresponding to relative improvements over baseline of 7.1% and 1.7%. For the Advanced-level WST scores, the mean absolute magnitudes of T2-T1 improvement were 15.6% and 4.4% for the WSTP and EC groups respectively, corresponding to relative improvements over baseline of 30.1% and 6.9%. These relative improvements are similar in magnitude to previous WSTP training RCTs for other populations of manual wheelchair users –32.There were slight improvements in the WST scores of the EC group. This phenomenon has been previously reported –32, but In choosing to study community-dwelling veterans , we antiSupporting the hypothesis that any training-induced improvements would be retained at follow-up one year later, the within-group paired t test between T2 and T3 for the WSTP group showed no significant decline.ascends 15 cm curb and performs 30 s stationary wheelie, both at the Advanced level) that met our criterion of ≥ 20% difference in success rate between T1 and T2. For both of these, the T2 success rates were higher. No skills met this criterion for the EC group. There were no skills in either group for which the success rate dropped by ≥ 20% between T2 and T3, supporting the retention hypothesis. At T1, the success rates for 8 (25%) of the 32 skills  were <75% (our criterion for a low success rate) for both the WSTP and EC groups. This is comparable with earlier US studies of people with SCI [The success rates for individual skills were consistent with the findings for the total and subtotal WST scores. For the WSTP group, there were 2 (6%) of the 32 skills , it is of interest that wheelchair skills can be taught or relearned even years after injury and that the participants made positive comments about their training experiences. This may be because the early phases of rehabilitation can be busy and wheelchair skills training is only one of many issues that require attention. Wheelchair users may have more time and be more receptive to wheelchair skills training after they are settled in their communities but our study suggests that there remains room for improvement many years after injury. Performing training in the home environment was effective and appreciated by the participants.The CHART data provided insights about the participation levels of our participants. The six subscale scores and the total score were all high. In comparison with the Hosseini et al  CHART daThere were a number of study limitations, some of which have already been mentioned. Although an initial sample size of 106 is fairly large for a training study on participants with SCI who were followed for one year after training, the moderate number of drop-outs likely reduced the power of the study. However, we explored whether missing data due to drop-outs skewed our findings. We compared the demographic and clinical characteristics of the participants who completed the study and those who dropped out and found only level of education to differ, with the drop-outs having a lower level of education. As for the ITT analyses, neither of the two methods used provided results that were appreciably different from the results of the participants who completed the study.We did not ask participants about any wheelchair skills training that they had received before the study, either as components of their initial rehabilitation or subsequently from the VA or another source. However, the VA provides the full continuum of care to veterans with SCI, including initial comprehensive rehabilitation. Wheelchair training is typically provided as an integral part of initial rehabilitation . Some veWe also did not assess the appropriateness or fit of the wheelchairs that our participants used. However a recent study reported that people with SCI with the VA as the payer source consistently received high quality and appropriately customized wheelchairs and the VA was the only payer group for which all beneficiaries received wheelchairs that met standard of care .There was a slight statistically significant difference in the T1-T2 latencies (longer for the WSTP group) and an almost significant difference (p = 0.052) for the T2-T3 latencies (longer for the EC group). However, the magnitudes of the differences were small and it is unlikely that they affected the conclusions that can be drawn from the study. Although the demographic and clinical data of our participants differed slightly from those reported in other US studies of participants in SCI Model Systems sites ,25, suchThe failure to blind testers to group allocation was a limitation; blinding of participants to the nature of their training would not have been possible. The failure to blind testers was due to budgetary constraints. The assessments were often carried out in the participants’ home environments by the same persons who conducted the training. The objective nature of the main outcome measure (the WST) was felt to mitigate this limitation. In the future, if similar constraints are an issue, we recommend achieving tester blinding by using the WST-Q by telephone.Although we did not collect data on the cost of the WSTP intervention as we used it, it is likely that home training is more expensive to the healthcare system than hospital-based training due to the travel time and costs. Also, one-on-one training is likely to be more expensive than group training  because Given the high initial WST scores, there may have been a ceiling effect. In future studies, we recommend using the WSP Goal Attainment Score (GAS)  as a comFuture studies will need to address these limitations. Additionally, research explorations of the low prevalence –20 and dIn addition to manual wheelchair skills training, there is growing evidence of the need for and effectiveness of powered wheelchair skills training ,64. MostIn spite of the limitations of the current study and the need for future work, this is the first study to demonstrate the effectiveness and safety of one-on-one wheelchair skills training in the home environment for community-dwelling veterans with SCI.The implications of our findings in general are that such training should be made available to any person with SCI who uses a wheelchair, even if the person has been using a wheelchair for many years. As one means of accomplishing this objective, there should be more focus on wheelchair skills training in professional schools. The need for and effectiveness of such training has been previously reported –69. SCI Individualized wheelchair skills training in the home environment improves the advanced wheelchair skills capacity of experienced community-dwelling veterans with SCI by 30% over baseline scores although it has only a small impact on participation levels. These findings suggest the need to consider a more formal approach to wheelchair skills assessment and training within the VA systems of care.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.


