Background: Non-medical use of prescription drugs (NMUPD) is an increasing phenomenon associated with physical and psychological consequences. This study investigated the effects of distinct forms of stress on NMUPD.Methods: Data from 5308 young adult men from the Swiss cohort study on substance use risk factors (C-SURF) were analysed regarding NMUPD of sleeping pills, tranquilizers, opioid analgesics, psychostimulants, and antidepressants. Various forms of stress  during the period preceding the NMUPD assessment were measured. Backward log-binomial regression was performed and risk ratios (RR) were calculated.Results: NMUPD was significantly associated with the cumulative number of potentially traumatic events , with problems within the family , and the peer group . Factors describing family functioning in childhood showed very few significant associations. Sexual assault by acquaintances was associated only with use of sleeping pills ; physical assault by acquaintances was not associated with NMUPD. Physical  or sexual assaults  perpetrated outside the family context did show associations with several drug categories.Conclusion: NMUPD appears to be more consistently associated with discrete and potentially traumatic events and with recent social-environmental stressors than with long-lasting stressors due to family functioning during childhood and youth. Physical and sexual assaults perpetrated by strangers showed more associations with NMUPD than those perpetrated by a family member. The study protocol was approved by the Human Research Ethics Committee of the Canton Vaud (protocol number 15/07).Participants were enrolled at three of Switzerland’s six recruitment centres that conscript men for military service; these cover 21 of the country’s 26 cantons. Attending military recruitment is mandatory for all Swiss men at around the age of 19. Enrolling participants at these locations thus provided C-SURF with a representative sample of young Swiss men. The centres were involved solely to enrol participants; however, the study was conducted independently of the military (questionnaires were sent to the home addresses) and regardless of the fact whether participants provided military or civilian service or obtained an exemption. The present study used C-SURF data collected from the initial baseline assessment of those enrolled  and from the follow-up evaluation (assessment of traumatic experiences and NMUPD), which took place about 15 months later.2.2.An initial group of 7556 conscripts gave their written consent to participate. Of these, 5987  participated in the baseline assessment, and 5479 (91.5%) completed the follow-up assessment 15 months later. One hundred and seventy-one participants were excluded from the analysis due to missing data. The final sample consisted of 5308 participants. More information on the enrolment procedure, non-consent, and non-response bias was provided in previous studies by our group . Substances were: (1) sleeping pills, (2) tranquilizers, (3) opioid-based analgesics, (4) stimulants, or (5) antidepressants. For each substance group, several examples of specific pharmacological agents and their commonly commercialized product names were listed. Additionally, an any NMUPD group (use of at least one of the aforementioned prescription drugs) was constructed.2.4.2.4.1.www.c-surf.ch  was assessed at the 15-month follow-up using 12 stressful events of the Post-traumatic Diagnostic Scale  and two assessed whether participants believed that they had been raised in an emotionally supportive family environment . Responses were given on a five-point Likert scale . The means for each variable were dichotomized at a cut-off of 3 (≥ 3 was coded as 1) and recorded as a lack (coded 1) or presence (coded 0) of either parental monitoring (factor 4) or emotional support (factor 5). Questions for measures 1–5 stem from the European School Survey Project on Alcohol and Drugs  Recent, past 12-month prevalence of serious problems with family members assessed at baseline (which means about 15 months prior to follow-up) was scored as no problems with family, one or two problems, and three or more problems; (2) Serious problems with friends were measured in the same way. Long-lasting social-environmental stressors were measured as follows : (3) Perceived quality of participants’ relationships with their parents, before reaching the age of 18, were measured on a five-point Likert scale . The means of these responses were dichotomized at a cut-off of 3 (≥ 3 was coded as 1) thereby separating the responses into one group with satisfactory relationships (coded 0) and another with unsatisfactory relationships (coded 1). The perceived quality of parenting during childhood and youth was assessed by means of four questions. Two of these were related to whether there was a presence or absence of parental monitoring  were used to adjust for socio-demographic differences.2.6.pout ≥ .05) was used, starting with all IV and forcing confounding socio-demographics into the model. Before conducting regression models, multicollinearity was checked using the variance inflation factor (VIF) for each explanatory variable. No problem of multicollinearity was detected, as the highest VIF value  was well below the thresholds (≥ 5 or ≥ 10) generally considered as evidence of multicollinearity  estimates, we used log-binomial regressions instead of logistic regressions for multiple adjusted models yielding RR . Prevalence of sexual assault by a family member and by a stranger was 1.2 and 1.0%, respectively . Opioid 3.2.p < .01). Physical assault by a stranger was significantly associated with the NMU of psychostimulants  and opioid analgesics , whereas sexual assault by a stranger was significantly associated with the NMU of tranquilizers  and antidepressants .Multiple, log-binomial regressions  revealedAmong social-environmental stressors, only recent, past 12-months problems with the family (RR between 1.70 and 5.66) were consistently significantly associated with NMUPD of all classes, whereas lack of parental support was not associated with any.p < .05). Lack of parental monitoring was only significantly associated with use of stimulants , and having a parent who had suffered from a mental disorder was only significantly associated with the NMU of tranquilizers . Having problems with friends was significantly associated with the NMU of tranquilizers and opioid analgesics.Unsatisfactory relationships with parents were only significantly associated with the NMU of sleeping pills , which are related to a conflict-loaded, disruptive atmosphere within families, have shared variance with assaults, rendering the effect of assaults non-significant in multiple models. Our findings concerning the lack of association between sexual and physical assaults by family members and NMUPD in young men can only be generalized with caution as our analysis was based on relatively low numbers of occurrences resulting in low statistical power in comparison to the other investigated stressors. With regard to the finding that assaults perpetrated by strangers showed more frequently associations with NMUPD than assaults within the family it is possible that feelings of guilt and shame which contribute to higher rates of SU in men (Rahim & Patton, Our results further showed that stress resulting from problems with friends or families contributes substantially to NMUPD. These findings are in line with studies indicating that adolescents with strong bonds to their family or school are less likely to engage in NMUPD (Cerda et al., Parental monitoring of an adolescent’s choice of peers has been shown to be an important protective factor of SU (Van Ryzin, Fosco, & Dishion, Similar difficulties apply to the finding that the presence of a mental disorder in one’s parents was not significantly associated with NMUPD outcomes, with the exception of tranquilizer use. Stressors such as parents being treated for chronic pain or having their own problems with NMUPD or SU are well established risk factors for the initiation of NMUPD in adolescents (Boyd et al., One limitation of this study is the lack of measurements of the severity for most of the stressors, which makes it impossible to weight the specific impact of the independent variables. There was also no information available about whether the various traumatic events had a clinically relevant outcome, such as PTSD. Thus, the available data did not allow distinguishing between stressful, potentially traumatic, and manifestly traumatic events. In addition, since the reference period for stressful events preceded the reference period of NMUPD, it is adequate to interpret results in the direction that stressful events may predispose individuals to use such substances. However, as we were not able to determine the age at which the event occurred one cannot rule out the possibility that the findings may be partially attributable to a reverse causation, i.e. early NMUPD may contribute to being exposed to stress. Finally, another limitation applies to the fact that some responses might be biased by the fear that confidentiality might not be fully guaranteed as this study’s recruitment was organized at the beginning in collaboration with the military enrolment. There also might be underreporting of sexual assaults within families as this is highly intimate and shameful information which some study participants might not be willing to reveal.In conclusion, this study showed that NMUPD by young men was greatly influenced by prior exposure to one or more potentially traumatic events. Recent problems with family or friends were other stressors highly associated with NMUPD. Interestingly, stressors describing inherent family functioning showed very few associations with NMUPD.


