Long-term conditions (LTCs) are the biggest contributor to health loss in New Zealand. The economic cost and burden on the health system is substantial and growing. Self-management strategies offer a potential way to reduce the pressure on health services. This study evaluates a comprehensive self-management programme (the BetaMe programme) delivered by mobile and web-based technologies for people with Type 2 diabetes (T2DM) and pre-diabetes. The primary aim of this study is to evaluate the effectiveness of the BetaMe programme versus usual care among primary care populations in improving the control of T2DM and pre-diabetes, as measured by change in HbA1c and weight over 12 months.n = 430). Eligible participants will be 18 to 75 years old, with T2DM or pre-diabetes, with an HbA1c of 41–70 mmol/mol up to 2 years prior to study commencement. Eligible participants who consent to participate will be individually randomised to the control arm  or intervention arm . The programme consists of a 16-week core followed by a maintenance period of 36 weeks. It incorporates (1) individualised health coaching, (2) goal setting and tracking, (3) peer support in an online forum and (4) educational resources and behaviour-change tools. The primary outcome measures are change in HbA1c and weight at 12 months. Secondary outcomes are changes in waist circumference, blood pressure, patient activation and diabetes-specific behaviours. All outcomes will be assessed at 4 and 12 months for the total study population and for Māori and Pacific participants specifically. All primary analyses will be based on intention-to-treat. Primary analysis will use linear mixed models comparing mean outcome levels adjusted for initial baseline characteristics at 12 months.Participants will be recruited through two primary healthcare organisations and a Māori healthcare provider in New Zealand  contains supplementary material, which is available to authorized users. Long-term health conditions  are by far the largest contributor to health loss in New Zealand (NZ), accounting for 88% of disability-adjusted life years . The ecoSelf-management measures, such as a healthy diet, regular exercise and weight management, are key to preventing the progression of pre-diabetes to T2DM, controlling T2DM and reducing complications –13. For Self-management strategies for T2DM have resulted in improvements in glycaemic control –18, weigThe BetaMe Programme is an integrated self-management programme for T2DM and pre-diabetes developed by Melon Health Ltd.  with a sBetaMe is delivered using both mobile and web-based platforms in a 12-month programme with four key components: health coaching, provision of evidence-based resources, peer support and goal tracking. Each component has demonstrated benefits for improved outcomes for people with T2DM or pre-diabetes . Baseline and follow-up measures at 16 weeks included HbA1c, weight, waist circumference, pre-diabetes status (defined as HbA1c 41–49 mmol/mol) and blood pressure. Of the 117 patients, 108 completed the programme (92%). Of these, 91% reduced their HbA1c, 94% lost weight (mean = 4.2 kg), 87% had reduced waist circumference (mean = 4.2 cm) and 78% had final HbA1c levels below the pre-diabetes range .The feedback from pilot participants was extremely positive. Participants strongly endorsed both the coaching and community aspects of the programme (77% found these the most helpful aspect). Most (84%) reported that they were likely to recommend BetaMe to someone else, while only 2% said they were unlikely to do so. In a recent US review of digital diabetes prevention and self-management solutions in the workplace, the BetaMe programme ranked top for patient engagement and was in the top five overall .In this study, we will undertake a randomised controlled trial to compare the BetaMe programme with usual primary care for patients with T2DM and pre-diabetes, examining the impact on HbA1c and weight as well as waist circumference, blood pressure and a number of self-reported measures of effectiveness at 12 months. This work will address several gaps in the current knowledge base around comprehensive self-management programmes for diabetes. First, most existing studies examine only one part of what is considered to be a self-management programme. For example, a 2011 meta-analysis of 22 trials with 1657 participants concluded that there was evidence that mobile-phone applications led to significant improvements in glycaemic control (a mean reduction in HbA1c of 6 mmol/mol over a mean 6 months’ duration) [The primary aim of this study is to evaluate the effectiveness of the BetaMe programme versus usual care among primary care populations in improving the control of T2DM and pre-diabetes, as measured by change in HbA1c and weight over a 12-month period.Improving HbA1c and weight outcomes at 12 months for Māori and Pacific participants specifically (combined across T2DM and pre-diabetes groups);Improving short-term weight management among those with T2DM and pre-diabetes ;Improving other health-related outcomes, including waist circumference, blood pressure, health-related quality of life and self-reported self-management The secondary aims, which will be explored using secondary analysis of the data, are to evaluate the effectiveness of the BetaMe programme versus usual care in:The study will be a two-arm, parallel-group, individually randomised controlled trial for people with pre-diabetes and T2DM, comparing the BetaMe programme with usual primary care (active control), with investigator-blinded assessment of outcomes , and a large Māori healthcare provider (Whai Oranga o Te Iwi). These providers collectively include around 800,000 patients. The research team will work directly with the two PHOs to identify and recruit practices that are interested in the study and able to commit to the study timeframes and process requirements. Practice recruitment will be through an opt-in process.Participants with pre-diabetes or T2DM will initially be identified using clinical data held by participating PHOs and practices. Those identified as having T2DM and meeting the eligibility criteria below will be included. Those with HbA1c in the 41–49-mmol/mol range in the 2 years prior to commencing the study, and with no recorded diagnosis of T2DM, will be classified in the pre-diabetes group.Have a current HbA1c of 41–70 mmol/mol Are not currently on insulin treatment for diabetesAdults aged between 18 and 75 years, enrolled in participating practicesHave daily Internet access (on any of computer/tablet/smartphone)Are able to provide informed consentPatients will be eligible for inclusion if they meet the following inclusion criteria:Are pregnantHave cognitive impairment likely to be sufficient to hinder their understanding of the study or the programme, as this may negatively affect their self-management behaviour Are unable to read and write in EnglishAre unable to use phone or computer due to physical disability such as poor eyesight or manual dexterityPatients will not be eligible for the study if they:The PHOs or practices will generate a list of current people with T2DM and pre-diabetes, using an HbA1c result of 41–64 mmol/mol within the past 24 months. Individuals that are retested at baseline and return an HbA1c result of 65–70 mmol/mol remain eligible for the study. Patients who are known not to meet the other eligibility criteria will be removed from this list. The remaining patients will be invited by the practice for the research team to contact them.A research nurse will then contact participants to explain the study, confirm eligibility and obtain informed consent. Baseline data will be collected at this point including HbA1c (for those who do not have an HbA1c test result in preceding 3 months), weight, height, waist circumference and blood pressure (using standardised approaches); along with a health-related quality of life measure, a validated measure of chronic condition self-management; and a validated measure of diabetes-specific behaviours (e.g. frequency of blood sugar monitoring). Details of the measures and questionnaires are in Tables BetaMe is Internet-based and delivered using both mobile and web-based platforms by Melon Health Ltd. . The BetHealth coaching. The health coaches are all either qualified personal trainers or registered nurses trained in motivational interviewing, cognitive behavioural therapy, and nutrition, and have completed the Heart Foundation Pacific Heartbeat Community Nutrition course. Health coaches arrange an initial one-on-one session with participants. The purpose of this first 1 h session is for health coaches to work with participants to develop goals tailored to their needs and values. The session follows a set format. Māori and Pacific coaches are available. Once these goals have been set and agreed on, participants’ goals are entered into their profile on the BetaMe system. Coaches will continue to work with participants, and will message participants weekly to check on their progress, reinforce information from the modules and arrange further meetings at frequencies that suit the participant . These subsequent meetings are generally around 15–20 min in length. All meetings with the coaches will be by audio or video, as per the participant’s preference. Support and clinical governance of the health coaches is provided by a clinical psychologist, general practitioner and an experienced health coach. Coaches also regularly interact with one another via an online forum (separate from patient forums) to share any problems they are having and solutionsHealth literacy component. There are eight key education modules introduced to participants via the web and mobile platform with attached resources . The key messages in the modules are reinforced in corresponding email newsletters, discussions on the newsfeed initiated by the community manager and by the participant’s coach. Every 2 weeks, a new module is released that presents participants with a range of topics that offer key messages to help them to learn about and manage their own health. The modules cover risk factors for T2DM and pre-diabetes, healthy eating and physical activity, identifying triggers for less healthy decisions and establishing healthy habits, sleep and shift work, meal planning, problem solving, healthy coping and mindfulness . They will be provided with a set of digital scales and a tape measure so they can take their own measurements and track their progress. They will be able to share these data with their health coach at any time, or with their physician by email or at their next appointmentPeer support. The programme also includes peer support with real-time chat within a private and secure online social network monitored by a community manager who is a registered nurse trained in nutrition. This person monitors the community to ensure nothing unsafe  is shared, and that people are supported and supportive. They will also reinforce key messages, highlight local events in the area, start conversations and welcome new people to the community. The community manager also provides a point of contact for both technical support, and for support more generally, particularly after the