Several neurodegenerative diseases are driven by the toxic gain-of-function of specific proteins within the brain. Elevated levels of alpha-synuclein (α-Syn) appear to drive neurotoxicity in Parkinson's disease (PD); neuronal accumulation of tau is a hallmark of Alzheimer's disease (AD); and their increased levels cause neurodegeneration in humans and model organisms. Despite the clinical differences between AD and PD, several lines of evidence suggest that α-Syn and tau overlap pathologically. The connections between α-Syn and tau led us to ask whether these proteins might be regulated through a shared pathway. We therefore screened for genes that affect post-translational levels of α-Syn and tau. We found that TRIM28 regulates α-Syn and tau levels and that its reduction rescues toxicity in animal models of tau- and α-Syn-mediated degeneration. TRIM28 stabilizes and promotes the nuclear accumulation and toxicity of both proteins. Intersecting screens across comorbid proteinopathies thus reveal shared mechanisms and therapeutic entry points.DOI:http://dx.doi.org/10.7554/eLife.19809.001 Behind many neurodegenerative diseases are specific proteins that abnormally accumulate inside neurons and damage the cells. In Parkinson’s disease, the protein alpha-synuclein accumulates; in Alzheimer’s disease, the protein tau is one of the toxic culprits; and in other neurodegenerative diseases, alpha-synuclein and tau both accumulate. Genetic studies suggest that accumulation of the two proteins may be linked, but little is known about the factors that regulate the levels of these proteins inside neurons.Rousseaux et al. set out to identify how these proteins are regulated in the hope of finding new ways of targeting them and reducing their toxicity. Screening a subset of human genes led to one that encodes a protein called TRIM28, which regulates the levels of both alpha-synuclein and tau. When the TRIM28 protein was depleted in human and mouse cells, the levels of alpha-synuclein and tau also went down. This effect was specific because the levels of other proteins with the potential to cause neurodegeneration remained unaffected.Models of neurodegenerative disease in fruit flies and mice were then used to explore how TRIM28 affects the levels of tau and alpha-synuclein in animals. In each case, the proteins’ levels dropped when TRIM28 was suppressed and this in turn protected the neurons from damage. Rousseaux et al. went on to show that TRIM28 affected how alpha-synuclein and tau were cleared in cells. Overexpressing TRIM28 revealed that it could encourage both alpha-synuclein and tau to accumulate in the nucleus of cells over time.Finally, Rousseaux et al. compared post-mortem brain tissue from people who had neurodegenerative conditions that are driven by or associated with tau and alpha-synuclein with tissue from those who did not. The cell nuclei in the diseased tissue had much more TRIM28 associated with alpha-synuclein and tau than those in the healthy tissues.Overall, the findings show that TRIM28 promotes the accumulation and damaging effects of both alpha-synuclein and tau. The next steps will be to understand how TRIM28 does this. It will also be important to determine if this effect can be targeted, whilst leaving others roles of TRIM28 intact, in order to explore it as a potential target to treat or prevent neurodegenerative diseases.DOI:http://dx.doi.org/10.7554/eLife.19809.002 SNCA) is now thought to mediate neurotoxicity not only in Parkinson’s disease (PD), but also in Parkinson’s Disease Dementia (PDD) and Lewy Body Dementia (LBD) where it accumulates in Lewy bodies (MAPT) accumulates not only in neurons affected by Alzheimer’s disease (AD), but in PD and PDD, while mutations that increase the stability of tau result in frontotemporal dementia with parkinsonism (FTDP-17 (or FTD) , previously characterized as missing all but 12 bp of exon 1 and the first 324 bp of intron 1. We confirmed that Trim28LOF-1 is a loss of function allele of bonus because it failed to complement Trim28LOF-2, which has already been described as a bonus LOF allele . Tyrosine hydroxylase and Nissl staining in both the SNc and Striatum was performed as previously described . Brains were frozen in O.C.T. compound (Tissue-Tek) and 30 µm free-floating sections were cut from the striatum and Substantia Nigra escribed . UnbiaseAn experimenter blind to the treatment or mouse genotype injected 6–8 week old α-Syn and tau transgenic mice (mThy1-α-Syn and P301S tau) and their respective wild-type littermate controls bilaterally with virus harboring TRIM28-mCherry or mCherry as a control into the CA1 region of the hippocampus . One monImmunoprecipitation of protein complexes was performed as previously described . Briefly4 cells) were transfected with 250 ng of ‘Prey’ plasmid . Cells were monitored for fluorescence for 48–72 hr (fluorescence begins around 24–30 hr) and cells were either collected for flow cytometry analysis  or fixed for fluorescence microscopy. Cells visualized for fluorescence microscopy were permeabilized and stained for DAPI to visualize nuclei and epifluorescence from the complementation was monitored.Bimolecular fluorescence complementation (Split-YFP assay) was performed as previously described with some modifications . N-termiThe relative amount of nuclear versus cytoplasmic stained protein (α-Syn or tau) was performed in primary granule neurons previously infected (for 7 days) with lentiviral constructs overexpressing mCherry, TRIM28:mCherry or TRIM28-Mut:mCherry on coverslips. The nucleus was outlined using DAPI and the cytoplasmic outline was determined by DIC (not shown). For each biological replicate, at least 15 cells were sampled and the nuclear fraction of each cell was calculated as the intensity of the nuclear localized fluorophore divided by the intensity of the fluorophore spanning the cytoplasm and the nucleus  and multiplied by 100. Three independent coverslips per condition were tested and statistics were based on these three biological replicates. Alternatively, postmortem medial frontal gyrus tissue was stained for α-Syn or tau together with TRIM28 and nuclei were visualized using DAPI. Positive nuclear co-localization was set as a threshold of staining co-localizing with DAPI. At least 150 cells over the span of 5 fields (at 40x magnification) were counted per human case.Nuclear/Cytoplasmic fractionation was carried out in freshly spun down cells 7 days following infection . Briefly, the NE-PER Nuclear and Cytoplasmic Extraction Kit  was used to isolate nuclear and cytoplasmic fractions of primary neurons or tissue. Of note, the protocol of the NE-PER kit was modified to incorporate an additional PBS-wash of the nuclear pellet following the cytosolic extraction as we found it increased purity of the preparation. Probing with antibodies specific to each compartment (i.e. Lamin A and histone H3 for the nuclear fraction and Vinculin and GAPDH for the cytoplasmic fraction) was used as a measure of extraction purity and for normalization.Tissue from patients with PD, AD, PSP and control subjects were obtained from the Neuropathology Core at the Johns Hopkins Udall Centre. Tissue was obtained from consenting donors and use conformed to JHMI Institutional Review Board approved protocols. Neuropathological assessment conformed to the National Institute on Aging-Reagan consensus criteria and similar post-mortem intervals were used between