As part of an ongoing effort to generate complete genome sequences of hand, foot and mouth disease-causing enteroviruses directly from clinical specimens, two complete coding sequences and two partial genomic sequences of human bocavirus 1 (n=3) and 2 (n=1) were co-amplified and sequenced, representing the first genome sequences of human bocaviruses from Vietnam. The sequences may aid future study aiming at understanding the evolution of the virus. Parvoviridae, subfamilyParvovirininae and genusBocaparvovirus. The virus genome is ~5.3 Kb in length. HBoV-1 was first discovered in 20051. Since then three additional HBoV species, namely HBoV-2, HBoV-3 and HBoV-4, have been discovered4. While the clinical significance of HBoV remains unknown, worldwide their prevalence in respiratory/gastrointestinal tracts varies between 0–26%6. In Vietnam, the reported prevalence of HBoV was 2–17%10. Currently, there is relatively limited sequence information, especially at genome-wide level, of HBoV from Vietnam, although such knowledge may be essential for the development of sensitive, specific diagnostic PCR for the local viral strains, and may aid future investigation documenting the circulation and spread of the viruses at global scale. Human bocaviruses (HBoV) are non-enveloped, single stranded DNA viruses of the familyHerein we report the recovery of two complete coding sequences (CDS) and two partial genomic sequences of HBoV from swabs of Vietnamese children enrolled in our ongoing hand, foot and mouth disease (HFMD) research program in Ho Chi Minh City. The research program aims to look at various disease aspects, including pathogen evolution and its potential implication for vaccine development and implementation.11. In brief, 110 µl of selected swabs were centrifuged at 13,500 rpm for 10 minutes to remove host cells or large cellular components. After DNAse treatment, viral nucleic acid (NA) was then isolated from 100 µl of supernatant using QIAamp viral RNA kit , and recovered in 50 µl of elution buffer (provided with the kit). Ten microliter of the isolated NA was subjected to cDNA synthesis using Super Script III kit  and FR26RV-Endoh primer (primer sequences can be found elsewhere11). The cDNA was then converted to double-stranded DNA using exo-Klenow (Invitrogen), and subsequently pre-amplified using Platinum PCR supermix (Invitrogen) and FR20RV primer11. PCR product was then purified and subjected to library preparation using Nextera XT DNA sample preparation kit  and was finally sequenced using MiSeq reagent kits (Illumina) in an Illumina MiSeq platform (Illumina)11.Whole-genome sequencing of the dominant pathogens (including coxsackievirus A6 (CV-A6), CV-A10 and CV-A16) were performed on 296 RT-PCR positive swabs using an in-house MiSeq-based approach11 to generate the complete genome sequences of the targeted enteroviruses using Geneious software v 8.1.5 , the remaining reads were then subjected to publicly available metagenomic pipelines; Taxonomer12 and Sequence-based Ultra-Rapid Pathogen Identification (SURPI)13 to explore the contents of non-enteroviral sequences in the tested swabs. Evidence of bocavirus sequences were found in four swabs . A reference-based mapping approach using Geneious software (Biomatters)11 was then employed to recover the HBoV genomes from the corresponding dataset. Subsequently, 2 CDS  were successfully assembled with a mean coverage of 1,922 and 3,745, respectively. In the other two datasets from the remaining 2 swabs only partial genomic sequences of HBoV, each with 2870 bp in length and a mean coverage of 15.4 and 448.7, were recovered.After reference-based mapping14 and Neighbor-joining available in Geneious (Biomatters), respectively  under accession numbers KY050743, KY050744, KY072816 and KY072817.The clinical samples used in this study were derived from an on-going HFMD study in three referral hospitals in Ho Chi Minh city, Vietnam. The study was reviewed and approved by the local Institutional Review Boards and the Oxford Tropical Research Ethics Committee (OxTREC), University of Oxford, Oxford, United Kingdom. Written informed consent was obtained from parent or legal guardian of each participant. All previous comments have been thoroughly addressed and the manuscript has significantly improved.We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The authors have carefully addressed all comments raised in the previous review.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I suggest this manuscript can be indexed.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Comparison of the genome sequences of human bocaviruses between from Vietnam and the others should be much more discussed.To date, all of the HBoV genotypes contain the episomal structure. Then, it would be better to analyze it in the paper.The genome of HBoV is organized in three ORFs: ORF1 encoding NS1 protein; ORF2 encoding NP1 protein; ORF3 encoding VP1 and VP2 proteins. So it was suggested that the Phylogenetic trees of nucleotide and amino acid sequences of the HBoV genes should be constructed.Human bocavirus (HBoV) was first identified in 2005, and regarded as a causative pathogen of respiratory tract diseases. The paper reported the recovery of two complete coding sequences and two partial genomic sequences of HBoV from swabs of Vietnamese children enrolled in HFMD research program in Ho Chi Minh City. The experiments were well designed and performed. In addition, the results were described almost properly. In this meaning, the manuscript is sound and suitable for the indexing. However, as I mentioned below, this manuscript has still some points to be clearly explained.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Comparison of the genome sequences of human bocaviruses between from Vietnam and the others should be much more discussed. Response: We have now discussed this in the discussion section. The second sentence of the discussion reads “Phylogenetically, the four HBoVs from Vietnam were closely related to other HBoV strains sampled from various countries worldwide, reflecting a wide distribution of these HBoV lineages at global scales”.To date, all of the HBoV genotypes contain the episomal structure. Then, it would be better to analyze it in the paper. Response: We thank the referee for this comment. Please forgive our ignorance but we understood that episomal structure is formed by repeated sequences at the 5’ and 3’ ends, which unfortunately were not fully sequenced. Therefore the analysis could not be done reliably.The genome of HBoV is organized in three ORFs: ORF1 encoding NS1 protein; ORF2 encoding NP1 protein; ORF3 encoding VP1 and VP2 proteins. So it was suggested that the Phylogenetic trees of nucleotide and amino acid sequences of the HBoV genes should be constructed. Response: We have reconstructed additional phylogenetic trees according to the suggestion of Dr Xiu-ling Ji. We have therefore added those additional phylogenetic to this revised version, and added a sentence to elaborate it in the result section; “Similar results were obtained when the analyses were done for 3 individual open reading frames, ORF1, ORF2 and ORF3 ”. Although the article adds novel information on HBoV epidemiology in Vietnam and presents the sequences of current strains in this geographic region, it has numerous technical shortcomings. The methodology used is not sufficiently described. E.g., the authors state that coxsackieviruses were detected by whole genome sequencing. This technique, however, would detect genomic host DNA, coxsackie is an RNA virus. Moreover, primer sequences and PCR protocols are missing, and alignments are not shown. The reference list is extremely short, and the overall description of methods, results and discussion is weak.We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above. Response:. Please allow us to clarify that the method used was developed in our laboratory for amplification and sequencing viral sequences, and it has been published (citation #11). Although host DNA can also be simultaneously sequenced, investigation of its presence in the obtained reads is beyond the scope of the present Research Note. We thank Dr Verena Schildgen and Dr Oliver Schildgen for their constructive comments Given the method used was detailed in our previous publication (including primer sequences), we have chosen to briefly present it in this revised version as per the reviewers’ comment.  Likewise, we have updated our reference list (from 11 to 23 references), and added more text to the discussion section. Please also refer to our responses to the other reviewers regarding the updated result section, while we hope the reviewers appreciate that the format of a Research Note is concise. This paper describes the first complete genome sequences of human bocaviruses in Vietnam. Reporting complete genome sequences from potential local viral pathogens is vital to develop accurate diagnostic methods and to perform additional studies. However, depending on the scope of this journal, this paper would also be suitable for publication in the journal Genome Announcements.The authors show that they have detected 4 bocaviruses in enterovirus positive samples, as identified by RT-PCR, which demonstrates the strength of agnostic deep sequencing. However, could it be that the symptoms observed in these patients was caused by these enteroviruses and not by the bocaviruses detected in these samples? And which specific enteroviruses  were detected in these bocavirus positive samples? Three viruses were found in throat swabs while one virus was found in rectal swabs. Which virus was found in which sample? E.g. was the genome coverage in the rectal swab lower and does this perhaps also explain the different species detected — was species 2 found in rectal swabs and species 1 in throat swabs? And some minor points:In the abstract the authors mention that “The sequences may aid future study aiming at understanding the evolution of the pathogen”. However, in the introduction and conclusion they mention that the clinical significance of bocavirus infection remains unknown. I suggest to change the word pathogen by virus. The phylogenetic tree is difficult to read in the current resolution The paper is very compact and carefully written, however I feel that some essential information is missing:I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. The authors show that they have detected 4 bocaviruses in enterovirus positive samples, as identified by RT-PCR, which demonstrates the strength of agnostic deep sequencing. However, could it be that the symptoms observed in these patients was caused by these enteroviruses and not by the bocaviruses detected in these samples? And which specific enteroviruses  were detected in these bocavirus positive samples? Response: We agree with the referee that the observed signs/symptoms might have been caused by HFMD causing enteroviruses, and have discussed this in the discussion section added to this revised version. We have provided the information about the specific enteroviruses detected in the four HFMD in the text and Table 1 added to this revised version. The second sentence from the last of the result section now reads “All the four HFMD patients  in whom HBoV was detected had mild HFMD, and were enrolled in November 2013 – March 2014.”Three viruses were found in throat swabs while one virus was found in rectal swabs. Which virus was found in which sample? E.g. was the genome coverage in the rectal swab lower and does this perhaps also explain the different species detected — was species 2 found in rectal swabs and species 1 in throat swabs? Response: We found HBoV-1 in 3 throat swabs and HBoV-2 in 1 rectal swab. There was no correlation between genome coverage and sample types , although the sample size was small. We have presented those data in the original manuscript and have now modified the text slightly to further elucidate the referee’s comment and provided the details in Table 1. “Evidence of bocavirus sequences were found in four swabs . A reference-based mapping approach using Geneious software (Biomatters) was then employed to recover the HBoV genomes from the corresponding dataset. Subsequently, 2 CDS  were successfully assembled with a mean coverage of 1,922 and 3,745, respectively (Table 1). In the other datasets from the remaining two swabs only partial genomic sequences of HBoV, each with 2870bp in length and a mean coverage of 15.4 and 448.7, were recovered. Subsequent sequence alignment and phylogenetic analysis using MUSCLE and Neighbor-joining available in Geneious (Biomatters), respectively  revealed that all 3 Vietnamese HBoV recovered from the throat swabs belonged to HBoV-1 and had >98% of sequence similarity at nucleotide level with other HBoV-1. The other recovered from rectal swab belonged to HBoV-2 and had a close relatedness with a Thai strain CU54TH (GU048663) with a sequence similarity of 97.3% .” Additionally, we have also discussed the detection of HBoV-1 and HBoV-2 in throat- and rectal swab, respectively in the discussion section.In the abstract the authors mention that “The sequences may aid future study aiming at understanding the evolution of the pathogen”. However, in the introduction and conclusion they mention that the clinical significance of bocavirus infection remains unknown. I suggest to change the word pathogen by virus. Response: We have replaced the word “pathogen” by “virus” as recommended.The phylogenetic tree is difficult to read in the current resolutionResponse: We have improved the resolution of the phylogenetic trees.