first 16 weeks of the programme when the health coaching module is completed.The BetaMe intervention consists of four separate components; health coaches, provision of evidence-based educational resources, goal tracking and peer support:In the first 16 weeks of the programme, participants will set goals, work with health coaches, receive the evidence-based modules and be encouraged to use goal tracking and participate in the peer support programme. After that time, participants will be encouraged to continue tracking and participating in peer support, for the remainder of the 52-week programme. Information from the modules is available for the entire 52-week period.At the time of randomisation, contact details for participants randomised to the intervention arm will be forwarded to Melon Health Ltd. (with participant permission). A health coach will then contact the participant within five working days to initiate the programme. Those randomised to the intervention arm will begin the BetaMe programme whilst continuing to receive usual clinical care from their primary healthcare team. They will also receive resources produced and provided by Diabetes NZ.Melon Health’s platform is built to maximise patient confidentiality and general security of the application at all levels. The system exceeds New Zealand privacy requirements and meets US HIPAA  security rules for health data, with regular auditing by external security specialists.The platform currently integrates with over 350 wearables and biometric sensors through its open API (application programming interface), and platform is accessible through a web-based application or Android/iOS applications.Participants in the control arm will receive usual care. Although this may vary somewhat between practices and GPs, the key interest is the extent to which the BetaMe programme may improve outcomes beyond that expected in the context of usual care. For people with T2DM, this would typically involve an annual diabetes review to monitor glycaemic control and assess the patient for the presence of any diabetes complications and a review of treatment. In addition, it could include education and advice on lifestyle factors, such as diet and exercise, provided by a trained practice nurse or diabetes specialist nurse. For people with pre-diabetes, usual care would typically include lifestyle advice and education about diet and exercise, and an annual HbA1c.https://www.diabetes.org.nz/pamphlet-ordering/). This is to increase retention of control arm participants in the study, and to ensure that all patients have basic information on T2DM and pre-diabetes.The study team will also provide participants in the control arm with standard information about pre-diabetes and T2DM and its management, from resources produced and provided by Diabetes NZ . To ensure balance in numbers allocated to each study arm, randomisation lists will be blocked.Based upon their baseline HbA1c test results (pre-diabetes or diabetes range), participants will be randomised into the intervention or control arm  using a computer-generated randomisation schedule stratified by participating practice and ethnicity (and separate for the two HbA1c-level groups). Randomisation will be completed within two working days of the completed baseline assessment. To ensure allocation concealment, randomisation will be centrally determined from within the REDCap electronic data capture platform The randomisation allocation list will not be visible to the team member(s) requesting the allocation, and this team member(s) will only have details on the patient ID, practice, and ethnicity when requesting allocation for any patient. The exact length of each randomisation block will be kept in a separate document held by the principal investigator (PI), project manager and project statistician, and thus will not be available to the team member(s) responsible for determining allocation.Given the nature of the intervention, it is not possible to keep participants blind to their intervention status. Research nurses taking objective measurements will be kept blind to the intervention status of participants to the extent possible. However, when the nurse meets with the participant at the 4- and 12-month follow-up visits, it is not possible to guarantee that the nurses remain blind to the intervention allocation of the participant. The results dataset will be stored with the study arm identity blinded by code. The key to this code will be held independent of the analysis dataset by the PI, a second member of the research team (MM), and an independent member of the internal Data Monitoring Group. Data cleaning and analysis will be conducted by a statistician who is blind to group allocation. Once analysis is complete, the results will be unblinded.The co-primary outcomes are changes in HbA1c and weight at 12 months. HbA1c provides the most sensitive measure of glycaemic control among those with T2DM. High levels are predictive of higher levels of complications and mortality. Weight (and waist circumference) are associated with T2DM, cardiovascular disease, several cancers and overall mortality –30. We wChange in waist circumference at 12 monthsChange in blood pressure at 12 months. High blood pressure (common amongst people with diabetes) is associated with a higher risk of cardiovascular disease and mortality, and is amenable to lifestyle interventions , 36Secondary outcomes are:Medications (regular prescription medications), and changes to medications and dosesChanges in self-management of chronic conditions using the Partners in Health Scale. This is a validated tool for self-reported self-management of chronic conditions Diabetes-specific behaviours and outcomes will be measured using two validated instruments: the Summary of Diabetes Self-Care Activities tool, which assesses participants’ self-care activities ; and theHealth-related quality of life measured using the five-level EuroQol five dimensions questionnaire (EQ-5D-5 L) . Short-tAdditional self-reported outcomes include:Outcomes will be assessed for all participants (regardless of whether they complete the intervention) by trained nurses at baseline, 4 months and 12 months Tables .All primary analyses will be based on intention-to-treat principles . Demographic and baseline characteristics will be summarised using descriptive statistics. Both HbA1c and weight will be analysed as continuous variables. For the HbA1c primary outcome, analysis will be conducted both combined and separately for pre-diabetes and T2DM. For weight, the primary analysis will combine participants with pre-diabetes and T2DM. Analysis will use linear mixed models, comparing mean outcome levels between intervention and control arms at the 12-month endpoint, adjusted for initial HbA1c level (accounting for baseline differences in outcome) and for other important baseline covariates . This analysis will include the 4-month measurements, providing some information for those individuals subsequently lost to follow-up and hence missing final outcome data . As the study involves repeated measures data, these models will include random effects for individuals to account for correlation between measurements from the same person at different follow-up times.The study is powered in the pre-diabetes group to detect a difference of 2.5 mmol/mol between study arms . This rAnalysis of weight differences between study arms will use the same analytical methods as for the HbA1c outcome. For these weight analyses, those with pre-diabetes and T2DM will be pooled prior to analysis as the relevant intervention target weight loss is the same for these two groups. Analysis will be stratified on patient group to account for the pooled approach, and we will also report weight differences by study arm within each patient group. The comparison of weight loss in the combined patient group is powered to detect a difference between study arms of 5 kg . This rn = 234 × 0.2 = 46 Māori participants) gives an anticipated margin of error on the intervention effect of ± 4.5 mmol/mol for HbA1c in the diabetic group.We will also estimate differences in HbA1c and weight endpoints by study arm for each of the stratified randomisation ethnic groups , and will report the mean intervention effect and 95% confidence interval for each group. Proportions of patients meeting HbA1c and weight-loss targets will also be analysed by ethnic group with logistic regression (adjusted for baseline covariates), and adjusted absolute risk differences in treatment outcomes will be computed to quantify treatment effectiveness . AssuminAnalysis of self-reported outcomes will be conducted in a similar way.Results will be actively disseminated . All data are checked by research nurses at the time of their entry and those variables that are used for stratified randomisation  are re-checked by the research manager prior to randomisation. Participants’ data will be de-identified by a unique code number and information linking that code to a particular patient  will be kept in a secure location with password protection. A master list which links the patients’ NHI and trial code number will be kept so any information about the study, including results, can be communicated to participants. Analysis will be carried out on de-identified data. All trial-specific data for the study will be securely stored by the research team for 10 years once the trial has ended.An independent Data Monitoring Group is not required for this study because the study is low risk in terms of likelihood of life-threatening complications or serious adverse events, the study team has no professional or financial links with the company that is delivering the intervention, and it will not be necessary to instigate stopping rules because there will be no interim analyses of efficacy . An inteAt the end of the study, there will be a process evaluation, which will include assessments of implementation of the BetaMe programme and mechanisms of action. Data will be collected from three sources: (1) from the mobile/web platform to identify key usage patterns; (2) with consent from the participants, health coaches will record audio interactions with participants and will log key reasons for contact (a random subset of the coaching sessions will be assessed by the research team for consistency with the log); and (3) participants will be invited to provide individual feedback on the acceptability and usefulness of the intervention at the end of the study period.Focus groups, lasting approximately 1 h each, will be held in each region to obtain in-depth feedback about the intervention and how participants interacted with it. Usage will be described, and linked to outcomes using standard quantitative analytic approaches . Focus group data will be coded thematically, with key issues identified.http://www.ANZCTR.org.au/ACTRN12617000549325.aspx. The full protocol is available at this site, including consent forms and detailed documentation. Any important protocol modifications will be reported to this organisation.The trial is registered with the Australian New Zealand Clinical Trials Registry on 19 April 2017. Registration number: ACTRN: ACTRN12617000549325. See This trial’s