samples. For this study, tissues from the frontal cortex  or pons (for PSP and their respective controls) was utilized. Please see t-test was used whereas for multiple comparisons, ANOVA followed by the appropriate post hoc analysis were utilized. The summary of all statistical analysis is presented in Experimental analysis and data collection were performed in a blinded fashion whenever possible. The sample size was chosen based on previous studies using the models described in the study in order to ensure adequate statistical power. Pre-determined exclusion criteria were used in the nuclear/cytoplasmic biochemical fractionation by determining purity of the prep. If the fractionation was not adequate , sample was re-fractionated or omitted from the study. Tissue sections with high background staining/staining artifacts were removed and/or re-stained for the analysis. Randomization together with blinding was used for histological quantification as well as human case studies. Randomization was done using Microsoft Excel and blinding was performed by assigning new non-descript codes to cases. Unblinding was only done following analysis. Outliers for histology were removed if there was a lack of detectable viral expression in the animal and if the sample met the Grubb’s outlier test p-value. p values were determined using the appropriate statistical method via GraphPad Prism, as described throughout the manuscript. For simple comparisons, two-tailed Student’s  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "TRIM28 regulates the nuclear accumulation and toxicity of both α-synuclein and tau" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:In a shRNA screen, Rousseaux et al. identify TRIM28 as a regulator of α-synuclein and tau, two proteins involved in neurodegeneration. The authors' data suggests that deceasing levels of TRIM28 decreases levels of these two proteins and their toxicity, and conversely increasing levels has the opposite consequences. The authors go on to further investigate the role of TRIM28 on the intracellular localization of synuclein and tau, potential physical interactions, and potential effects of TRIM28 on the half-lives of these proteins. Overall the manuscript presents interesting and novel data.Essential revisions:1) In several places the addition of experimental details would greatly enhance the readability of the manuscript. Several places where details would help are listed in individual reviews.2) The authors show data that TRIM28 acts on synuclein and tau stabilization. However, there is much literature on other functions of TRIM28 in gene regulation and epigenetic control. Data from experiments described in this manuscript could be consistent with an indirect function of TRIM28 on synuclein and tau. This should be acknowledged and commented on.3) The half-life experiment examining TRIM28's effect on synuclein and tau should examine transcript levels of these genes in this experiment (and in experiments utilizing primary neurons or transgenic mouse brains). Since vinculin is used as a control, it's half-life should also be described. Also how do the author's rule out that translational changes  are not affected by TRIM28.4) In terms of determining whether the ligase activity of TRIM28 is necessary for nuclear localization of tau and synuclein the authors examine mutant TRIM28. These results need to be interpreted in light of whether the mutant protein still interacts with tau and synuclein.5) Finally, the authors suggest that changes in TRIM28 levels and localization may be a "driving force" in nuclear accumulation of synuclein and tau. However, the reviewers suggest that this data does not suggest causality. Additional important questions regarding this experiments (see Reviewer 2 comments) should be addressed and the conclusions from TRIM28 localization changes in diseased human brain need to be reinterpreted in light of reviewer's comments.Reviewer #1:In a shRNA screen Rousseaux et al. found that TRIM28 regulate the localization of α-synuclein and tau. Further, the authors demonstrated that TRIM28 regulates the toxicity of these proteins in both the fly eye and mouse brain. Although the manuscript fails to elucidate mechanism, given that α-synuclein and tau are associated with neurodegenerative disorders, these findings are of interest to the neurodegeneration field. However additional data would strengthen the authors' findings. For example, the authors have obtained a conditional allele of Trim 28, what is the effect of complete deletion on the pathology associated with α-synuclein or tau expression. Does deletion of Trim28 have additional actions on the transcriptional profile that could secondarily affect stability of α-synuclein and tau?Using a Dox inducible cell line of α-synuclein and tau, the authors suggest that Trim 28 changes the stability of these proteins. Do the transcript levels change with Trim28 changes? What is the effect of Trim28 on endogenous levels of α-synuclein and tau. Is there a change in localization of these proteins in these cells which could explain the difference in protein decay?Other points:Overall the manuscript would benefit with more description of experiments within the text (this reviewer had to look in legends and in methods often). Examples are given below.Drosophila should be included in the text of the results. In panel B, were an n=15 used for each experiment and were there two experiments performed? What tissue is used in panel C? How much of a "partial" loss of function are the two Trim28 alleles i.e., how much wild type Trim28 remains?Reviewer #2:The current study by Rousseaux et al. identifies a novel regulator of both α-Synuclein and Tau protein levels, TRIM28. Using a number of in vitro and in vivo models the authors convincingly show that knock-down of TRIM28 is sufficient to reduce α-Synuclein and Tau levels and their associated neurotoxicity. Conversely, overexpression of TRIM28 increases α-Synuclein and Tau half-lives, and accordingly further exacerbates their neurotoxicity in vivo. Overall the authors are commended for very nicely executed, elegant experiments particularly in vitro and in the animal models and this work is of sufficient novelty and interest for a broad audience in the neurodegenerative disease field.That being said, we have reservations regarding the interpretation of the patient data presented in Reviewer #3:In this manuscript the authors report the results of a focused genetic screen looking for genes that co-regulate levels of tau and synuclein, proteins that contribute to multiple neurodegenerative diseases, and the identification of TRIM28. Decreasing levels of TRIM28 decreases tau and synuclein levels and toxicity, while overexpression has the opposite effect. The focus is on possible direct physical interactions that might mediate localization of tau and synuclein to the nucleus. Overall the findings are interesting and novel.1) The authors tested changes in levels of other degeneration-causing proteins and did not see any change. They conclude that TRIM28 is likely to be a key postranslational regulator of tau and synuclein. However, the list of proteins tested is small. There is a rather large literature on TRIM28 and its roles in multiple cellular processes. TRIM28 has also been shown to be a global regulator of many aspects of gene regulation and epigenitic control. I would encourage the authors to consider and comment