Background: Trauma-focused psychotherapies for posttraumatic stress disorder (PTSD) have been demonstrated to be efficacious, but also have considerable non-response and dropout rates. Intensive treatment may lead to faster symptom reduction, which may contribute to treatment motivation and thereby to reduction of dropout.Objective: The aim of the current study was to investigate the feasibility and preliminary effectiveness of an intensive five-day inpatient treatment with Eye Movement Desensitization and Reprocessing (EMDR) and trauma-informed yoga for patients with PTSD.Method: A non-controlled pilot study with 12 adult patients with PTSD was conducted. At baseline the PTSD diagnosis was assessed with the Clinician-Administered PTSD Scale (CAPS-5) and comorbid disorders with the Mini International Neuropsychiatric Interview (MINI). Primary outcome was self-reported PTSD symptom severity  measured at the beginning of day 1 (T1), at the end of day 5 (T2) and at follow-up on day 21 (T3). Reliable change indexes (RCI) and clinically significant changes were calculated.Results: From T1 to T3, PTSD symptoms significantly improved with a large effect size (Cohen’s d = 0.91). Nine of the 11 patients who completed treatment showed reliable changes in terms of self-reported PTSD. At T3, two of the patients no longer met criteria for PTSD as measured with the PCL-5. One patient dropped out after the first day. No serious adverse events occurred.Conclusions: The majority of patients in our pilot study experienced symptom reduction consistent with reliable changes in this five-day inpatient treatment with EMDR and yoga. Randomized controlled trials – with longer follow up periods – are needed to properly determine efficacy and efficiency of intensive clinical treatments for PTSD compared to regular treatment. This is one of the first studies to show that intensive EMDR treatment is feasible and is indicative of reliable improvement in PTSD symptoms in a very short time frame. Posttraumatic stress disorder  and eye movement desensitization and reprocessing therapy (EMDR) are demonstrably the most efficacious treatment methods for PTSD to date and are recommended as treatments of choice in several guidelines  and being motivated for brief intensive inpatient treatment. Exclusion criteria were: non-Dutch or non-English speaking, acute suicidality, acute psychosis and severe substance dependency.The pilot study was conducted at Foundation Centrum ‘45, a national centre for specialized assessment and treatment of people with complex posttraumatic psychopathology in Oegstgeest, the Netherlands. For this study, patients stayed at the clinic for five nights and five days. Prior to the intensive treatment week, patients had a maximum of three preparatory sessions in which a treatment plan was made confirming their motivation for the programme, and stating the admission date, treatment goals and adherence to rules at the clinic. Also, psycho-education was given about PTSD and the rationale behind the treatment programme. A stress signalling plan was made in which the patient’s coping mechanisms with low, middle and high stress levels were identified so that the therapists at the clinic were informed about individual coping strategies. In addition to this stress signalling plan, some simple stress regulation techniques were discussed for use at the clinic. Patients also selected the three to five traumatic memories which were most directly related to their PTSD symptoms, to be processed during the intensive week. Finally, patients were shown the clinic of Centrum ‘45. A baseline assessment took place before admission to the clinic (T0). Participants were assessed at the beginning of the first day (T1), at the end of the fifth day  and at day 21 (T3). To determine the feasibility of the programme, patients were asked to fill out a visual analogue scale (VAS) after every EMDR session, to monitor tolerability of the session.1.2.On the first day of the programme, patients received a short physical examination by a nurse specialist as part of standard clinical care, after which they made a case conceptualization with their EMDR therapist of the specific traumatic memories to be processed and the order in which they would be addressed. Depending on the duration of this discussion, patients started with EMDR in this first session or in the next one. Patients had their second EMDR session in the afternoon. During the next four days, patients had two 90-minute EMDR sessions a day. At the end of each day, the patients received one hour of trauma-sensitive yoga. If possible, patients were admitted in pairs and, if this was the case, they received their yoga session together. All other treatment components were offered individually. Patients were encouraged to exercise between sessions, either lightly by walking or more intensively with fitness. At the beginning and the end of the day, they had a 15-minute supportive talk with a mental health nurse. One week after treatment, patients received a follow-up 90-minute EMDR session.The EMDR sessions were aimed at reducing emotional distress associated with the most upsetting traumatic memories, following the Dutch version of the EMDR protocol  to 4 (extreme/incapacitating) using information on both frequency and intensity of symptoms obtained during the interview. Items with a score of ≥ 2 are counted toward diagnosis. Psychometric studies of the CAPS-5 are currently underway; initial experience suggests that it has strong interrater reliability and is generally more user-friendly and efficient than previous versions  together with the CAPS-5 were administered  was used. The reliability and validity of the MINI are well established  to 4 (extremely). A total severity score (range 0–80) can be obtained by summing the item scores. A provisional PTSD diagnosis can be made by counting each item rated 2 (moderately) or higher. A cut-off score of 33 appears to be reasonable until further psychometric work is available  by various mental health symptoms in the past week. The Dutch version of the BSI has satisfactory reliability and validity  to 10 (extremely hard to tolerate). Furthermore, we investigated symptom increase at T2 and T3. Serious adverse events and harms, as defined as the totality of possible adverse consequences of an intervention or therapy  were calculated using means and standard deviations of total scores of the PCL-5 at T1–T2 and T1–T3.To interpret the effectiveness of the treatment programme with regard to PTSD symptoms, treatment outcome was categorized into recovered, improved, unchanged and worsened from T1 to T2 and from T1 to T3. This categorization of treatment outcome was based on criteria of clinically significant change and the Reliable Change Index (RCI). Because of the small sample size this method was preferred over standard quantitative methods, since problems such as a lack of statistical power and biased results due to outliers are likely to occur using standard quantitative methods in small samples. Clinically significant change was defined as a shift from a clinical level of symptoms to a subclinical level of symptoms, following the PCL-5 cut-off score. The RCI was used to establish whether the difference between two test scores obtained at two measurement occasions reflects statistically reliable change, and was calculated conform the method described by Jacobson and Truax . RCI val2.Demographic and clinical characteristics of the 12 patients are listed in d = 0.63). SD = 2.21) on the PCL-5 between T1 and T3. This effect corresponds with a large effect size (Cohen’s d = 0.91). Of the nine patients who showed a reliable change after treatment, two patients showed changes corresponding with a loss of their probable PTSD diagnosis and thus fulfilled the criteria for clinically significant change.Three of the 12 patients fulfilled the criteria for treatment resistance and had received between 16 and 46 sessions of trauma-focused psychotherapy before enrolment in the intensive programme (BEPP and/or EMDR). Two of these patients responded to the treatment programme with symptom reduction consistent with reliable changes, and one of them prematurely dropped out of the programme. Two other patients had started trauma-focused treatment in an outpatient setting and prematurely stopped this previous treatment because of severe suicidal intent and increase in psychotic symptoms or severe dissociative reactions. Both patients responded to the programme, showing PTSD symptom reduction corresponding with reliable changes.2.1.SD = 1.10; range 0–10). None of the patients dropped out because of non-tolerability. The patient who dropped out indicated that he wanted to focus on problems in his relationship. No serious adverse events occurred in the patients who participated in the programme. Three patients reported a mild temporary increase in PTSD symptoms not corresponding with reliable change at T2, and PTSD symptom improvement consistent with reliable changes at T3. One patient reported symptom improvement consistent with reliable change at T2, but not at T3.Tolerability of the sessions is shown in 3.This pilot study aimed to investigate the feasibility and preliminary effectiveness of a five-day intensive treatment consisting of EMDR, yoga and supportive components. The effects at the last treatment day were modest in terms of PTSD symptom reduction and indicative of a medium effect in terms of effect size. At follow-up, two weeks after treatment, treatment effects were large in terms of effect size and nine of 11 patients reported improvement with regard to self-reported PTSD symptoms corresponding with reliable changes. Two patients no longer met criteria for a probable PTSD diagnosis according to the PCL-5. The other seven patients reported a significant improvement in self-reported PTSD symptoms but still met criteria for a probable PTSD diagnosis. Two patients experienced no change in self-reported PTSD symptoms and one patient dropped out after the first day due to a change in personal treatment goals. None of the patients dropped out due to intolerability and no serious harms or adverse events occurred. Regarding feasibility, the inpatient treatment with EMDR and yoga proved to be acceptable for the large majority of patients who completed the programme and drew benefit from it.d = 0.91) is encouraging. This effect should be placed in perspective, however, compared to previous meta-analyses of