Universal Trial Number (UTN) is U1111–1189-9094.Long-term conditions pose a great burden to patients, family/whānau and health services. Self-management interventions are a potential way to address this burden with evidence that they can be effective in improving physical and mental health. Important gaps in evidence remain, and are a barrier to implementation. The BetaMe programme is an integrated, evidence-based, comprehensive, self-management programme delivered by mobile and web-based technology. A pilot of 117 patients with pre-diabetes was extremely successful with 78% of people reducing their HbA1c level below pre-diabetes range over 4 months. However, the study was volunteer-based and did not incorporate a comparison arm.This paper describes the protocol of a study that will address the question of whether this kind of programme is likely to be effective in reducing HbA1c and weight among people with T2DM and pre-diabetes in a primary care environment. It addresses shortcomings of previous studies and includes an explicit aim to assess the effectiveness of this kind of programme among Māori and Pacific people who have higher rates of T2DM and pre-diabetes than other ethnic groups in NZ.If found to be effective, this programme would allow healthcare providers to deliver an intervention that is person-centred and supports the self-care of people with T2DM, pre-diabetes and potentially other LTCs.This paper is based on protocol version 1.2, dated 1 August 2017. Recruitment to the study began on 9 June 2017. Approximate date of recruitment completion: February 2018.Additional file 1:SPIRIT Checklist. (DOC 120 kb)Additional file 2:Health literacy modules. (DOCX 14 kb)Additional file 3:Dissemination policy. (DOCX 18 kb)


Hypergravity induced changes in hippocampal transcriptome were more similar to acute injection than chronic diffusion of corticosterone or aging.Centrifugation is a widely used procedure to study the impact of altered gravity on Earth, as observed during spaceflights, allowing us to understand how a long-term physical constraint can condition the mammalian physiology. It is known that mice, placed in classical cages and maintained during 21 days in a centrifuge at 3G gravity level, undergo physiological adaptations due to hypergravity, and/or stress. Indeed, an increase of corticosterone levels has been previously measured in the plasma of 3G-exposed mice. Corticosterone is known to modify neuronal activity during memory processes. Although learning and memory performances cannot be assessed during the centrifugation, literature largely described a large panel of proteins  which expressions are modified during memory processing. Thus, we used the Illumina technology to compare the whole hippocampal transcriptome of three groups of C57Bl6/J mice, in order to gain insights into the effects of hypergravity on cerebral functions. Namely, a group of 21 days 3G-centrifuged mice was compared to (1) a group subjected to an acute corticosterone injection, (2) a group receiving a transdermal chronic administration of corticosterone during 21 days, and (3) aged mice because aging could be characterized by a decrease of hippocampus functions and memory impairment. Our results suggest that hypergravity stress induced by corticosterone administration and aging modulate the expression of genes in the hippocampus. However, the modulations of the transcriptome observed in these conditions are not identical. Hypergravity affects  Stress is known to modulate memory by affecting the hippocampus functions. As reviewed recently  suggested that the memory processes could be affected  and housed in standard cages  under standard conditions  with free access to standard food and water. Mice were habituated to animal room for 2 weeks prior to beginning testing.Mice were divided into different groups described as follows: 24 mice (six cages) were placed in the centrifuge at 3G for 21 days (3G group = hypergravity); 24 mice were placed in normogravity (1G group = confinement) in the centrifuge room and confined in the same gondola as used in the centrifuge. The cages were supplied with enough food and water to allow an uninterrupted 21 days centrifugation/confinement period. This part of the experiment was performed in the animal facility containing the centrifuge .A group of 10 mice were implanted subcutaneously with Matrix-Driven Delivery (MDD) pellets  containing 10 mg of corticosterone, allowing the constant and continuous delivery of corticosterone during 21 days (CC group). Likewise, a control group of 10 mice were implanted with the placebo pellets during 21 days (PL group), in order to rule out a putative confounding effect of implantation and surgery in the CC group. One mouse treated with placebo died before the end of the protocol. Furthermore, 10 mice  received a single intraperitoneal injection of corticosterone , 1 h before euthanasia as described previously  was also included in the study to compare the effect of aging to hypergravity and corticosterone-induced stress.CC, PL, AC, and AGE mice were housed in the animal facility of our laboratory, in Bordeaux, France.All animal groups are summarized in Table The project has been validated by the French Ministry of Research in accordance with the European Community and French guiding principles (C2EA-50 (Bordeaux) C2EA-14 (Marseille)). The principal investigator is authorized by French authorities to perform animal experiments (n° C33-01-029).Mice were euthanized in the same time window (9–11 a.m.) by a lethal dose of pentobarbital, brains were extracted from the skull and blood was collected. Hippocampi were dissected and placed in 2 mL tubes containing 1 mL of tri-reagent  and 10–12 ceramic bead  and frozen in dry ice. Samples of group 1G and 3G were transported to Bordeaux in dry ice. All samples were prepared for transcriptomic analysis as described below.* (sequence mode single read 1 × 50 bp), and sequences were mapped. The quantities of sequences obtained in each sample were sufficient to perform a statistical analysis  in tri-reagent , isolation of the total RNA from each hippocampus was performed following the supplier procedures. RNA integrity and purity were verified by using RNA HighSens Analysis Kit  and the concentration of RNA was measured with spectrophotometry  for each hippocampus. We determined that the total RNA in one hippocampus was not sufficient to be sequenced by Illumina. Thus, the RNAs from three to eight mice were pooled in order to generate three samples for each experimental condition  in the CFX96 thermocycler (Bio-Rad). The specificity of the amplification products was confirmed by melting curve analysis. All samples were analyzed in duplicates. PCR efficiency was calculated from the slope of the standard curve. Gene expression levels were calculated using the 2-dCt method normalized by reference gene (stox1) that is steadily expressed in transcriptomic analysis and we have verified also by RT-qPCR.Reverse transcription (RT) reaction was carried out using the iScript advanced kit (Bio-Rad). Obtained cDNA were amplified using the primers designed with Quantprime software  following the instructions of the supplier: samples and standards solutions were diluted (1:100) in the buffer and placed in the plate in duplicates. The reaction buffer containing anti-corticosterone antibody was added and after 2 h of incubation the chemoluminescent subtract was added. The chemoluminescence was measured with the Optima apparatus .p-values were corrected using the Benjamini and Hochberg procedure controlling false discovery rate (FDR). Supplementary statistical analyses were performed with Graphpad prism software . Data were expressed as means ± S.E.M.; n represents the number of tested animals. The samples were compared pair by pair with t-test and with one-way ANOVA. The P < 0.05 were considered as significant and indicated by ⋆ in figures.The first steps of transcriptomic data analysis were performed by GATC. To validate the statistical analysis, https://david.ncifcrf.gov), in order to identify the associated pathways and function of the different genes modified in the experimental conditions. The query was made as proposed: sp-pir-keywords for functional categories and biocarta, KEGG pathway, and panther pathway for signaling pathways. Finally, MGI database was used to suggest the putative phenotype outcome of changes induced by hypergravity.The list of proteins was analyzed using Database for Annotation, Visualization, and Integrated Discovery . We also noticed that in the 3G group the variability of individual measurements was increased .As described previously in mice centrifuged in the same device , and the p-value was ameliorated by the use of the modified Thompson tau test , as reported in Figure To compare the effects of hypergravity on the transcriptome to those observed in stress conditions, we treated mice chronically with corticosterone pellets. The levels of corticosterone in blood of animals treated with chronic corticosterone and placebo were evaluated .The acute intraperitoneal injection of corticosterone induced an increase of blood corticosterone concentration close to 10-fold more than in the 3G group  and showed opposite effects on the expression of two transcripts compared respectively to normogravity and PL controls  C57BL6 mice exposed for 21 days to hypergravity showed an increase of their corticosterone plasma levels, (2) the previously reported variability of the individual adaptation to gravity modifications was indeed present  to determine general rules in biological adaptation to gravity levels. Finally, these experiments could also reinforce the theory of the gravity continuum proposed and discussed in the biology and gravity fields , yet in a lesser extent than expected. As reported recently, in order to reach a 1.8- to 2-fold increase of the blood corticosterone concentration in mice after 21 days, a dose of 40 mg/Kg/day would be required  close to the ones observed in aging and chronically-treated mice. This result suggests that containment alone can be sufficient to induce a rise in corticosterone levels. Future experiments should clarify if this increase can impact the memory performance by itself , and aging. In order to validate our experimental procedure—pooling RNAs from different mice in one sample used in transcriptomic—and to test the biological variability, some gene expressions were analyzed by RT-qPCR.The data obtained confirmed the effects observed in transcriptomic arrays, suggesting that the experimental design  is not able to induce massive bias . This result is probably due to an important dispersion of individual physiological status/history but also to the absence of selection of the mice based on learning and memory performances, for example. Moreover, the number of gene affected by aging depends also on the age of the mice , Kl, Clic6, Kcne2, F5, Slc4a5, and Aqp1 were more highly expressed in the choroid plexus compared to the brain parenchyma  and Agence Nationale pour la Recherche (AdapHyG n° ANR-09-BLAN-0148). YP was granted by ANR and AP's doctoral fellowship was granted by CNES and Region Aquitaine.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.