on these activities and the possibility that effects on tau and synuclein might have an indirect component.2) The authors may want to define the term "druggable", as the definition may be different depending on the investigator.3) For the fly experiments involving tau overexpression in the eye, it would be useful to know that there are no effects on other GMR promoter-dependent phenotypes. This would act as a stand in for changes in transcriptional regulation. The climbing assay has similar issues in that a specific promoter is being used and TRIM28 is known to have effects on transcriptional regulation.4) Next, they moved to mice overexpressing synuclein in dopaminergic neurons. This experiment has a nice control in that there is a GFP included as an IRES element. This figure  shows th5) Finally, the authors used a TRIM28 heterozygote to look for dose-dependent effects on phosphorylated synuclein. Can they comment on why this assay was used as opposed to that in the above panels? Is this simply more sensitive? Does it reflect a particular pathology? If so, can they provide a reference for this biology?6) The authors did a half-life experiment suggesting that TRIM28 expression stabilizes synuclein and tau. It would be important to show that other proteins are not stabilized, and to know what Vinculin's half-life is in order for it to be a useful control. For example, if its half-life is very long, its levels would not change much even if it was being affected. Can the authors rule out effects on translation from existing transcripts? I just point this out because if translation is not inhibited , there could be effects on protein abundance over time that are translational, and would otherwise be scored as alterations in protein half-life.7) The authors asked if TRIM28's ligase activity is required for nuclear localization of tau and synuclein. They did this by expressing a mutant version of TRIM28 that has two cysteine mutations in the ring domain. As these cysteines play a structural role in the ring domain, these mutations could disrupt the overall folding/conformation of this domain. It is important to determine if mutant versions of TRIM28 still interacts with tau and synuclein. This would be an important experiment to distinguish between ligase-dependent mechanisms of regulation versus physical binding and recruitment to the nuclear compartment. If binding is lost it would be difficult to conclude more than that interactions are important.8) The authors saw increased levels of TRIM28 in an insoluble fraction in human disease brains and that tau and synuclein were enriched in nuclei. From this they suggested that "The aberrant change in TRIM28 levels and distribution could therefore be a driving force in the nuclear accumulation of α-Syn and tau. " To test this they look for colocalization of TRIM28 in nuclei of disease brains and see significantly more cells that have colocalization of synuclein, tau and TRIM28 than in controls. This result is interesting. But could it just be that TRIM28 becomes trapped in insoluble nuclear complexes with synuclein and tau in disease brains? I am not sure I see a link that is necessarily causal. Can the authors discuss why an increase in number of nuclei with TRIM28, synuclein and tau should be taken to indicate that TRIM28 is driving the process rather than being carried along for the ride as an interacting protein? Essential revisions:1) In several places the addition of experimental details would greatly enhance the readability of the manuscript. Several places where details would help are listed in individual reviews.We have expanded on our description of experimental details throughout the manuscript, particularly in the Results section.2) The authors show data that TRIM28 acts on synuclein and tau stabilization. However, there is much literature on other functions of TRIM28 in gene regulation and epigenetic control. Data from experiments described in this manuscript could be consistent with an indirect function of TRIM28 on synuclein and tau. This should be acknowledged and commented on.We now acknowledge and comment on a putative indirect mechanism in the Discussion section.3) The half-life experiment examining TRIM28's effect on synuclein and tau should examine transcript levels of these genes in this experiment (and in experiments utilizing primary neurons or transgenic mouse brains). Since vinculin is used as a control, it's half-life should also be described. Also how do the author's rule out that translational changes  are not affected by TRIM28.SNCA and MAPT transcripts  In terms of determining whether the ligase activity of TRIM28 is necessary for nuclear localization of tau and synuclein the authors examine mutant TRIM28. These results need to be interpreted in light of whether the mutant protein still interacts with tau and synuclein.We tested the binding affinity of α-Syn and tau for TRIM28 in the context of this E3-ligase mutant and found that, while TRIM28 E3-ligase mutant was itself less stable (as previously noted in 5) Finally, the authors suggest that changes in TRIM28 levels and localization may be a "driving force" in nuclear accumulation of synuclein and tau. However, the reviewers suggest that this data does not suggest causality. Additional important questions regarding this experiments (see Reviewer 2 comments) should be addressed and the conclusions from TRIM28 localization changes in diseased human brain need to be reinterpreted in light of reviewer's comments.We agree that human data from post mortem tissue rarely suggests causality  and have thus toned down the interpretation in the Results and Discussion.Reviewer #1:In a shRNA screen Rousseaux et al. found that TRIM28 regulate the localization of α-synuclein and tau. Further, the authors demonstrated that TRIM28 regulates the toxicity of these proteins in both the fly eye and mouse brain. Although the manuscript fails to elucidate mechanism, given that α-synuclein and tau are associated with neurodegenerative disorders, these findings are of interest to the neurodegeneration field. However additional data would strengthen the authors' findings. For example, the authors have obtained a conditional allele of Trim 28, what is the effect of complete deletion on the pathology associated with α-synuclein or tau expression. Does deletion of Trim28 have additional actions on the transcriptional profile that could secondarily affect stability of α-synuclein and tau?We thank the reviewer for their constructive feedback. Given that complete deletion of TRIM28 is embryonic lethal, we employed Trim28 heterozygous mice as a model to genetically control for TRIM28 levels. Although conditional null mice have transcriptional changes , we suspect that a heterozygous mouse should have much less effects on global transcriptional changes, though we cannot rule this out without performing a large scale profiling experiment on the brain which is beyond the scope of this study.Using a Dox inducible cell line of α-synuclein and tau, the authors suggest that Trim 28 changes the stability of these proteins. Do the transcript levels change with Trim28 changes? What is the effect of Trim28 on endogenous levels of α-synuclein and tau. Is there a change in localization of these proteins in these cells which could explain the difference in protein decay?SNCA and MAPT transcript levels in the Dox-inducible cell line in response to TRIM28 expression. We