regular weekly treatments and other intensive treatment formats  since index trauma. Time since trauma may reflect other factors related to chronicity that affect treatment outcome  in patients who had suffered childhood  trauma, had high levels of comorbidity and psychosocial stressors and had not previously benefitted from other trauma-focused treatments. The programme by Bongaerts et al. consisted of two times four consecutive days (with a break of three days between) of three hours of EMDR, so a total of 24 hours of trauma therapy; they also found large effects . These pilot studies thus imply that an even more intensive treatment programme or a two-week programme may be suitable for PTSD patients with a more complex clinical picture.Like in the pilot studies by Hendriks et al.  with TF-This pilot study has some important limitations. The first limitation is the lack of a control group, which would have been helpful to assess the effects of time and of nonspecific factors on PTSD symptoms. Also, the sample was small so this warrants caution in generalization of the results. Furthermore, although we assessed both PTSD diagnosis with the latest DSM-5 instrument, the CAPS-5 and also assessed self-reported PTSD symptom severity with the PCL-5 at three time points, the lack of a long-term assessment inhibits conclusions about the stability of the treatment effects. Even though the EMDR sessions were carried out according to the manual by well-trained therapists and tolerability was monitored over sessions, it would be recommended to monitor treatment integrity of each EMDR session. For further research, we also recommend the use of clinician-rated outcome measures for PTSD and comorbidity and optimizing measurement timing. Finally, treatment was offered in a clinical setting, with short individual supportive talks, the encouragement to perform mild exercise and trauma-informed yoga sessions. These additional interventions may have contributed to the treatment effect we found. Especially trauma-sensitive yoga has shown promising results as stand-alone intervention (Van der Kolk et al., An important clinical implication is that offering EMDR treatment in this intensive programme seems to be a feasible and fast way to significantly reduce symptoms of PTSD. Patients who fulfilled our definition of treatment resistance, and who had received previous ineffective trauma-focused treatment, drew significant benefit from this intensive treatment. Another clinical implication of this pilot study is that the treatment format can be further developed and optimized. Other intensive treatment programmes indicate that patients may benefit more from a slightly longer (Bongaerts et al., 4.In conclusion, this is the first study to investigate the feasibility and preliminary effectiveness of an intensive five-day inpatient treatment with two EMDR sessions and one trauma informed yoga session per day for patients with PTSD. In terms of our objectives, we see it as promising that nine out of 12 patients reported improvement corresponding with reliable changes with regard to PTSD and that none of the patients dropped out due to intolerability. The intensive treatment programme appeared to be effective for patients who were previously unable to benefit from evidence-based treatment, as some of the participants had previously undergone unsuccessful trauma treatment or experienced severe worsening of symptoms in outpatient treatment but responded well to the intensive treatment programme. Although the effect size in our pilot study was large at two weeks post-treatment, we note that the effect size was not as large as in other intensive treatments (e.g. Ehlers et al.,


LRRK2 gene are the most common genetic cause of Parkinson’s disease (PD) [LRRK2 gene, remains enigmatic—as does the nature of the dysfunction caused by mutations that leads to degeneration in the human brain.Mutations in the ase (PD) . DespiteLRRK2 parkinsonism is characterized by a clinical presentation that is almost indistinguishable from idiopathic Parkinson’s, and the LRRK2 locus is associated with genome-wide associated risk for idiopathic Parkinson’s linked to the promoter region of the gene [LRRK2 parkinsonism is the Lewy body, a significant minority of cases present with other forms of protein aggregate pathology. These include neurofibrillary tangles consisting of aggregated tau and consistent with a diagnosis of progressive supranuclear palsy (PSP), TDP-43 positive inclusions similar to those observed in frontal–temporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), and glial cytoplasmic inclusions normally associated with multiple system atrophy (MSA) [the gene . These athe gene . Althoughy (MSA) . There ihy (MSA) . The impDirectly relevant to this phenomenon, there is a substantial body of evidence linking the pathological propagation of misfolded protein aggregates to neurodegeneration . For ParLRRK2 mutation carriers, I propose that LRRK2 is involved in the regulation of cell-to-cell protein aggregate spreading and propagation. This hypothesis posits that the normal function of LRRK2 has a direct influence on the ability of protein aggregates to transfer between cells, acting to repress propagation, and that coding mutations in LRRK2 act as a gain of function to modify this physiological role. The cellular consequence of mutations in LRRK2 is, therefore, an amplification of protein templating and concomitant cytotoxicity leading to neurodegeneration and disease progression. A key element of this hypothesis is that the role that LRRK2 plays in the regulation of protein aggregate propagation is autonomous of the protein constituents of these aggregates. An initial stochastic misfolding event involving alpha-synuclein, therefore, would be amplified by the presence of a mutation in LRRK2 leading to Lewy body or glial cytoplasmic inclusion pathology. Were the initial misfolding event to involve tau, however, this would lead to the propagation of neurofibrillary tangle pathology—and likewise for TDP-43. This hypothesis provides a potential explanation for the puzzle of pleomorphic pathology.To explain the pleomorphic pathology observed in LRRK2 gene [Given the centrality of protein aggregation to our current understanding of neurodegeneration, it is unsurprising that multiple studies have sought to examine a link between LRRK2 and the formation/propagation of protein aggregates. The majority of these investigations have focused on alpha-synuclein, studying the nature of protein aggregation and how LRRK2 can influence this. In murine models for LRRK2 and alpha-synuclein dysfunction, several studies have shown that the nature and frequency of alpha-synuclein aggregates can be modulated by knockout of the RK2 gene , 10, altRK2 gene , 11. TheRK2 gene . The putLRRK2 has been implicated in a wide range of cellular processes, however, there is increasing evidence linking it to the regulation of vesicular transport and catabolic processes in the cell . In partLRRK2 gene), alongside highly specific brain-penetrant inhibitors of LRRK2 kinase function, it will be possible to directly test this hypothesis. If correct, the prediction would be that disease-associated mutations in LRRK2 act to perpetuate and amplify protein aggregate propagation, whereas inhibition of LRRK2 kinase activity or loss of function would act to reduce aggregate spread.There are a number of experiments that would test whether LRRK2 has a conserved role in the regulation of cell-to-cell propagation of protein aggregates involved in neurodegeneration. Cell and animal systems have been developed to model the propagation of protein aggregates in the brain, including alpha-synuclein, tau and TDP-43. These provide a platform for investigating how LRRK2 biology intersects with protein aggregate spreading across the range of pathologies observed in LRRK2 neurodegenerative disease. By combining these model systems with genetic models for LRRK2 , may result in differential vulnerability, and based upon this it is likely that a similar combination of factors determines selective neuronal vulnerability in LRRK2 PD.Second, if it is the case that LRRK2 plays a generic role in the propagation of protein aggregates in the human brain, why is it that individuals with mutations in LRRK2 mutation carriers are limited to those consisting of alpha-synuclein, tau and TDP-43. If there is a conserved role for LRRK2 in the transmission of protein aggregates, why do patients not present with aggregation of amyloid beta or the accumulation of misfolded prion protein? The answer to this may lie in the predominantly intracellular nature of alpha-synuclein, tau and TDP-43 aggregates, and again this could be tested using models for amyloid beta or prion protein propagation.A third issue is why the pathological aggregates observed in LRRK2 mutation carriers presenting with Parkinson’s remains a mystery. As outlined above, the hypothesis that mutations in the LRRK2 gene drive an increased propensity for pathologically associated protein aggregates to convert from isolated stochastic misfolding events to widespread accumulation and cytotoxicity provides a plausible explanation for this phenomenon.In summary, the origin of the pleomorphic pathology observed in