Both adult and larval zebrafish have been demonstrated to show behavioural responses to noxious stimulation but also to potentially stress- and fear or anxiety- eliciting situations. The pain or nociceptive response can be altered and modulated by these situations in adult fish through a mechanism called stress-induced analgesia. However, this phenomenon has not been described in larval fish yet. Therefore, this study explores the behavioural changes in larval zebrafish after noxious stimulation and exposure to challenges that can trigger a stress, fear or anxiety reaction. Five-day post fertilization zebrafish were exposed to either a stressor (air emersion), a predatory fear cue  or an anxiogenic (caffeine) alone or prior to immersion in acetic acid 0.1%. Pre- and post-stimulation behaviour (swimming velocity and time spent active) was recorded using a novel tracking software in 25 fish at once. Results show that larvae reduced both velocity and activity after exposure to the air emersion and alarm substance challenges and that these changes were attenuated using etomidate and diazepam, respectively. Exposure to acetic acid decreased velocity and activity as well, whereas air emersion and alarm substance inhibited these responses, showing no differences between pre- and post-stimulation. Therefore, we hypothesize that an antinociceptive mechanism, activated by stress and/or fear, occur in 5dpf zebrafish, which could have prevented the larvae to display the characteristic responses to pain. Nociception is the sensory mechanism used to perceive actual or potential tissue damage. The neurons that mediate nociceptive information are called nociceptors. Fish can perceive and respond to a wide range of stimuli, including mechanical , thermalPain can be considered as an evolutionarily developed defence response to an aversive or noxious stimulus . AdminisAntinociception associated with fearful experiences involving a confrontation with a predator or a predator being in close proximity is well studied in mammals , but verUnder European legislation for experimental animals, fish are only protected when able to feed freely and in zebrafish this is six days post fertilization thus younger zebrafish are not covered by the legislation and can be considered as replacement for the use of adult fish. The objective of this study was to evaluate the behavioural responses of non-protected 5dpf zebrafish to stressful, fearful and anxiety-eliciting situations during noxious stimulation and thus validate their use as a replacement for adult forms. To determine the impact of stress larvae were exposed to air emersion, a standard stressor, and then exposed to a noxious chemical. For fear, larvae were exposed to conspecific alarm substance prior to the noxious event and for anxiety, caffeine was administered via immersion prior to noxious exposure. To confirm that these responses were mediated by stress, fear and anxiety, drugs that block the physiological stress response  and reduce anxiety and fear (diazepam) were employed. We showed that stress and fear or anxiety resulted in stress induced analgesia and reduced the responses to noxious treatment. These effects seem to be modulated by etomidate via a block on the stress response or via the anti-anxiety effects of diazepam.All experiments were conducted according to the guidelines of research ethics as approved by the Ethics Committee at the University of Liverpool (40/3534). Five days post-fertilisation (dpf) zebrafish larvae of AB wild type were used for the purposes of this experiment. Eggs were provided by the in-house breeding programme. Adult zebrafish were held in breeding pairs and eggs collected the day after. Eggs were then kept in 3L plastic tanks  in a closed aerated recirculation system supplied with filtered, aerated freshwater at a temperature of 28.5 ±0.5°C and on a 12 h: 12 h light:dark cycle until 5 dpf. These were then selected for experiments. Water quality parameters were kept ideal for this species . Any animals not used in the present study were either held as stock for other experiments or were humanely killed before reaching 6dpf by being placed in an Eppendorf on dry ice for use in another study investigating genomics.Larvae movements were analysed by placing them individually to eliminate group interactions in a custom-built plastic plate of 25 wells  with a 53μm mesh bottom , which allows water to be flushed in and out. The plate was positioned above an infrared light  to facilitate the tracking of the fish. The experimental tank was supplied from a glass sump tank (45 x 35 x 40cm) with filtered and maintained at a constant temperature of 28.5±0.5°C and with aeration provided via an air stone (12cm) and air line from a compressed air supply.Video of spontaneous free-swimming was recorded at 25 fp/s. In summary, behaviour of each larvae tracked using a digital monochrome infrared-sensitive camera  with an attached lens  mounted above the plate. Videos were acquired without compression, via IDS software  via connection to a laptop computer . For video analysis, a novel tracking software based on an object automated detection, tracking and monitoring algorithm was developed for this project. Data files generated by the tracking software were then processed with a bespoke algorithm in MATLAB, which can detect various behavioural larvae patterns larvae based upon standard motion features including average velocity (mm/s) and time spent active (%). Previous studies have demonstrated that these two parameters are affected by noxious, potentially painful stimuli in larval zebrafish ,36.Testing occurred between 09:00 and 16:00 using a randomized trial design to eliminate systematic effects due to time of day. In all experiments, larvae were caught at random and gently pipetted from the rearing tank and placed in the individual wells (25 well plate) to acquire video recordings. Animals were then allowed 30 minutes to acclimate to the experimental arena. All groups of larvae were recorded for 10 minutes to assess the pre-stimulation behaviour (video recording 1) and recorded again at the end of the experimental procedures for another 10 minutes to assess the post-stimulation behaviour (video recording 2). For each of the treatments described below see , 10 grou-1 right before the fish were placed in the apparatus. All experimental procedures are shown in To test that the acetic acid is actually evoking a nociceptive-like response, a group of fish (group AC) was exposed to acetic acid 0.1% by adding the acid  to the tank water using a syringe right after video recording 1. This concentration was shown to be effective in previous studies ,36. Pilo-1 of etomidate  in the tank water immediately before the larvae were placed in the apparatus and then exposed to air emersion after video recording 1. The sole effect of etomidate 0.5 mg l-1 was tested in a third group of fish (group ET), following the same procedure above but without the air emersion challenge. Finally, a group of larvae (group AE+AC) were subjected to the air emersion challenge and then exposed to acetic acid 0.1% right before video recording 2. To determine the effect of any potential handling stress, a control group, which underwent the same experimental procedure as the AE group but left undisturbed, was included (group CO/AE).To determine to what extent stress modulates the response to pain in larvae, a series of experiments were carried out. A first group of fish was exposed to a standard stressor, air emersion, for 1 minute by adjusting the water level in the experimental tank (group AE) right after video recording 1. The effect of an anaesthetic agent known to inhibit production of cortisol in fish and suppress physiological stress, etomidate , was tes-1 of alarm substance produced as proposed by Maximino [-1 of the substance in the tank immediately before video recording 1 and then exposed to the alarm pheromone and recorded again for 10 minutes (video recording 2). The sole effect of diazepam 5 mg l-1 was tested in a group of animals (DI), following the same procedure above but without the alarm pheromone. Another group of larvae (AP+AC) was exposed for 10 minutes to a concentration of 3.5 ml l-1 of alarm pheromone and the appropriate amount of acetic acid 0.1% was subsequently added to the tank water. Finally, to determine the effect of any potential handling stress, a control group, which underwent the same experimental procedure as the group AP but left undisturbed, was included (CO/AP). An additional control consisting of 0.005% (v/v) DMSO in tank water was also used to rule out any undesirable effects of DMSO, following the same general procedure but adding the appropriate amount of DMSO to the tank water right before video recording 2.To determine the impact of the alarm pheromone or alarm substance, a predator cue, on the pain response of larvae, a first group of fish (group AP) was exposed for 10 minutes to a concentration of 3.5 ml lMaximino  and then-1 of caffeine  for 10 minutes and then immediately recorded (video recording 2). The effect of diazepam (in 0.005% DMSO) on the caffeine reaction was tested in a third group of larvae (CF+DI) by dissolving 5 mg l-1 of this substance in the tank water 10 minutes prior to video recording 1 and then exposed to caffeine for 10 minutes and recorded again for 10 minutes (video recording 2). The sole effect of diazepam 5 mg l-1 was determined in a group of animals (DI), following the same procedure as above but without the addition of caffeine. Anxiolytic effects of diazepam are known to occur within 10 min of exposure in zebrafish [The stimulatory effect of caffeine on the pain response of larval zebrafish was determined with a set of experiments: the first group of fish was exposed to 100 mg lebrafish ,40.