found that the RNA decay rates were not altered upon TRIM28 overexpression suggesting that TRIM28 acts post-translationally . Examples are given below.We have clarified certain experiments throughout the text. We have answered these point-by-point below.We used primary cerebellar granule neurons from P7-9 pups. This has been clarified throughout the text and in the Methods section. Each dot in the bar graphs represents an individual experiment .Drosophila dsTrim28 is under the control of a UAS promoter and is expressed using the Gal4/UAS system. To avoid potential artifacts due to titration of Gal4 we routinely use a neutral UAS (UAS-eGFP), in order to achieve similar expression of tau in both the control (UAS-tau/UAS-GFP) and the experimental (UAS-tau/UAS-dsTrim28).The genotypes for the panels in Drosophila transgenic lines have not been previously published. A detailed description of how we generated them is included in the Drosophila Methods under the “Materials and methods” section as a “human wild-type 4 repeat tau under the control of a UAS promoter”.These tau Drosophila retina expressing tau/GFP or tau/dsTrim28).The legend for LOF-1 is a loss of function allele of bonus because it fails to complement Trim28LOF-2, which has already been described as a bonus null allele . These alleles show a 50% reduction in heterozygosis.The description of the Trim28 alleles used is in the Methods section. Trim28We have clarified this in the Results section. Both native and transgenic α-Syn are highly expressed in the hippocampus and its clear phosphorylation in this location is more readily quantifiable .We cannot definitively say that there is neuronal loss following Trim28 overexpression as NeuN staining on paraffin sections hasn’t worked nicely in our hands. That said, previous reports suggest that NeuN loss in this mouse model is accompanied by astrocyte accumulation . Given that we observe an increase in astrocytic number in the CA1 (with a concomitant decrease in CA1 thickness), this would be suggestive of cell loss. We have not tested whether neurons themselves are dying as the mechanism through which α-Syn and tau cause the demise of neurons  in these models is not entirely clear.We have clarified this in the Results section to explain the system in more detail. SH-SY5Y cells stably expressing the pINDUCER constructs (G418 selected) were used.We agree that these results suggest that TRIM28 forms a complex with α-Syn and tau but does not mean that they directly interact. While we have had difficulty in purifying recombinant TRIM28 to test the direct interaction, we have now tested the E3-ligase mutant in the context of the immunoprecipitation experiment. We find that, while the C65A/C68A mutant is less stable, it can still form a complex with α-Syn and tau .Reviewer #2:et al.αin vitroin vivoααin vivoin vitro[…]Overall, looking at the biochemical data we do not see a clear increase in overall TRIM28 levels under disease conditions. The authors indeed find a statistically significant shift of TRIM28 to the insoluble form in these conditions, but we wonder how TRIM28 could participate in actively bringing α-Synuclein/Tau into the nucleus if it's bound up in aggregates, presumable inactive. By staining , the nucWe thank the reviewer for their appreciation of this work and its impact. We agree that pathological analysis of human tissue data in the context of TRIM28 can be difficult to interpret and have thus toned down our conclusions to make them more modest (Results and Discussion).Reviewer #3:[…]1) The authors tested changes in levels of other degeneration-causing proteins and did not see any change. They conclude that TRIM28 is likely to be a key postranslational regulator of tau and synuclein. However, the list of proteins tested is small. There is a rather large literature on TRIM28 and its roles in multiple cellular processes. TRIM28 has also been shown to be a global regulator of many aspects of gene regulation and epigenitic control. I would encourage the authors to consider and comment on these activities and the possibility that effects on tau and synuclein might have an indirect component.We now comment more on the potential indirect effect of TRIM28 on α-Syn and tau in the Discussion section.2) The authors may want to define the term "druggable", as the definition may be different depending on the investigator.We have clarified our definition for “druggable” in the first paragraph of the Results section.3) For the fly experiments involving tau overexpression in the eye, it would be useful to know that there are no effects on other GMR promoter-dependent phenotypes. This would act as a stand in for changes in transcriptional regulation. The climbing assay has similar issues in that a specific promoter is being used and TRIM28 is known to have effects on transcriptional regulation.To address the reviewers concern we have performed qPCR on animals carrying Trim28 LOF alleles used in this study. All the results show that partial knockdown of Trim28 does not decrease the levels of Gal4 .4) Next, they moved to mice overexpressing synuclein in dopaminergic neurons. This experiment has a nice control in that there is a GFP included as an IRES element. This figure  shows thPlease see answer to point #1.5) Finally, the authors used a TRIM28 heterozygote to look for dose-dependent effects on phosphorylated synuclein. Can they comment on why this assay was used as opposed to that in the above panels? Is this simply more sensitive? Does it reflect a particular pathology? If so, can they provide a reference for this biology?We used this approach as a complementary approach to the dual-virus approach. This animal model recapitulates certain facets of PD that are not seen in the dual virus model such as pathological phosphorylation of α-syn. As such, we wanted to test whether reduction of TRIM28 could not only decrease cell toxicity but also pathological phosphorylation of α-syn. We have now referenced this model in more detail in the Results section as well as in the Materials and methods.6) The authors did a half-life experiment suggesting that TRIM28 expression stabilizes synuclein and tau. It would be important to show that other proteins are not stabilized, and to know what Vinculin's half-life is in order for it to be a useful control. For example, if its half-life is very long, its levels would not change much even if it was being affected. Can the authors rule out effects on translation from existing transcripts? I just point this out because if translation is not inhibited , there could be effects on protein abundance over time that are translational, and would otherwise be scored as alterations in protein half-life.SNCA and MAPT RNA decay is unaffected by TRIM28 overexpression status. These data are now presented as We have now tested RNA decay kinetics in the pINDUCER system following TRIM28 overexpression. We find that 7) The authors asked if TRIM28's ligase activity is required for nuclear localization of tau and synuclein. They did this by expressing a mutant version of TRIM28 that has two cysteine mutations in the ring domain. As these cysteines play a structural role in the ring domain, these mutations could disrupt the overall folding/conformation of this domain. It is important to determine if mutant versions of TRIM28 still interacts with tau and synuclein. This would be an important experiment to distinguish between