SOD1-G93A mice, disassembly of the neuromuscular synapse precedes motor neuron loss and is sufficient to cause a decline in motor function that culminates in lethal respiratory paralysis. We treated SOD1-G93A mice with an agonist antibody to MuSK, a receptor tyrosine kinase essential for maintaining neuromuscular synapses, to determine whether increasing muscle retrograde signaling would slow nerve terminal detachment from muscle. The agonist antibody, delivered after disease onset, slowed muscle denervation, promoting motor neuron survival, improving motor system output, and extending the lifespan of SOD1-G93A mice. These findings suggest a novel therapeutic strategy for ALS, using an antibody format with clinical precedence, which targets a pathway essential for maintaining attachment of nerve terminals to muscle.In amyotrophic lateral sclerosis (ALS) and animal models of ALS, including  Amyotrophic lateral sclerosis – often shortened to ALS – is a disease that starts with difficulties moving and progresses to paralysis of many muscles, including those used for breathing. The disease is usually lethal, with patients rarely surviving more than a few years after diagnosis. There is no cure or effective treatment for the disease. It begins with the breakdown of the connections, or synapses, between the muscles and the nerve cells that connect with them. After this, the nerve cell itself breaks down. Many therapeutic approaches have focused on attempts to prevent the nerve cells from dying, but few target the initial degeneration of the synapse.Cantor et al. asked if intervening when the synapse has already begun to break down could slow the progression of the disease in mice with ALS. Their approach involved using an antibody to bind to a receptor protein called MuSK, which plays an important role in maintaining the synapse between muscle and nerve cell. The antibody boosted the receptor’s activity, helping to preserve synapses, including those that connect nerve cells to the diaphragm muscle.The experiments showed that the antibody treatment led to fewer synapses breaking down, and kept more of the nerve cells alive. Healthier connections between the nervous system and the diaphragm improved the function of this muscle. As a result, the mice given the antibody treatment had a slightly extended lifespan, compared with those given no treatment.The findings suggest a possible new way to develop treatments for ALS, which could be used in combination with other therapies, such as those aimed at improving the health of the nerve cells. Together, this could improve quality of life for the majority of patients with ALS. Similar strategies could be used to develop treatments to preserve synapses in other neurodegenerative diseases, such as Alzheimer’s, Parkinson’s and Huntington’s disease, as well as some kinds of dementia. Preserving synapses early on, before the significant loss of nerve cells, could help to slow the progression of these diseases, improve the patients' quality of life and extend their lifespans too. SOD1, C9orf72, TDP43, and FUS  is a neurodegenerative disease that progresses relentlessly from a subtle decline in motor function to lethal respiratory paralysis within a few years of diagnosis . The dis and FUS . More coAlthough motor neuron cell death is a hallmark feature of ALS, the loss of neuromuscular synapses occurs prior to the loss of motor neurons and is the primary cause of motor paralysis in both familial and sporadic forms of ALS . The detSOD1 gene, including SOD1-G93A mice, recapitulate the hallmark features of ALS and provide the most thoroughly studied animal model for ALS  that recognize mouse MuSK and raised the idea that a subset of these antibodies may function as MuSK agonists in vivo . We studAgrin stimulates MuSK phosphorylation by binding Lrp4, which promotes association between Lrp4 and MuSK, requiring the first of three Ig-like domains in MuSK . In contTo determine whether agonist antibody #13 could engage MuSK in vivo, we intraperitoneally (IP) injected varying amounts of the MuSK agonist antibody on a human IgG1 backbone, or a control human IgG1 antibody to ragweed pollen, into wild type mice. Several days later, we stained whole mounts of the diaphragm muscle to determine whether the agonist antibody engaged MuSK at the synapse. We measured the pharmacokinetic properties of the injected antibody and found that the half-life of the injected antibody in blood was ~12 days . The antSOD1-G93A mice, on a C57BL/6 background, with 21–26 copies of the human SOD1-G93A gene . We next measured the reliability of synaptic transmission at the neuromuscular junction by repetitively stimulating the phrenic nerve at a physiological frequency (20 Hz). We found a rapid and severe decline in the amplitude of the CMAP, indicative of synaptic dysfunction and denervation, in SOD1-G93A mice chronically injected with the control antibody to GP120. In contrast, the decline in CMAP amplitude was far less severe in SOD1-G93A mice treated with the MuSK agonist antibody, demonstrating that the MuSK agonist antibody improved neuromuscular function , following phrenic nerve stimulation. We studied nd-stage . We stimnd-stage . We founfunction . Moreoveype mice . These CSOD1-G93A mice reduced motor neuron death. During embryonic development motor neuron death is regulated by innervation and reduced when motor neurons make additional synapses with muscle , whereas male SOD1-G93A mice injected with the control antibody had an average lifespan of 157.5 days (SOD1-G93A mice by 7 (p<0.05) and 10 days (p<0.001), respectively , linear regression (CMAPs), or two-way ANOVA (failures). Statistical analyses were conducted using Prism 7.0 software (GraphPad Software). The number (n) of mice used to calculate the mean, SEM values and the confidence limits  are indicated in the figure legends.The investigators were blinded from knowing whether mice were treated with the MuSK agonist or control antibody while acquiring and initially analyzing data. Data are presented as mean ±SEM. Statistical comparisons between groups were analyzed using an unpaired, two-tailed Student’s SOD1-G93A gene was routinely quantified by TaqMan real-time PCR and normalized to GAPDH (Life Technologies Assay# Mm00186822_cn). All mice included in this study had 21–26 copies of hSOD1-G93A (20) was placed on the cage floor so that mice had ready access to nourishment. Others have measured the lifespan SOD1-G93A mice by placing mice on their side and sacrificing mice if they were unable to right themselves in 15 s. Because we were concerned that this assay reported on limb muscle function and may not be temporally aligned with the time of death, we used a variant assay, which provided an accurate measure of longevity. When mice were unable to right themselves to eat or drink over the course of several hours, they invariably succumbed within a day; we defined this time as disease end-point and sacrificed mice at this time. Mice were housed and maintained according to Institutional Animal Use and Care Committee (IACUC) guidelines.The copy number of the human OD1-G93A . DietGelDiaphragm muscles were stained with Alexa 594-conjugated α-bungarotoxin (α-BGT)  to mark AChRs and rabbit antibodies to Neurofilament-L  and Synapsin 1/2  to label axons and nerve terminals, as described previously . At fullSpinal cords were dissected from mice perfused with 4% formaldehyde. Frozen sections (20 μm) of the lumbar region were stained with antibodies to choline acetyltransferase (ChAT) . We defined motor neurons as cells in the ventral horn of the lumbar spinal cord that were positive for ChAT, excluding ChAT-positive preganglionic and Pitx2-positive neurons. We only counted ChAT-stained cells with a clearly defined nucleus in order to avoid double-counting motor neurons in multiple sections. We analyzed ~10 sections, evenly spaced in the lumbar enlargement, which together contained >50 motor neurons in each mouse.Chimeric antibodies were produced by transferring cDNAs encoding the variable regions of MuSK agonist antibody #13 to expression vectors containing the mouse kappa and IgG2a constant region. MuSK agonist antibodies were produced in CHO cells and purified by Protein A and size exclusion chromatography. The activity of the reverse chimera antibody for stimulating clustering of AChRs in C2 myotubes was similar to that for the human agonist antibody to MuSK . Fab fraWe used a solid-phase binding assay to measure binding between the MuSK agonist antibody and the extracellular (ecto) region (E22 to T494), the first three Ig-like domains (E22 to I103) or the Frizzled-like domain (D312 to K456) from mouse MuSK . MaxisorLrp4 mutant muscle cells, were differentiated and treated with either neural Agrin (1 nM) or antibodies (10 nM). MuSK was immunoprecipitated from lysates, and MuSK expression and MuSK tyrosine phosphorylation were measured by probing Western blots, as described previously  artificial cerebrospinal fluid solution  at a rate of ~10 ml/min at room temperature (~20–24°C). The phrenic nerve that innervates the left hemi-diaphragm muscle was stimulated by drawing the distal part of the left phrenic nerve into a suction electrode . Recordings were accepted for analysis only when the CMAP amplitude (peak-to-peak) was unchanged following 1 Hz stimulation. The amplitudes of the evoked CMAPs at higher frequencies were expressed as a percentage of the first evoked CMAP for the entire duration of stimulation. Recordings were fed to an A/D interface  and acquired with Clampex  at a sampling rate of 50 kHz. Data were analyzed off-line using Clampfit . We defined CMAP failures as the absence of an evoked response discernable from the background noise recorded prior to the stimulation. 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 favorably evaluated by Didier Stainier (Senior Editor) and five reviewers, one of whom, Jonathan Glass (Reviewer #4), is a member of our Board of Reviewing Editors. The following individuals involved in review of your submission have agreed to reveal their identity: Mark Rich (Reviewer #1); Victor Faundez (Reviewer #3).Thank you for submitting your article "Preserving Neuromuscular Synapses in ALS by Stimulating MuSK with a Therapeutic Agonist Antibody" 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:G93A SOD1 mouse model of ALS. The authors show that intraperitoneal administration of an agonist antibody to MuSK started at symptom onset (P90) delays denervation of the diaphragm, decreases motor neuron loss in the lumbar spinal cord, and modestly prolongs survival. The data are well-presented and compelling, providing nice proof of principle for this mechanism of action for further therapy development as a pharmacological approach to prevention of denervation in ALS and other denervating diseases. Furthermore, this intervention is agnostic to the underlying cause of denervation, and may be applicable to both genetic and non-genetic causes of ALS.Denervation of skeletal muscle is an early and prominent pathological feature of animal models as well as human ALS. Here, Cantor, et al., extend Dr. Burden's previous publication demonstrating that genetic manipulation of MuSK expression reduces progressive denervation in the The reviewers were generally positive about the data presented, but raised some concerns. The importance of improving survival by only several days in this animal model was questioned. You mention in your final paragraph in the Discussion that "this antibody cannot override the many pathological pathways that occur in the motor neuron and other non-neuronal cells", and that this would represent an addition to a "combinatorial" intervention. You