-1 of caffeine for 10 minutes [Another group of fish (CF+AC) was exposed to 100 mg l minutes ,31 and t2), time active (%) and total distance moved (mm) did not fulfil the requirements of a normally distributed population  and of the homogeneity of variance , however initial data analysis showed that only the velocity (mm/s) and the time spent active (%) were affected by noxious treatment. Therefore, for the purposes of this study, only the average velocity (mm/s) and the time spent active (%) were used as indicators of the larval behaviour. Observations where larvae showed no movements at all both before and after stimulation and those in which the tracking process could not successfully track both before and after exposure were excluded from the data set . Any significant variation in the behavioural response  of the fish during the day that could mask the impact of the different treatments used was determined. Observations in the control treatment in the three experiments were sorted into six groups depending on the time of the day when they were taken  and a Kruskal-Wallis test (P<0.05) was used to determine potential differences between groups. No differences were found between the six hours of the day in the velocity or the time active in the control groups on the stress , alarm pheromone  and caffeine challenges . A Wilcoxon signed-rank test (P<0.05) was performed to assess any potential difference between the pre- (baseline) and post-stimulation average velocity and amount of time spent active on the same larvae in every Experiment. The change in average velocity and activity from pre-stimulation to post-stimulation states in each challenge was determined using a Kruskal-Wallis test (P<0.05). When Kruskal-Wallis test revealed any significant difference, post hoc Mann-Whitney U comparisons were made to compare each treatment group to both the control group using a Bonferroni correction, resulting in a significance level set at P<0.0031 (0.05/16).Statistical analyses were performed using SPSS version 22.0.0.1 software. Behavioural data, namely average velocity (mm/s), average acceleration (mm/s-1) and a reduction in the general activity (%)  evoked a decrease in the swimming speed and the time spent active compared with the baseline values  Figs  and 3. He values . Howeverues Figs  and 3. N-1 prior to exposure to the alarm substance did not exhibit a change in behavior after alarm substance . The air emersion challenge significantly decreased both the swimming speed and the time spent active compared to control larvae , whereas fish exposed to 0.5 mg l-1 of etomidate prior to the challenge (AE+ET) showed no variation in any of the parameters . The group administered with etomidate only (ET) showed no significant change in neither velocity nor the time active  relative to the control group  and in the time spent active . Fish exposed to acetic acid 0.1% showed a significant increase in the swimming speed  and a reduction in the time active  relative to the control group (CO/AE). However, no changes were observed with exposure to lidocaine 5 mg l-1 10 minutes prior to the alarm substance had a similar median velocity and time spent active  compared with controls (CO/AP). Both the group treated with the organic solvent DMSO  and the diazepam+DMSO  did not show a significant change in the two parameters measured. The differences in behaviour observed after exposure to the alarm substance were not evident after immersion in acetic acid 0.1%, with similar median velocity and time spent active .With regard to the alarm pheromone challenge, larvae exposed to acetic acid swam faster but for less time  than those left undisturbed (CO/AP), whereas treatment with lidocaine  prevented any changes relative to controls after exposure to the acid . When larvae were exposed to 100 mg l-1 of caffeine, no significant reduction in the time median velocity or time spent active were observed . Immersion in diazepam 5 mg l-1 before exposure to caffeine had no significant effect compared with the control group . Fish treated with both DMSO  and diazepam+DMSO only  displayed similar velocities and times active. The behavioural change observed in the caffeine-exposed fish was not apparent in those exposed to acetic acid 0.1% too, with similar median velocity , while there were no differences in the time active .Statistical analysis revealed that exposure to acetic acid exerted an increase in the swimming velocity and a reduction in the time spent active  relative to controls (CO/CF). These changes in the control group were not observed when compared with larvae administered with 5mg lThe present study has shown for the first time that larval zebrafish are significantly affected by stress, fear, and anxiety treatments that modulate the response to a noxious stimulus. Exposure to acetic acid for 10 minutes elicited significant behavioural changes in 5dpf zebrafish, with fish showing higher median swimming velocity (mm/s) and lower median time spent active (%) compared with the control group. Fish exposed to air emersion and diazepam swam slower and for less time, whereas those administered with caffeine only had a lower median velocity. Animals exposed to air emersion, diazepam and caffeine prior to the acetic acid challenge did not display these changes.Larvae exposed to a solution of 0.1% of acetic acid showed a significant behavioural response from the baseline values (pre-stimulation), characterised by higher swimming velocity and a reduction in the time spent active. Although a reduction in the activity has been previously observed in larval zebrafish in our laboratory  and in fSciaenops ocellatus) and goldfish (Carassius auratus) larvae, although this conclusion is based on survival and recovery times and not on physiological indicators such as cortisol.Short-term air exposure has been demonstrated to increase cortisol levels in fish ,48 and sThe alarm reaction consists of a set of behaviors that may protect fish from nearby active predators. This substance is synthesized in the epidermal cells of the fish and then released if there is cell membrane damage, which induces a strong fear response in nearby individuals ,52. WherCaffeine acts as a stimulant at low doses and there is ample evidence that it elicits anxiety-like behaviours in zebrafish –31 at siOur results show that the behavioural responses observed in the group exposed to a potentially noxious stimulus, i.e. immersion in acetic acid 0.1%, is inhibited by previous exposure to either air emersion and alarm substance. These results may suggest an antinociceptive effect of stress, and fear or a mechanism that inhibit nociception in situations of stress and fear, which results in stress-induced analgesia. This phenomenon has been described in mammals ,57,58 anWe hypothesize that an antinociceptive mechanism elicited by stress, fear and anxiety could have prevented the larvae to display the characteristic behavioural response caused by the noxious acetic acid.The novel results presented in this study demonstrate that 5dpf zebrafish showed altered behavioural responses after exposure to a potentially noxious stimulus and that these responses were inhibited by stress- and fear-eliciting situations. Therefore, this suggests the presence of a modulation mechanism of nociception in larval zebrafish, which is activated under potentially threatening or aversive situations. We believe that these findings are also relevant to welfare and handling in experiments involving the use of fish. Since these unprotected 5dpf larval zebrafish display similar behavioural responses to both noxious treatment and the various drugs tested in the present study, they could be proposed as a suitable alternative to replace adult zebrafish in nociception and analgesic studies. These results may propose the question as to whether 5dpf zebrafish should be protected if they show similar responses to potentially painful stimuli, however, further evidence is required such as brain processing mechanisms, altered future motivational state and an ability to learn to avoid noxious stimuli  before this question can be fully answered.