ligase-dependent mechanisms of regulation versus physical binding and recruitment to the nuclear compartment. If binding is lost it would be difficult to conclude more than that interactions are important.We thank the reviewer for the good suggestion. We have tested the interaction of α-Syn and tau with either wild type TRIM28 or E3 ligase mutant TRIM28. We find that, while the TRIM28 E3 mutant itself is less stable, it can still interact with α-Syn and tau thus suggesting that the nuclear localization is at least partly mediated through its E3 ligase activity .8) The authors saw increased levels of TRIM28 in an insoluble fraction in human disease brains and that tau and synuclein were enriched in nuclei. From this they suggested that "The aberrant change in TRIM28 levels and distribution could therefore be a driving force in the nuclear accumulation of α-Syn and tau. " To test this they look for colocalization of TRIM28 in nuclei of disease brains and see significantly more cells that have colocalization of synuclein, tau and TRIM28 than in controls. This result is interesting. But could it just be that TRIM28 becomes trapped in insoluble nuclear complexes with synuclein and tau in disease brains? I am not sure I see a link that is necessarily causal. Can the authors discuss why an increase in number of nuclei with TRIM28, synuclein and tau should be taken to indicate that TRIM28 is driving the process rather than being carried along for the ride as an interacting protein?We agree that post-mortem data only offers a snapshot of time and cannot suggest causality. Thus, per the suggestions of reviewers #1 and 2, we have toned down our claim that TRIM28 drives the nuclear localization of α-Syn and tau in human disease.References:, et al. (2008). KAP1-mediated epigenetic repression in the forebrain modulates behavioral vulnerability to stress. Neuron 60, 818-831.Jakobsson, J., Cordero, M.I., Bisaz, R., Groner, A.C., Busskamp, V., Bensadoun, J.C., Cammas, F., Losson, R., Mansuy, I.M., Sandi, C.


The anti-allergic cromones were originally synthesized in the 1960s by Fisons Plc, and the first drug to emerge from this program, disodium cromoglycate was subsequently marketed for the treatment of asthma and other allergic conditions. Whilst early studies demonstrated that the ability of the cromones to prevent allergic reactions was due to their ‘mast cell stabilizing’ properties, the exact pharmacological mechanism by which this occurred, remained a mystery. Here, we briefly review the history of these drugs, recount some aspects of their pharmacology, and discuss two new explanations for their unique actions. We further suggest how these findings could be used to predict further uses for the cromones. It arose from a study of the anti-spasmodic properties of the Egyptian medicinal herb Khellin. The experimental trail leading to the discovery of the anti-allergic properties of this compound and its clinical validation by the Fisons’ pharmacologist, Roger Altounyan (himself an asthmatic), which entailed considerable self-experimentation, have become the stuff of pharmacological legend and will not be recounted here , as well as on eosinophils and basophils. However, its significance to asthma and allergy or to mast cell mediator release is yet to be elucidated, thus it is unclear how these actions of the cromones could be translated into therapeutic effects. Nevertheless, one of the downstream effects of GPR35 activation is activation of PKC so this mechanism would certainly contribute to the observed changes of phosphoprotein abundance in cells.Anx-A1/FPR system, which regulates cell activation in many cell types  and IgE antigen, leading to mast cell activation (The mechanism by which glucocorticoids (and other factors) promote the actions of Anx-A1 has also been investigated in detail . In manythe cell . Anx-A1 the cell . Recent tivation . This adtivation . InteresProteolysis is vital in determining the extent of secreted Anx-A1 action and disturbances of this process may be a trigger in some diseases. In Wegener’s granulomatosis (for example), extreme PMN activation could be trailed down to abnormal Anx-A1 cleavage by the PR3 protease .2 and histamine than wild type cells in response to degranulating stimulants  Btivation .27 residue showed that the effect seen was very rapid with a maximum activation of PKC and Anx-A1 phosphorylation occurring within 5 min. Whilst the cromones alone had little effect on Anx-A1 phosphorylation, they greatly potentiated the effect of the glucocorticoids . This was evident by the redistribution of GFP-tagged Anx-A1 within the U937 cells, extracellular accumulation of the protein and, dramatically, by the observed inhibition of TxB2 generation. For example, the inhibition of TxB2 release in U937 cells treated with 2 nM of dexamethasone alone was only <20%, but this inhibition was increased to almost 100% in the presence of 20 nM nedocromil at 5 min time point. Cromolycate also has similar glucocorticoid potentiating ability.The regulation of PKC activity may be accomplished through several potential mechanisms. The duration of action of PKC is limited by phosphatases such as PP2A. PKCα (and some other isoforms) interact with PP2A following activation in many cell types, including macrophage  modulatory/targeting sub-unit, which determines the specificity of the assembled enzyme complex. C-terminal carboxymethylation of the catalytic sub-unit at Leuomplexes . The enze action  which maly clear .2+ which might possibly be a target for the drugs which could conceivably interact with this metal being carboxylic acids (unpublished data). Interestingly, previous authors had already investigated the possibility that cromones inhibited PP2A, but were unable to detect an effect in their system . This mechanism was also observed to operate in vivo. Using Anx-A1 null mice, the ability of nedocromil to inhibit leukocyte migration in a model of peritoneal inflammation in mice was lost whilst they were fully active in the wild type  exhibit cross-tachyphylaxis, or share a similar pharmacology, with cromones .1 antagonist ketotifen. When tested, ketotifen was found to have the same effect on the Anx-A1 system as nedocromil and cromoglycate suggesting a commonality of mechanisms of action  plays a key role in terminating its activation after an appropriate time period . One migFigure 3.The possible mechanism of action by cromone in mast cells is summarized in To conclude, we have reviewed evidence supporting the hypothesis that Anx-A1 is indeed a crucial endogenous regulator of mast cell function, which might reciprocally inhibit mast cell activation. Anx-A1 is secreted in increased amounts parallel with mediators during mast cell activation and thus, acts to control the extent of mast cell degranulation and activation response.We also described a different, GPR35-dependent mechanism, which could also transduce some of the effects of the cromones. In trying to reconcile these two ideas, it is worth noting that the effects on the Anx-A1 system that was reported here was very rapid (within 5 min) and that we have not looked at other actions of the cromones that may require a longer latent period, which could be mediated by an alternative GPR35 mechanisms.All the authors contributed to the writing of the manuscript. AS prepared the final version and prepared the paper for publication.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.