might expand on this message by referring to the models that show little effect on survival when motor neuron cell bodies alone are protected without protection of NMJs.There was also concern about the translational potential of this approach using repeated injections of an antibody. Specifically, there was a concern that repeated injections would generate an immune response to the antibody, causing a myasthenia-like disorder or even accelerated denervation. Please address this issue in your revised manuscript.Essential revisions:SOD1-G93A myofibers show reduced AChR clustering associated with markedly reduced MuSK and phospho-MuSK levels in vitro and in vivo  In the Introduction, the authors state "Although defects in the MuSK signaling pathway are not associated with ALS". However, it was recently reported that 2) The authors initially present pharmacokinetic data using a human antibody, and then transition to a mouse reverse chimera for chronic dosing. The human PK data is used to guide the dosing strategy. While the potency of these antibodies is identical for inducing AChR clustering in vitro, the blood levels of mouse antibody achieved at 10 mg/ml are only in the range of 1-2 µg/ml , whereas the human antibody produced initial levels from 50-100 µg/ml . If this is the case, the authors need to show NMJ saturation levels and validate MuSK phosphorylation in vivo with the mouse dosing paradigm, as these levels are considerably lower.SOD1-G93A mice. If the blood levels achieved with the mouse antibody are really 50-100 times lower, it becomes quite important to validate that receptors are being saturated .3) The authors do not confirm that the reverse chimera antibody is triggering MuSK phosphorylation in the treated 4) Do the authors have any data on NMJ morphology or physiology in muscles other than diaphragm? […] The reviewers were generally positive about the data presented, but raised some concerns. The importance of improving survival by only several days in this animal model was questioned. You mention in your final paragraph in the Discussion that "this antibody cannot override the many pathological pathways that occur in the motor neuron and other non-neuronal cells", and that this would represent an addition to a "combinatorial" intervention. You might expand on this message by referring to the models that show little effect on survival when motor neuron cell bodies alone are protected without protection of NMJs.SOD1-G93A mice, respectively. As suggested, we have added a statement and reference in the Discussion referring to models that show a similar effect on survival when motor neuron cell death is prevented without protecting neuromuscular synapses: “Motor neuron cell death is a critical feature in ALS, but elimination of Bax, which prevents apoptotic cell death, fails to preserve neuromuscular synapses and increases survival of SOD1-G93A mice by only 20 days . Together with our studies, these findings give credence to the idea that combinatorial therapeutic interventions, including those that preserve neuromuscular synapses, will be necessary to fully address the complex pathology and symptoms of ALS and contribute to an improved quality of life for patient and family.”Our study shows that preserving neuromuscular synapses increases survival by 7 to 11 days in female and male There was also concern about the translational potential of this approach using repeated injections of an antibody. Specifically, there was a concern that repeated injections would generate an immune response to the antibody, causing a myasthenia-like disorder or even accelerated denervation. Please address this issue in your revised manuscript.Repeated injections of therapeutic antibodies are commonly used to treat a variety of diseases, including arthritis, multiple sclerosis, asthma, osteoporosis, cancers, Crohn’s disease, muscular dystrophy; see: https://en.wikipedia.org/wiki/List of therapeutic monoclonal antibodies). Moreover, it is possible to reduce a potential immune response to the variable regions of the injected antibodies by having multiple, differing agonist antibodies available.Essential revisions:1) In the Introduction, the authors state "Although defects in the MuSK signaling pathway are not associated with ALS". However, it was recently reported that SOD1-G93A myofibers show reduced AChR clustering associated with markedly reduced MuSK and phospho-MuSK levels in vitro and in vivo  and an impaired response to Agrin. The authors describe the same defects in muscle structure and Agrin-responsiveness in dynein-deficient muscle cultures. Because decreasing dynein expression results in general defects in muscle structure, we believe that the link between dynein, mutant forms of SOD1 and MuSK expression is not particularly strong.Vilmont et al. show that cultured myotubes from 2) The authors initially present pharmacokinetic data using a human antibody, and then transition to a mouse reverse chimera for chronic dosing. The human PK data is used to guide the dosing strategy. While the potency of these antibodies is identical for inducing AChR clustering in vitro, the blood levels of mouse antibody achieved at 10 mg/ml are only in the range of 1-2 µg/ml , whereas the human antibody produced initial levels from 50-100 µg/ml . If this is the case, the authors need to show NMJ saturation levels and validate MuSK phosphorylation in vivo with the mouse dosing paradigm, as these levels are considerably lower.10 values. We have corrected this error in the revised version by displaying log values along the y-axis: the corrected Figure 4—figure supplement 1 shows similar blood values for the agonist antibody in mice injected with either the human antibody or the reverse chimera.We inadvertently failed to indicate that the values displayed along the y-axis in Figure 4—figure supplement 1 are log3) The authors do not confirm that the reverse chimera antibody is triggering MuSK phosphorylation in the treated SOD1-G93A mice. If the blood levels achieved with the mouse antibody are really 50-100 times lower, it becomes quite important to validate that receptors are being saturated .See response to comment #2.4) Do the authors have any data on NMJ morphology or physiology in muscles other than diaphragm?We did stain limb muscles to show that the MuSK agonist antibody bound to synapses in limb muscles as well as the diaphragm muscle, but all of our histological and physiological experiments were carried-out in the diaphragm muscle.


Recent advances in air pollution sensors have led to a new wave of low-cost measurement systems that can be deployed in dense networks to capture small-scale spatio-temporal variations in ozone, a pollutant known to cause negative human health impacts. This study deployed a network of seven low-cost ozone metal oxide sensor systems (UPods) in both an open space and an urban location in Boulder, Colorado during June and July of 2015, to quantify ozone variations on spatial scales ranging from 12 m between UPods to 6.7 km between open space and urban measurement sites with a measurement uncertainty of ~5 ppb. The results showed spatial variability of ozone at both deployment sites, with the largest differences between UPod measurements occurring during the afternoons. The peak median hourly difference between UPods was 6 ppb at 1:00 p.m. at the open space site, and 11 ppb at 4:00 p.m. at the urban site. Overall, the urban ozone measurements were higher than in the open space measurements. This study evaluates the effectiveness of using low-cost sensors to capture microscale spatial and temporal variation of ozone; additionally, it highlights the importance of field calibrations and measurement uncertainty quantification when deploying low-cost sensors. Surface level ozone is well established as harmful to human health, causing impaired lung function in both healthy and sensitive populations ,2,3. TheCriteria pollutants, including ozone, are continuously monitored in the U.S. at standard regulatory air quality monitoring (AQM) stations. These measurements are of high quality but are sparsely distributed and unsuited for providing high spatial resolution variations in concentration ,6,7. RecMultiple studies have evaluated the feasibility of deploying sensor networks to measure ozone in urban settings, and to measure spatial variability of air pollutants; different field calibration methods have been tested in an effort to improve data quality ,8,11,13.The study described here contains three spatio-temporal process scales as defined by Diem . We analIn order to demonstrate the utility of low-cost sensor systems for assessing open space and urban microscale spatial variability, we deployed a fleet of seven metal oxide ozone sensor systems in the Boulder, Colorado area. The sensor systems used for this study were based on the UPod platform . This st2S, a reducing gas, but this is not a common occurrence in most environments [The ozone sensors used were the MiCS-2611 heated metal oxide sensors, manufactured by e2v, now SGX Sensortech . Heated ronments . The senronments . In thisronments . Data frThe deployments took place at an urban site and an open space site. The urban site was on the University of Colorado Boulder campus , located in south Boulder, Colorado,  40 km northwest of Denver. The major road nearest to the UPods was Broadway, which had a 2015 Annual Average Daily Traffic Count (AADT) of 30,000 . The opeThe deployments spanned 25 June to 14 July for the campus UPods and 30 June to 12 July for the SBC UPods. For all data analysis, only the time period when all UPods were sampling, 30 June to 12 July, was included. The deployment locations of all UPods are summarized in detail in The calibration portion of the study consisted of a collocation period when UPods sampled similar air as a reference monitor in order to generate a calibration for the sensors, often termed a field normalization. Three of the UPods, C1, C2, and C3, were collocated with the Boulder Atmospheric Observatory (BAO) tower surface ozone monitor from 15 June to 18 June, and four of the UPods, SBC1, SBC2, SBC3, and SBC4, were collocated at the SBC reference station 15–25 June.The BAO Tower was run by the National Oceanic and Atmospheric Administration (NOAA), which operated a continuous UV absorption Thermo-Scientific Ozone Monitor  with an inlet that was 6 m above the ground . Ozone osR, by the sensor signal in clean air at 298 K, oR. The regression