This study was conducted to evaluate the safety and efficacy of a new more thermostable Flolan (epoprostenol) solution prepared with the reformulated pH 12.0 diluent in Japanese patients with pulmonary arterial hypertension (PAH) receiving higher doses of Flolan than those typically administered in Western countries.This open-label, single-arm study was conducted in 10 Japanese PAH patients. During the run-in period, patients were intravenously infused with Flolan (45 ng/kg/min or higher doses) solution prepared with the existing pH 10.5 diluent. The patients were then switched to a new more thermostable Flolan solution prepared with the reformulated pH 12.0 diluent and observed for a 4-week treatment period. As a primary endpoint, safety after switching to the new Flolan solution was evaluated. Secondary endpoints included hemodynamics and the necessity for dose adjustment of Flolan in these patients.All 10 patients completed the study period. Observed adverse events were nausea and hepatic function abnormal in 1 patient each, and both events were mild. No patients required dose adjustment due to the change from baseline in mean pulmonary artery pressure (mPAP) measured 3 hrs after switching to Flolan solution prepared with the reformulated diluent. No major changes from baseline in mPAP, pulmonary vascular resistance, or right atrial pressure were observed at 24 hrs and at 4 weeks after switching to the Flolan solution prepared with pH 12.0 diluent. Although some patients showed increases in cardiac output (CO) from baseline at 24 hrs and 4 weeks, no patients required dose reduction as a result of an increase in CO.Neither safety/efficacy concerns nor any dose adjustments of Flolan after switching to a more thermostable Flolan solution prepared with the reformulated pH 12.0 diluent could be required in Japanese patients with PAH receiving higher doses of Flolan.NCT02705807ClinicalTrials.gov Identifier:  Pulmonary arterial hypertension (PAH) is a rare disease characterized by increased pulmonary artery pressure (PAP), leading to right heart failure and death . SeveralThe stability of Flolan solution is improved at ambient temperatures by increasing the pH of the diluent used to prepare the solution from 10.5 (existing diluent in the range of pH 10.2 to 10.8) to 12.0 (reformulated diluent in the range of pH 11.7 to 12.3). The Flolan solution freshly prepared with the reformulated pH 12.0 diluent can be stored at 2°C to 8°C for up to 8 days, and can be administered up to 24 hrs at up to 35°C or up to 72 hrs at up to 25°C. Therefore, the use of a cold pack and frequent changes of the medication cassette are not required, hopefully leading to increased convenience for patients with PAH. The change in the new Flolan formulation is limited to the diluent: the amount and formulation of the active ingredient, epoprostenol, remains the same. Therefore, the new formulation is not expected to impact either the safety or the efficacy of Flolan, and its clinical profile is expected to be the same as that prepared with the existing diluent formulation. In fact, a recent study of Flolan conducted at centers in Western countries demonstrated that no dose adjustment was required for switching from solution prepared with the existing diluent to solution prepared with the reformulated diluent and that, furthermore, the safety and tolerability of Flolan solution prepared with the reformulated diluent were similar in 16 Caucasian PAH patients , 10. It The objective of this study was to evaluate the safety, efficacy on hemodynamic parameters, and necessity of dose adjustment after switching to Flolan solution prepared with the reformulated diluent in Japanese patients with PAH receiving higher doses of Flolan.The protocol for this trial and supporting TREND checklist are available as supporting information; see This single-center, open-label, single-arm phase 4 study was conducted in 10 Japanese patients with PAH between May 2016 and July 2016 . Patients were eligible for inclusion in this study only if all of the following inclusion criteria were met and none of the exclusion criteria were present.2) < 90% as measured by pulse oximetry at screening, regardless of whether or not supplemental oxygen was provided, 4) emergently hospitalized or visited the emergency room for a condition related to PAH or additional treatment for PAH in the prior 3 months, 5) suffering from a clinical condition not expected to remain stable for the duration of the study, and 6) having participated in a clinical study involving another investigational drug or device within 4 weeks before screening.Main inclusion criteria: 1) male or female Japanese patients at least 18 to 75 years of age at the time of screening, 2) had to be on Flolan therapy for PAH, 3) receiving Flolan at 45 ng/kg/min or higher for PAH, 4) had to be on stable doses of their Flolan treatment with existing diluent for a minimum of one month prior to screening though it was acceptable to adjust within 10% of dose during the last one-month period, and 5) had to be on stable doses of any current PAH treatments other than Flolan in the 30 days prior to screening. The main exclusion criteria: 1) receiving Flolan for a condition or in a manner outside the approved indications, 2) congestive heart failure arising from severe left ventricular dysfunction, 3) a resting arterial oxygen saturation  up to 24 hrs and 4 weeks after switching to the Flolan solution prepared with the reformulated diluent. Safety endpoints were evaluated during the treatment and follow-up periods. Efficacy endpoints were also evaluated during the treatment period.GlaxoSmithKline K.K.  provided only the reformulated diluent for Flolan used during the treatment period. Vials of Flolan lyophile (epoprostenol 0.5 mg and 1.5 mg) used during all of the study periods and the existing diluent of Flolan used during the run-in period were products already on the market. Since the reformulated diluent became commercially available in June 2016, we used this newly marketed product during the follow-up period. To prepare the Flolan solution, patients diluted 1 or more vials of Flolan lyophile with 2 vials of the reformulated diluent, producing 100 mL of medication for each day of the treatment period. Medication cassettes were filled with this reconstituted formulation prior to either refrigerated storage or immediate continuous intravenous infusion of Flolan.PAH standard treatments that could be used in combination were as follows, but not limited to them: vasodilators such as angiotensin-converting enzymes inhibitors and calcium channel blockers, cyclic guanosine monophosphate-specific phosphodiesterase type 5 inhibitors such as sildenafil citrate and tadalafil, and endothelin receptor antagonist such as ambrisentan and bosentan hydrate. During the study, no other prostanoids or their inducers were permitted.The incidence of adverse events (AEs), clinical laboratory data, 12-lead electrocardiogram (ECG), vital signs, and oxygen saturation were evaluated for the primary endpoint. Multiple occurrences of the same events in 1 patient were counted only once. Individual AEs were coded by MedDRA version 19.0 and classified according to System Organ Class and Preferred Term. Clinical laboratory  values were measured at the screening visit, baseline, and 4 weeks after switching to Flolan solution prepared with the reformulated diluent. Vital signs  and oxygen saturation were measured at screening, baseline, 1, 3, 24 hrs, and 4 weeks after switching. The 12-lead ECG was obtained during the run-in period, and at baseline, 24 hrs, and 4 weeks after switching.Secondary endpoints were evaluated: 1) the number of Flolan dose adjustments based on the change in mPAP from baseline at 3 hrs after switching to the new Flolan solution prepared with the reformulated diluent, 2) the reason for the Flolan dose adjustments after switching to the Flolan solution prepared with the reformulated diluent, 3) change in N-terminal pro-B-type natriuretic peptide (NT-proBNP) from baseline to 4 weeks after switching to Flolan solution prepared with the reformulated diluent, 4) World Health Organization (WHO) functional class by visits and change from previous visit, and 5) changes in hemodynamic parameters: mPAP, right atrial pressure (RAP), pulmonary vascular resistance (PVR), and cardiac output (CO) from baseline to 24 hrs and at 4 weeks after switching to Flolan solution prepared with the reformulated diluent.Hemodynamic parameters such as mPAP, RAP, PVR, and CO were evaluated by RHC test at 1, 3, and 24 hrs, and at 4 weeks after switching to Flolan solution prepared with the reformulated diluent. The WHO functional classification was evaluated at screening, baseline, and 4 weeks after switching to Flolan solution prepared with the reformulated diluent. Blood samples were collected at baseline, 24 hrs, and 4 weeks after switching to Flolan solution prepared with the reformulated diluent in order to measure the plasma concentration of NT-proBNP.The sample size (10 patients) for this study was determined based on the feasibility of performing an exploratory study, and not hypothesis testing. Therefore, there was no sample size rationale and the formal statistical analysis was not conducted. All the 10 patients completed the 4-week treatment and 1-week follow-up periods in this study. Data were summarized by using descriptive statistics. The intent-to-treat (ITT) population was selected as the group to be analyzed. The ITT population comprised patients who received at least 1 dose of Flolan solution prepared with the reformulated diluent. The ITT population was used for all safety and efficacy evaluations. As primary endpoints, frequency tabulation or summary statistics  were calculated for safety. As secondary endpoints, the frequency of Flolan dose adjustments, the frequency of WHO functional class by visits, and the change from the previous visit were calculated. Summary statistics were calculated for the measured values and changes from baseline in NT-proBNP and the hemodynamic parameters .The patient disposition is shown in Patient characteristics are shown in The status of exposure to the investigational drug is shown in An overview of AEs is shown in No deaths or AEs leading to discontinuation of the investigational drug were reported. Also, no cardiovascular events were observed. As to the other clinical chemistry tests including thyroid function, hematology tests, and urinalysis, no obvious changes from the baseline values were detected at 4 weeks after switching to Flolan solution prepared with the reformulated diluent. As to vital signs, no effects on systolic and diastolic blood pressures or heart rate were observed. There were no abnormal findings on the 12-lead ECG at 4 weeks after switching to Flolan solution prepared with the reformulated diluent. There was no obvious change from baseline in oxygen saturation at any of the evaluation time points after the baseline measurement.No dose adjustments were performed based on mPAP changes from baseline, as determined at 3 hrs after switching to Flolan solution prepared with the reformulated diluent. In this study, the dose of Flolan was slightly reduced in 2 patients at 1 hr after switching to Flolan solution prepared with the reformulated diluent by the investigator due to increased CO .Changes from baseline in mPAP, RAP, PVR, and CO are shown in Nine of 10 patients were evaluated by the investigator at screening as WHO functional class II, and no change was observed at baseline. One patient was evaluated as class I by the investigator at screening and at baseline, but the evaluation was class II 4 weeks after switching to Flolan solution prepared with the reformulated diluent.Changes from baseline in plasma NT-proBNP concentration at 24 hrs and 4 weeks after switching to Flolan solution prepared with the reformulated diluent are shown in This study was conducted to evaluate the safety, efficacy, and necessity of dose adjustment after switching to a more thermostable Flolan solution prepared with the reformulated diluent in Japanese patients with PAH receiving higher doses of Flolan than those used in Western countries.The mean dose of Flolan for 1 hr from switching to Flolan solution prepared with the reformulated diluents was 83.28 ng/kg/min. The doses of Flolan were similar throughout the treatment period. These were higher as compared with those administered overseas, generally 20 to 40 ng/kg/min, or the western study, 36 ng/kg/min , 13, 14.As to safety, the results obtained in the present study revealed no unknown safety concerns associated with switching to Flolan solution prepared with the reformulated diluent in Japanese PAH patients receiving higher-dose Flolan.As to efficacy, given that mPAP measurements and changes are important as hemodynamic parameters reflecting the therapeutic efficacy of investigational drugs and PAH disease conditions, the frequency of and reasons for Flolan dose adjustments based on changes from baseline in mPAP were established as secondary endpoints. In this study, there were essentially no changes in mPAP and only small changes in other hemodynamic parameters including PVR and RAP, suggesting minimal impact of switching to Flolan solution prepared with the reformulated diluent. Inter-patient variations in changes from baseline in the NT-proBNP concentrations at 4 weeks after switching to Flolan solution prepared with the reformulated diluent were observed, but there seemed to be no noteworthy changes. One patient had WHO functional class of change, which was considered by the investigator to be an individual variability rather than being due to disease progression. These results suggested that there would be neither dose adjustments nor any impacts on efficacy of switching to Flolan solution prepared with the reformulated diluent in Japanese PAH patients receiving higher-dose Flolan.The limitation of this study is that there were only 10 patients. This was an exploratory study, and secondary endpoints such as hemodynamic parameters, NT-proBNP, and WHO functional class were not statistically analyzed due to the small sample size. Therefore, its evidence level is not comparable to that of other clinical studies with more patients that have performed formal statistical analysis. We could not draw definitive conclusions about the safety and efficacy of a new Flolan prepared with the pH 12.0 diluent.Use of the pH 10.5 diluent to prepare Flolan solution necessitates frequent preparation and the use of cold packs by patients due to the thermo-instability of epoprostenol, thus placing a substantial burden on patients with PAH, especially those with a low quality of life and/or complications. Flolan solution prepared with the pH 12.0 diluent allows drug administration without frequent preparation or the use of cold packs. This study suggests that Flolan solution prepared with the reformulated diluent could provide a convenient treatment option for patients while maintaining efficacy and safety.Neither safety/efficacy concerns nor dose adjustments of Flolan after switching to the new more thermostable Flolan solution prepared with the reformulated pH 12.0 diluent were required in Japanese patients with PAH receiving higher doses of Flolan than those generally used in Western countries.S1 TREND Checklist(PDF)Click here for additional data file.S1 Protocol(PDF)Click here for additional data file.