Generalized tonic-clonic seizures (GTCSs) are the main risk factor for sudden unexpected death in epilepsy (SUDEP). Experimental and clinical data strongly suggest that the majority of SUDEP results from a postictal respiratory dysfunction progressing to terminal apnea. Postictal apnea could partly derive from a seizure-induced massive release of endogenous opioids. The main objective of this study is to evaluate the efficacy of an opioid antagonist, naloxone, administered in the immediate aftermath of a GTCS, in reducing the severity of the postictal central respiratory dysfunction.The Efficacy of Naloxone in Reducing Postictal Central Respiratory Dysfunction in Patients with Epilepsy (ENALEPSY) study is a multicenter, double-blind, randomized, placebo-controlled trial conducted in patients with drug-resistant focal epilepsy who are undergoing long-term video-electroencephalogram (EEG) monitoring (LTM) in an epilepsy monitoring unit (EMU). We plan to randomize 166 patients (1:1) to receive intravenous naloxone (0.4 mg) or placebo in the immediate aftermath of a GTCS. Because inclusion in the study needs to take place prior to the occurrence of the GTCS, and because such occurrence is observed in about one-fourth of patients undergoing LTM, we plan to include a maximum of 700 patients upon admission in the EMU. The primary endpoint will be the proportion of patients whose oxygen saturation is <90 % between 1 and 3 min after the end of a GTCS. Secondary outcomes will include the following: the proportion of patients who show postictal apnea, the occurrence and duration of postictal generalized EEG suppression, the total duration of the postictal coma, postictal pain, and the number of patients who have a second GTCS within 120 min after the intravenous injection.The demonstration of naloxone’s efficacy on the severity of postictal hypoxemia will have two primary consequences. First, naloxone would be the first and only therapeutic approach that could be delivered immediately to reverse postictal apnea. Second, demonstration that an opioid antagonist can effectively reduce postictal apnea would pave the way for an assessment of a preventive therapy for SUDEP targeting the same pathophysiological pathway using oral administration of naltrexone.NCT02332447. Registered on 5 January 2015.ClinicalTrials.gov identifier: The online version of this article (doi:10.1186/s13063-016-1653-1) contains supplementary material, which is available to authorized users. Sudden unexpected death in epilepsy (SUDEP) is defined as sudden, unexpected, witnessed or unwitnessed, nontraumatic, and nondrowning death in patients with epilepsy, with or without evidence of a seizure and excluding documented status epilepticus, in which postmortem examination does not reveal a toxicologic or anatomic cause of death . SUDEP pThere is currently no effective treatment to prevent SUDEP, apart from optimizing antiepileptic drugs (AEDs) , 4. As uAlthough the exact pathophysiologic mechanisms that lead to SUDEP remain unknown , 7, mostAnimal studies suggest that seizure-related release of endogenous opioid peptides participate in termination of seizures . In patiOur hypothesis is that SUDEP results partly from postictal apnea promoted by a GTCS-induced massive release of endogenous opioids, and that an opioid antagonist could represent an effective preventive treatment for SUDEP . This coBefore evaluating the efficacy of chronic administration of naltrexone, it is legitimate to perform a proof-of-concept study by testing the acute effect of an equivalent injection treatment  in the immediate aftermath of GTCS recorded in the hospital during video-EEG monitoring of patients with refractory epilepsy. One-third of these patients develop postictal respiratory dysfunction and hypoxemia , which s2).The Efficacy of Naloxone in Reducing Postictal Central Respiratory Dysfunction in Patients with Epilepsy (ENALEPSY) study is a multicenter, double-blind, randomized, placebo-controlled trial  in 1 of the 15 participating epilepsy monitoring units (EMUs) in France  with drug-resistant focal epilepsyPatients undergoing long-term video-EEG or video-SEEG monitoring in one of the participating centers to record and characterize their seizuresPatients who provide written informed consent to participate in the studyPatients affiliated with the French health care systemAbout 25 % of patients with drug-resistant partial epilepsy who are undergoing long-term video-EEG monitoring develop at least one focal secondary generalized tonic-clonic seizure (sGTCS) , 20, 21.Children (<18 years)Pregnant or breastfeeding womenHypersensitivity to naloxoneHistory of severe heart disease Ongoing opioid treatment, including both pure agonists and partial agonistsAddiction to opioids, heroin, or any similar substanceParticipation in another drug clinical trial within the last 2 monthsPatients fulfilling one or more of the following exclusion criteria will not be eligible for inclusion:Patients who have an sGTCS during the long-term video-EEG or video-SEEG monitoring while being supervised by a nurse or a physician will be randomized to receive intravenous naloxone (0.4 mg) or placebo.As indicated in the exclusion criteria, naloxone will not be allowed for patients receiving ongoing opioid treatment, including both pure agonists and partial agonists. Therefore, patients receiving ongoing opioid treatment will be excluded. However, in the context of presurgical evaluation, especially for patients who undergo invasive recording with SEEG, the management of pain might require temporary administration of an opioid agonist. In such patients, short-lasting administration of opioid treatment following inclusion will be authorized, except for extended-release formulations of morphine. However, this will result in temporary ineligibility for randomization during the 12 h following the last opioid administration. If pain management requires administration of opioid treatment during 3 consecutive days, the patient will be excluded from the study.Naloxone is a specific opioid antagonist without partial agonist activity. Intravenous administration has been approved in France since 1977 for the treatment of acute opioid overdose. In this indication, the recommended starting dose is 0.4 mg . In acutTo avoid any follow-up or measurement bias, the patient, the supervising nurse, or the physician and the investigator will not be aware of the nature of the administered treatment. To respect the blinding and to ensure indistinguishability, preparations of both naloxone and placebo will be centralized by the pharmaceutical department of