Equations (1)–(4) relate sR/oR to the reference instrument concentration (C), temperature (T), and absolute humidity (H); T and RH terms were included to account for the cross-sensitivities of heated metal oxide sensors to those parameters [p1, p2, p3, p4, and p5 were computed each time a model was generated.Ozone sensors in each UPod were calibrated using the reference data from the site where they were collocated with reference instruments. Multiple linear regression was used to generate a model to convert the raw sensor signal into a concentration (in ppb). We found the sensor resistance as a function of the logged voltages then normalized the sensor resistance, rameters ,19. RH wrameters . The coeR2), the root-mean-square error (RMSE), and the distribution of the fit residuals with concentration, humidity, and temperature. Residuals were calculated by subtracting the calibrated UPod data from the reference data during the collocation time period. Regression analysis is based on the assumption of normally distributed residuals so calibration equations that generated approximately normally distributed residuals were deemed better fits than those that did not.The fit of the calibration models to the reference data was evaluated using the coefficient of determination . The fit statistics for the model applied to the first half represents the validation. A similar validation procedure was not completed at the BAO site because a wind storm limited our data collection during that time period.We generated a calibration dataset for each collocation period by applying the calibration models to the UPod data during collocation periods at both SBC (20 June to 25 June) and BAO Tower (15 June to 18 June). The two calibration datasets represent the best possible agreement of UPod ozone measurements; differences in UPod measurements during the deployment that are less than the calibration differences would be too small to resolve.R2 and RMSE were calculated for each model. An example of model selection process for UPod SBC1 is summarized in R2 (between 0.91 and 0.97) as well as similar RMSE . The calibration RMSE represents the smallest potential uncertainty in the sensor measurements given that the reference instrument data are assumed to be the truth, i.e., contain no uncertainty. Contributions of terms in Equation (4) to s/RoR for SBC UPods during the data validation period are summarized in s/RoR had a higher R2 with concentration than with temperature or humidity, but temperature did play a larger role at higher concentrations. Correlations between s/RoR and temperature and ozone concentration were higher for SBC3 than for SBC1 or SBC2, due to the sensor-to-sensor variation in calibration models. For SBC3, the CT term in Equation (4) likely did not contribute as much to s/RoR as it did for SBC1 and SBC2. This demonstrates the importance of calibrating each sensor individually to accurately capture the influence of the different terms in each calibration model.Raw data from each of the 7 UPods was fit to Equations (1)–(4) and the R2 and RMSE and the results are shown in 2 and VOCs), it is likely that this spike in the June 16 data was caused be a power issue affecting the sensors. SBC4 did not perform as well as the other SBC UPods with an R2 of 0.73 and an RMSE of 12 ppb. Additional error analysis for SBC1, SBC2, and SBC3, was completed to evaluate the RMSE for higher ozone concentrations, since we are typically interested in higher ozone levels and their associated uncertainty. For concentrations >60 ppb, the average RMSE for the three SBC pods during validation was 5.7 ppb, verifying that the calibration models measured similar error with the reference instrument during high ozone measurements.The performance of the SBC calibration was evaluated using the validation dataset. The calibrated collocation data during the validation period for the four UPods  were compared to the reference data using the The poor performance of sensor SBC4 was investigated further; see R2 with reference data when the temporal drift term was included in the calibration model, however, the RMSE increased by 0.1 ppb compared to the Equation (4) model. Future studies with longer durations of multiple weeks to months may benefit from incorporating sensor baseline drift into their calibration models, but for the relatively short-term nature of this study, adding a term for temporal drift did not improve the calibration model performance.Sensor baseline drift over time is a common issue for heated metal oxide sensors ,19. We tTo frame the discussion of spatial variability, the average uncertainty of 5 ppb from the validation dataset was employed as this represents a more realistic estimate of best possible sensor agreement during the deployment. The 5 ppb average uncertainty was applied for both the SBC and campus UPods since a similar validation procedure was not completed for the BAO collocation. The issue with SBC4 is motivation to improve the hardware and software of low-cost tools like the UPod to minimize data losses and discontinuity in data as recorded by SBC4. Until low-cost sensing tools and quantification models become more robust, added redundancy and focus on validation of quantification methods are necessary.x, as well as by varying deposition rates between sites. The concentrations measured by the campus UPods were on average greater than the SBC UPods, which differed from a previous study in 2008 that measured higher annual average concentrations at SBC than in Boulder at a site slightly northeast of campus . This diR2 values that reflect the agreement between UPods were calculated for the deployment data to assess microscale and mesoscale correlation of UPods. Intra-site correlation between UPods was high at both locations with R2 values ranging from 0.88 to 0.93 for SBC UPods and 0.88 to 0.93 for campus UPods. Correlation between the two sites was lower with the R2 values for SBC and campus UPods between 0.54 and 0.63. In this study, correlation on the microscale was higher than on the mesoscale, however correlation is not the only important indicator of spatial variability. The Differences in the hourly ozone observed at each site can be seen in In both The time-averaging results are important when considering ozone spatial variability in a regulatory context, versus a human health context. Exceedances of the NAAQS for ozone are evaluated using 8-h averaged data, and this study found that spatial differences were smaller over the regulatory timescale. Ozone exposure can impact human health on one-hour timescales and the large differences observed in the hour data could lead to different exposure potentials on small spatial scales ,28. SpatThe spatial differences in high ozone measurements are investigated further in Overall, the results from this study indicate that there is both intra-open space and intra-urban spatial variability of ozone on very small spatial scales ranging from 12 m  to 6.7 km . Low-cost sensor systems such as the UPod used in this study are able to quantify these small variations and provide accurate results within a given uncertainty range. Measurements using low-cost sensors could be useful for future human health exposure studies to help quantify spatial variability of ozone in neighborhood settings. This study demonstrates the importance of performing collocations in the field to generate accurate calibrations; validation datasets are also important in evaluating calibration performance, and obtaining accurate uncertainty estimates that are needed to frame the deployment results.


P < 0.01. The rise in sCD14 was lower (P < 0.01) in the rifaximin group  than in the non-rifaximin group . TNFR1 decreased (P < 0.01) in the rifaximin group  but increased in the non-rifaximin group . Other markers remained unaffected. Rifaximin led to a reduction of inflammatory markers and bacterial 16S rRNA which may implicate BT in the inflammation in HSS.Cirrhosis is the dominant cause of portal hypertension globally but may be overshadowed by hepatosplenic schistosomiasis (HSS) in the tropics. In Zambia, schistosomiasis seroprevalence can reach 88% in endemic areas. Bacterial translocation (BT) drives portal hypertension in cirrhosis contributing to mortality but remains unexplored in HSS. Rifaximin, a non-absorbable antibiotic may reduce BT. We aimed to explore the influence of rifaximin on BT, inflammation, and fibrosis in HSS. In this phase II open-label trial (ISRCTN67590499), 186 patients with HSS in Zambia were evaluated and 85 were randomized to standard care with or without rifaximin for 42 days. Changes in markers of inflammation, BT, and fibrosis were the primary outcomes. BT was measured using plasma 16S rRNA, lipopolysaccharide-binding protein, and lipopolysaccharide, whereas hyaluronan was used to measure fibrosis. Tumor necrosis factor receptor 1 (TNFR1) and soluble cluster of differentiation 14 (sCD14) assessed inflammation. 