The purpose of this study was to introduce a technique to extract the remaining peritoneal gas in order to improve the post-laparoscopic shoulder pain.This study included 12 patients undergoing laparoscopic gynecologic procedures between February and March 2016 in Minimally Invasive Techniques Research Center, Pars Hospital, Tehran, Iran. For complete suction of the air from abdominal cavity, the air was first vacuumed from the pelvic cavity in Trendelenburg position and then the patients were put in anti-Trendelenburg position. In this position, as the remaining gas was shifting toward subdiaphragmatic area, the suction tube was shifted to a position next to the camera canal and the remaining air was suctioned. A 10 point visual analogue scale was used to measure the severity of patients’ post-operative shoulder pain.hr post-surgery. At 12 hr post-surgery, the mean VAS was 0.8±1.5. At 24 hr post-surgery, the mean VAS for shoulder pain was 0.3±0.8. Finally, 48 hr post-surgery, the VAS score for all patients was zero.The mean VAS for shoulder pain was 0.8±1.7 4 Our approach for emptying the abdominal cavity from residual gas after laparoscopic procedures seems to be useful in preventing post-operative shoulder pain among patients undergoing gynecological laparoscopic surgeries. Further studies are suggested to compare the effect of our proposed method with other methods. Several studies have shown a relation between the residual gas volume and the severity of post-operative shoulder pain  following induction of the general anesthesia as a prophylactic antibiotic. Laparoscopy was performed by direct trocarization and CO2 insufflation. Two 5 mm ports were inserted through outer upper margins of bilateral rectus muscle sheaths, a 10 mm port through umbilicus for telescope insertion, and then a suprapubic trocar as described before , nausea and/or vomiting, and the time to first gas passing. Also, the duration of laparoscopic procedure, post-operative hospital stay and any complications were recorded as well. A 10 point visual analogue scale (VAS) was used to measure the severity of patients’ post-operative shoulder pain at 4, 12, 24 and 48 hr post-operatively. The score 0 in VAS scale indicated “no pain” and the score 10 indicated the worst pain possible and patients were asked to give a score from 0 to 10 based on their severity of pain. The pain score was assessed by nursing staff, who were blinded regarding the aims and design of the study. The main objectives of the present study were to evaluate shoulder pain at 4, 12, 24 and 48 hr after the surgery and the demand for analgesic medications.The Minimally Invasive Techniques Research Center, Pars hospital ethics committee approved this case series study and informed written consent for conducting and later publication of the study results was obtained from all participants before the start of study. The study was conducted in Minimally Invasive Techniques Research Center, Pars hospital, Tehran, Iran, from February to March 2016 and included twelve patients with age range of from 17 to 59 years who underwent uncomplicated gynecological laparoscopic procedures. Patients with any history of pre-operative shoulder pain were excluded from the study. Also, patients with a history of pulmonary disorder, abdominal or pelvic pain, tubo-ovarian abscess and severe adhesions in abdominal or pelvic regions were excluded from the study. All patients entering the study were operated by the same surgical team. All patients were given intravenous Cefazolin  in abdominal cavity is considered to be the main cause of post-operative shoulder pain and some researchers have indicated a relation between the residual gas volume and the severity of post-operative shoulder pain  and none of patients needed repeating of surgery.A main limitation of many previous studies examining the effectiveness of drainage in reducing post-laparoscopic shoulder pain is the inclusion of patients with a wide range of surgical indications . Our stuOur approach for emptying the abdominal cavity from residual gas after gynecologic laparoscopic procedures seems to be useful in preventing post-operative shoulder pain and also reducing the duration of post-laparoscopic hospital stay. It is probable that this method could be used as a non-invasive technique for reducing post-laparoscopic shoulder pain. Further prospective studies are suggested to compare the effect of our proposed method with previous methods of pain reduction with or without drain placement to confirm its usefulness.


The authors compared apatinib—a tyrosine kinase inhibitor that targets the vascular endothelial growth factor receptor-2 (VEGFR-2)—with placebo among 273 randomly assigned patients and reported an overall survival advantage in favor of the experimental treatment. This study represents an important step forward in gastric cancer research, as it confirms the value of targeting VEGFR-2 beyond the already available evidence that supports the use of ramucirumab, an anti-VEGFR-2 antibody, in the second-line setting.3 However, there are several potential pitfalls that may limit the value of these results.Li et alv 16.5%). The authors conclude that this difference does not formally reach statistical significance, but, in our opinion, this could have had an unpredictable impact on overall survival. Indeed, PS is one of the main determinants of survival in advanced gastric cancer from first-line to third-line setting,6 and even a seemingly mild deterioration in the general condition, that is, a PS of 1 according to the Eastern Cooperative Oncology Group scale, might impair outcomes in such a fragile patient scenario.With regard to patient characteristics, an imbalance in performance status (PS) distribution is evident, with an excess of patients with a PS of 0 in the experimental arm compared with placebo , and grade 3 to 4 neutropenia and hand-foot syndrome were not reported.2 Whereas these adverse events are manageable when apatinib is administered as monotherapy, they may not suit well when apatinib is combined with cytotoxic agents used in gastric cancer, such as fluoropyrimidines and platinum compounds. With regard to cardiac safety, initial data with apatinib seems reassuring; however, comparisons with sunitinib and bevacizumab—as reported in the Discussion of the article by Li et al1 and for which we have greater knowledge about potentially severe cardiovascular events—seem at least premature. This is particularly true if we keep in mind that elderly patients (age > 70 years) were excluded from the trial, the number of patients between age 65 and 70 years was limited (37 patients), and the median age in the two arms (age 58 years) is lower than that observed in routine practice.1 In the RAINBOW trial with ramucirumab, despite a similar median age in the enrolled patient cohorts, the upper age limit was 84 years and the number of patients age ≥ 65 years was greater (249 patients). Neither in the REGARD trial7 nor in the RAINBOW trial8 did the age subgroup analyses report increased toxicity among older patients, with the exception of a relatively higher rate of grade ≥ 3 neutropenia in the RAINBOW trial.In terms of safety, apatinib resulted in a not negligible rate of grade 3 to 4 hand-foot syndrome (8.5%), with approximately one of two patients experiencing proteinuria  and 5.7% of patients experiencing grade 3 to 4 neutropenia. Is it really reasonable to conclude that apatinib “has a favorable safety profile in comparison with other antiangiogenic agents?”1 Quality-of-life data for ramucirumab monotherapy have been recently published; ramucirumab proved to delay the worsening of symptoms and the deterioration of PS.9 It should be recognized that the population enrolled in the apatinib trial had a far more pretreated disease, but patient characteristics of PS, age, and number of metastatic sites seem comparable between trials.2Finally, no significant improvement in quality of life was noted for apatinib.10 other studies should be awaited to confirm these data outside Asia.With an arguably increasing number of patients receiving ramucirumab in the second-line setting, it is difficult to anticipate the potential impact of apatinib in routine clinical practice. As these agents share the same target along the pathways that regulate tumor angiogenesis, the efficacy of apatinib in overcoming resistance to ramucirumab is unclear. Moreover, results with apatinib have been reported among Chinese patients. As a result of differences in the biologic background and treatment patterns between Eastern and Western patients with gastric cancer,To conclude, apatinib is another arrow in the bow against gastric cancer. Future trials and, we hope, the identification of predictive biomarkers will ultimately tell us how far we can hurl our hope in this challenging struggle.