Hospices Civils de Lyon. The study drugs will then be distributed to the pharmacies of other investigator sites and stored at room temperature. Because of urgent need of treatment in case of occurrence of GTCS, one or more study treatments will be stored at room temperature in each EMU according to the expected number of randomizations at each centerRandomization will be centralized, stratified by center, and balanced by block of patients. For each study center, randomization lists will be prepared by the clinical research unit of Hospices Civils de Lyon. The pharmaceutical department of Hospices Civils de Lyon will prepare numbered batches according to the randomization lists.2) will be centralized and evaluated in blinded fashion with other data by the coordinator investigator of the study, who will not be involved in the video-EEG monitoring of the included patient.In case of the occurrence of a GTCS, the affected patient will be randomized in a 1:1 ratio and assigned to the naloxone group or the placebo group. The batch number will be registered in the patient’s case report form. All digital data  into one of two groups: a single dose of 1 ml of either (1) naloxone or (2) placebo will be administered by the supervising nurse or physician, using direct intravenous injection. The evolution from a focal seizure to a GTCS being gradual, and the total duration of the seizure ranging from 2 to 3 min, the injection will be prepared during the course of the seizure. Given the assumptions about the role of endogenous opioid release in the spontaneous termination of seizures, naloxone will be administrated immediately after the end of the GTCS and not before. Specifically, treatment administration will be performed within the 2 min following the end of the GTCS.2, heartbeat and blood pressure, recovery of consciousness and pain, using a visual analogue scale. In patients with SpO2 < 85 % during >2 min after the injection of the study drug, O2 will be administered using a high-concentration breathing mask (15 L/min). Occurrence of one or more GTCSs during the 2 h following treatment administration will be monitored.Immediately after the administration of the treatment, the supervising nurse or physician will monitor respiratory movements to detect absence of chest expansion, SpO2) will be centralized and evaluated blind to other data. An automatic and objective analysis of SpO2 data will be performed using a specific detection algorithm that allows automatic detection and quantification of SpO2 and electrocardiogram variations.All digital data Report of adverse events observed throughout the studyAssessment of pain using a visual analogue scale immediately after recovery of consciousness following the postictal coma.Number of patients who have a second GTCS within 120 min after the intravenous injectionA clinical research assistant mandated by the sponsor will ensure the successful completion of the study and the collection of data generated by writing, documentation, recording, and report, in accordance with the standard operating procedures implemented at the Hospices Civils de Lyon and in accordance with good clinical practice and regulatory legislation. An independent Data and Safety Monitoring Board (DSMB) external to the trial investigators has been established specifically to monitor data throughout the study to determine if it is appropriate, from both scientific and ethical standpoints, to continue the study as planned. The DSMB will meet every year to review serious adverse events and propose continuing or stopping the study. The DSMB is made up of three experts in epilepsy , anesthesiology , and methodology in clinical research .2 is <90 % during at least 5 s between 30 s and 5 min after the onset of intravenous injection of the study drug in the immediate aftermath of a GTCS of 33 % in the control group [Sample size was determined using Fisher’s exact test with a two-sided alternative hypothesis. For a significance level of 5 % (two-tailed), assuming a proportion of patients whose SpOol group  and 11 %2. The proportion of patients whose SpO2 is <90 % during at least 5 s between 30 s and 5 min after the onset of intravenous injection of the study drug in the immediate aftermath of a GTCS will be presented in each group  with the corresponding exact 95 % CI. These proportions will be compared between the two groups using Pearson’s chi-square test (or Fisher’s exact test if conditions for the chi-square test are not fulfilled).The primary analysis will be based on the intention-to-treat principle, including all randomized patients with usable records for SpO2 having no major protocol deviation such as patients in whom the onset of intravenous injection of the study drug was within the 2 min following the end of the seizure (whereas those in whom the study drug was administered >120 s after the end of the seizure will be excluded). Secondary endpoints will be compared between groups using Pearson’s chi-square test, Fisher’s exact test, Student’s t test, or the Wilcoxon-Mann–Whitney test, depending on the variable studied. The significance level will be set at 0.05.A per-protocol analysis of the primary endpoint will also be performed in the population, which will comprise all randomized patients with usable records for SpOAlthough SUDEP is the leading cause of death in people with chronic refractory epilepsy, there is currently no effective treatment to prevent SUDEP, apart from optimizing AEDs , 4. As uThe demonstration of naloxone’s efficacy in reducing the severity of postictal hypoxemia will have two primary consequences. First, naloxone would be a therapeutic approach that could be immediately delivered to reverse postictal apnea, especially during long-term video-EEG monitoring. Although rare, SUDEP has occurred during video-EEG in several EMUs in Europe . In addiSecond, a demonstration that an opioid antagonist can effectively reduce postictal apnea would pave the way for an assessment of a preventive therapy targeting the same pathophysiologic pathway using naltrexone. Indeed, this orally administered opioid antagonist can be delivered over the long term with an excellent safety profile, including in patients undergoing alcohol withdrawal at high risk of seizure, in whom a proconvulsive effect has not been observed , 23. FolThe study protocol was designed in September 2013 and funded by the French Ministry of Health in 2014. The study was registered with ClinicalTrials.gov (NCT02332447) in January 2015. The first participant was enrolled on 26 June 2015. A total of 157 patients have been included so far, of whom 16 have been randomized.