16S rRNA reduced from baseline  to day 42 in the rifaximin group ,  Others have speculated that endotoxins result in release of endothelin-1 after cytokine activation. Endothelin-1 then leads to increased portal pressure by increasing vascular resistance.17 Use of antibiotics in managing spontaneous bacterial peritonitis (SBP) in cirrhosis is widely advocated.15 We know that SBP occurs because of BT in cirrhosis18 but it is unclear if SBP occurs in HSS.Bacterial translocation (BT) in cirrhosis contributes to portal hypertension and is the main cause of mortality.Schistosoma mansoni,19 but it was not established if patients had portal hypertension. Another study suggested that BT may play a role in postoperative infectious complications in patients with schistosomal portal hypertension.20 In a case–control study, we recently demonstrated that HSS is characterized by inflammatory cytokines that may be as a result of BT from the gut.2 Proof of causation is difficult to secure, so we attempted to confirm the occurrence of BT in HSS by giving rifaximin orally to reduce gut microbial contents. Rifaximin is a broad-spectrum antibiotic and is active against gram-negative and gram-positive bacteria including anaerobes and is minimally absorbable.21 Therefore, effects of downstream pathophysiology would implicate BT. We postulated that if the markers of BT during treatment with rifaximin are reduced, this would be strong evidence that bacteria are translocating from the gut and driving inflammation.BT remains unexplored in HSS and we are not sure whether it contributes to portal hypertension in these patients. A short report from Western Kenya suggested that there is increased endotoxaemia in patients exposed to We therefore set out to investigate if giving rifaximin to patients with HSS would reduce the markers of BT and consequently reduce the systemic inflammatory markers. We also explored the effect of rifaximin on hyaluronan (HA), a fibrotic marker in these patients.This was a phase II open-label randomized control trial, but all primary endpoints were measured in the laboratory by staff members who were unaware of the treatment allocation. Consent from the patients was obtained in writing and verbally. Randomization was carried out using a list of codes prepared by an independent advisor with a random number generator. Sequential patients were enrolled and their treatment allocation was decided by referring to the code list.Patients were recruited in the Department of Internal Medicine and Endoscopy Unit of the University Teaching Hospital, Lusaka, Zambia, between January 2014 and August 2016. Inclusion criteria were hematemesis and/or splenomegaly, varices, 18 years and above, and positive serology for schistosomiasis. Exclusion criteria were cirrhosis, seropositive for human immunodeficiency virus, hepatitis B virus, hepatitis C virus, or inability to give consent.Of the 186 patients who were evaluated, 85 patients fulfilled the criteria and were randomized to rifaximin 600 mg orally twice per day with standard care, or standard care only, and followed up for 42 days. Standard care for both groups comprised praziquantel 40 mg per kg body weight given orally in divided doses over 1 day and propranolol 40 mg three times per day, escalating the dosage upward as tolerated and aiming for reduction in resting pulse rate to below 60 beats/minute. Drugs were self-administered orally at home. Adherence to rifaximin was monitored by self-report.All patients were to complete a 42-day follow-up. The primary outcomes in this study were changes in plasma inflammatory, BT, and fibrotic markers over a 42-day period of rifaximin. The secondary outcomes were response to beta blockers, variceal rebleeding episodes, and mortality. Safety was assessed by adverse events and the laboratory tests.A questionnaire was administered at baseline and at the end of the follow-up period. The questionnaire captured demographic data including history of contact with water bodies. Medical history in the questionnaire included hematemesis, abdominal pain, abdominal swelling, yellowing of eyes, fever, and number of tablets of rifaximin missed during the clinical trial. Physical examination was done at baseline and at day 42 and this information was recorded in the questionnaire. Examination included all systems with much emphasis on the liver and the gastrointestinal system. The information in the questionnaire was useful in analyzing various outcomes of the clinical trial.2 Limulus amoebocyte lysate chromo was used to detect LPS in plasma . DNA was extracted using plasma Qiagen DNA extraction kit according to the manufacturer’s instructions. Quantitative polymerase chain reaction was used to quantify DNA of the 16S rRNA gene as an indication for BT. LBP was also used as a biomarker of BT,2 using enzyme-linked immunosorbent assay (ELISA)  at 2-fold dilution. HA was measured by ELISA (R&D Systems) at 80-fold dilution.Ten milliliters of blood was drawn from each patient at baseline and day 42 and collected in tubes containing heparin anticoagulant. Full blood count was measured using a Sysmex 800i analyzer, Kobe, Japan. The rest of the blood sample was centrifuged at 3,000 rpm at 4°C for 5 minutes. The plasma was aliquoted into triplicates and stored at −80°C. The inflammatory markers measured were tumor necrosis factor receptor 1 (TNFR1) and soluble cluster of differentiation 14 (sCD14). To measure BT, 16S rRNA, lipopolysaccharide (LPS), and lipopolysaccharide-binding protein (LBP) were used, whereas HA was used to measure fibrosis.22 using ELISA (R&D Systems) at 10-fold dilution. sCD14 was also determined by ELISA (R&D Systems) at 400-fold dilution. ELISA assays were run in duplicate with appropriate controls and analyzed on a Biotek EL 800 ELISA plate reader using Gen5 1.10 software.TNFR1 was used as a measure of tumor necrosis factor alpha activity in bloodAssuming 95% confidence interval, with a reduction in BT in HSS, a sample size of 40 patients in each group was sufficient with power of 80% to detect a significant difference.P value of less that 0.05 was considered significant.Data were entered into Microsoft Excel and imported to STATA version 13.1  and GRAPHPAD PRISM 6.01  for analysis. Intention to treat analysis was used. For description purposes, we used median with interquartile range because most of these data were not normally distributed. To compare the blood tests between the groups, we used the Mann–Whitney test for unpaired data. To compare measurements from baseline to day 42 within the group, we used Wilcoxon matched-pairs signed-rank test. A This study was approved by the University of Zambia Biomedical Research Ethics Committee (ref: 006-07-12) and Zambia Medicine Regulatory Board . The trial was registered internationally (ISRCTN67590499).One hundred and eighty-six patients were evaluated and of the 101 ineligible patients, most of them were seronegative for schistosomiasis ; althougThere was reduction of 16S rRNA, a marker of BT, sCD14, and TNFR1, inflammatory markers for 42 days of rifaximin as shown in There was good clinical effect of beta blockade (reduction in resting pulse pressure). No patient reported any episode of hematemesis.There were no major adverse events reported during a 42-day follow-up. One patient in the rifaximin group reported an episode of rectal bleeding that required no intervention. Another patient in the rifaximin group complained of abdominal discomfort a week after starting taking rifaximin, but this resolved without any intervention.11 Our results suggest that similar mechanisms may also be driving portal hypertension in HSS.The reduction in inflammatory markers and 16S rRNA copies after 42 days of intestinal decontamination with rifaximin provide evidence that BT may have a clinical association with inflammatory processes in HSS-related portal hypertension. We know that BT is associated with cirrhosis, and this drives the disease process by increasing portal hypertension.27 The minimal reduction of LPS is not inconsistent with this interpretation as LPS is a constituent of gram-negative bacteria only,28 and it might be expected that rifaximin would have a greater effect on gram-positive bacterial loads. Among the inflammatory markers that were reduced after 42 days of rifaximin treatment, sCD14 is the marker which other researchers have associated with BT.32 LPS elicits secretion of sCD14 from macrophages including Kupffer cells after binding of LPS to toll-like receptor 4 in the presence of LBP and sCD14.25 For this reason, activation of any of these markers could reflect BT. TNFR1, another inflammatory marker, also fell after 42 days of rifaximin treatment which is consistent with BT as a driver of systemic inflammation. Tarrats et al.33 have associated TNFR1 with liver fibrosis in animal models. LBP did not change much after 42 days of rifaximin in this clinical trial, which is somewhat at odds with the effect we saw in the case–control study.2 Other researchers have shown that LBP may also be elevated in gram-positive sepsis in the elderly and those who are obese.36 In this clinical trial, no patient had obesity and there was no extremity of age. The lack of effect of rifaximin on LBP may have a similar explanation to that for the lack of effect on LPS.The pronounced reduction of systemic inflammatory markers and the significant reduction of 16S rRNA copies might be explained by the contribution of BT as rifaximin is a nonabsorbable oral antibiotic.37 In an animal model of cirrhosis, fibronectin, another fibrotic marker, reduced after intestinal decontamination with rifaximin.38 It is not clear whether this could translate to humans but our data suggest not in the short term.The only fibrotic marker measured in patients in the clinical trial was HA which was not affected by rifaximin. We think that the duration of rifaximin was too short to have any impact on HA, and by the time these patients present to hospital with variceal bleeding fibrosis is well established and likely to be irreversible. HA is an important molecule which is associated with hepatic fibrosis in HSS.9/L or less as being associated with upper digestive bleeding even in asymptomatic HSS patients.39The pancytopenia in our patients is consistent with hypersplenism. There was profound thrombocytopenia in both groups but patients did not present with bleeding during follow-up although Arthur and others have described platelet counts of 100 × 1018 The reason for the low variceal rebleeding rate during the 42-day follow-up could be due to adherence to propranolol as confirmed by good beta blockade at the end of 42-day period in both groups compared with baseline. This probably reflects the importance of beta blockers in preventing variceal bleeding in schistosoma-related portal hypertension.40There were no major adverse events associated with rifaximin during the 42-day follow-up period which is in agreement with what others have said about the safety of rifaximin.It is a limitation of this study that it was an open-label clinical trial, which is prone to bias, although the laboratory personnel were blinded. Confirmation of compliance to rifaximin in these patients was not possible as rifaximin blood levels could not be measured. The effect of BT on clinical endpoints may also have been underestimated in this clinical trial because the patients were stable and seen as outpatients. They did not have acute variceal bleeds on recruitment and would be predicted to have lower rebleeding rates on follow-up.We showed that rifaximin led to the reduction of systemic inflammatory cytokines and bacterial 16S rRNA, a direct marker of BT. This suggests that BT may be involved in the pathophysiology of HSS and warrants the further study to demonstrate if rifaximin gives clinical benefit. It would be of interest to study patients recruited immediately after acute variceal bleeds, with clinical endpoints, perhaps as a multicenter placebo-controlled trial on a larger scale.