SHANK3 gene participated in the clinical trial for a period of 18 months. Starting 6 months before the trial, children were systematically assessed for cognitive, language and motor development and for adaptive, social and emotional behaviour every 6 months. The second, third and fourth assessments were followed by daily nose sprays containing either intranasal insulin or intranasal placebo for a 6-month period. A fifth assessment was done directly after the end of the trial. Intranasal insulin did not cause serious adverse events. It increased the level of developmental functioning by 0.4–1.4 months per 6-month period, but the effect was not statistically significant in this small group. We found a stronger effect of intranasal insulin, being significant for cognition and social skills, for children older than 3 years, who usually show a decrease of developmental growth. However, clinical trials in larger study populations are required to prove the therapeutic effect of intranasal insulin in PMS.Phelan-McDermid syndrome (PMS) or 22q13.3 deletion syndrome is a rare neurodevelopmental disorder with at least 60 children and 35 adults diagnosed in the Netherlands. Clinical features are moderate to severe intellectual disability and behavioural problems in the autism spectrum. Other researchers had observed a beneficial effect of intranasal insulin on development and behaviour in a pilot study in six children with PMS. To validate this effect, we conducted a randomized, double-blind, placebo-controlled clinical trial using a stepped-wedge design. From March 2013 to June 2015, 25 children aged 1–16 years with a molecularly confirmed 22q13.3 deletion including the  There are at least 60 children and 35 adults diagnosed with this syndrome in the Netherlands. The main clinical features of PMS are intellectual disability and behavioural problems in the autism spectrum.SHANK3 gene (OMIM 606230).4 Mutations in this gene have been reported in humans with intellectual disability and with autism ,5 and have been shown to cause deficits in social interaction and learning in animal models.7 SHANK3 is a scaffold protein located at axon terminals and postsynaptic densities of neurons that has an important role in synaptic maturation and neuronal signalling.9 Neurons derived from induced pluripotent stem cells of PMS patients showed reduced SHANK3 mRNA and protein expression, a decreased number of excitatory neurotransmitter-receptors and fewer excitatory synapses.9 As the molecular mechanisms underlying the syndrome are uncovered, research is gradually focusing on developing therapeutic strategies at the biological level. One strategy is the administration of insulin-like growth factor 1 (IGF-1), which has been reported to reverse deficits in synaptic signalling in heterozygous Shank3-deficient mice.10 In a randomized placebo-controlled crossover pilot study, nine children with PMS were treated with daily intraperitoneal IGF-1 injections over a period of 12 weeks. The results showed an improvement of social impairment and restrictive behaviour.11 A non-invasive therapeutic strategy is treatment with intranasally administered insulin. Intranasal application leads to uptake of insulin in the cerebral spinal fluid (CSF), bypassing the blood–brain barrier.12 Insulin administered by this route has been shown to improve memory, general cognition, functional abilities and mood in healthy adults, adults with Alzheimer's disease and adults with minimal cognitive impairment without causing systemic effects on blood glucose levels or other serious adverse events .14 Schmidt et al. hypothesised that intranasal insulin could compensate for the cognitive deficits in PMS and conducted an exploratory non-placebo-controlled study in six children with PMS.15 They reported an improvement of motor activity, cognitive function, nonverbal communication and autonomy after treatment with intranasal insulin, without an effect on blood glucose levels or SAEs. However, replication of these effects in a clinical trial with a larger number of participants is needed.The neurodevelopmental features in PMS are mainly caused by a deletion of the The aim of our study was to evaluate the effect of intranasal insulin on development and behaviour in a larger group of children with PMS using a randomized, double-blind, placebo-controlled clinical trial.16 Before the clinical trial, we statistically evaluated several different trial designs  and found the stepped wedge design showed the highest power for testing treatment effect.16 The stepped wedge design is especially appropriate for studies in which the intervention is expected to do more good than harm, a situation in which it is considered unethical to withhold treatment from a subgroup of participants  or to only provide treatment to all participants if the effect has been proven to be effective (as in wait-list design). With the stepped wedge design, the change from intranasal placebo to intranasal insulin occurs at different time points for each study group . Children with severe  brain damage or with a metabolic or neuromuscular disease were excluded because development and behaviour could be significantly influenced by these factors and less dependent on their SHANK3 deficiency, the hypothetical target of the treatment. Of the 38 children, two were lost to follow-up before the start of the trial, parents of 10 children decided not to participate in the trial and we excluded one child with severe brain damage. Ultimately, 25 children were included in the trial.The study was performed between March 2013 and June 2015. Patients were recruited from a group of 38 Dutch children with PMS who had been diagnosed in the Clinical Genetics department of the University Medical Centre Groningen or referred to our centre from other hospitals in the Netherlands. Inclusion criteria were a molecularly confirmed 22q13.3 deletion including Basic characteristics of participants, treatment exposure and safety endpoints were collected for all participants. The primary objective was to investigate whether intranasal insulin leads to a greater increase in the level of general developmental functioning compared with the change in developmental functioning without insulin. The secondary objective was to investigate whether intranasal insulin leads to an improved functioning of adaptive and social-emotional behaviour.t=0 months), participants started with either intranasal insulin (group A) or intranasal placebo (group B and C) according to schedule . One spray was 0.1 ml, containing 10 IU of insulin. The composition and packaging of the placebo solution was identical to the insulin buffer solution. In the placebo solution, insulin was replaced by human albumin  to a concentration of 3.47 mg/ml.According to schedule, patients received a set of nose sprays with either insulin or placebo solution each 6-month period. The recombinant human insulin solution for this study consisted of the licensed parenteral drug Humuline Regular 100 IU/ml . The dosage was based on the pilot study of Schmidt 17 and to children 20–60 IU/day (0.5–1.5 IU/kg/day).18 The daily dose for our study was calculated so that the maximal safe dose from previous studies was maintained, but maximal levels in the CSF would be achieved. Because head circumference in children corresponds to their brain volume,19 we calculated the insulin dose based on both estimated body weight20 and head circumference21  to find the optimal therapeutic dose tic dose . The med18 Therefore, peripheral glucose or insulin levels were not measured in study participants. Safety and tolerability were evaluated based on both expected and unexpected adverse events and SAEs as reported by parents to the investigators by email, phone and through their participant diaries. Previously reported, and therefore expected, adverse events were transitory nasal irritation, rhinitis, spontaneous nose bleeds and headache.13 As the placebo buffer solution consisted of the same components as the insulin buffer solution, the potential adverse effects of the placebo solution were expected to be similar.Several independent studies have shown that intranasal insulin administration did not alter systemic insulin concentration or glucose levels in children and adults.To measure the effect of intranasal insulin, development and behavioural functioning were assessed at 6-month time intervals by different tests (see next section). The rates of improvement of development and behaviour in the treatment period were then compared to the rates of improvement in the pre-treatment period.t=−6 and t=0 month assessments of the pre-clinical trial phase were used to determine baseline changes in general development and behaviour.The results from the t=6, t=12 and t=18 months). Immediately after each assessment, participants switched to a new set of nose sprays ,23 or Wechsler Preschool and Primary Scale of Intelligence,24 third edition, Dutch version (WPPSI-III-NL)25 depending on the developmental age of the children. Bayley-III-NL and WPPSI-III-NL outcomes are expressed in raw scores that were converted to a developmental age equivalent (DAE) as previously described.3Two licensed clinical child psychologists with professional experience, intensively trained in the administration of the instruments used in this study and experienced in working with children with special needs, assessed children at home or in their school environment. General development, our primary outcome parameter, was assessed by the Bayley Scales of Infant and Toddler Development,Experimentele Schaal voor de beoordeling van het Sociaal-Emotionele Ontwikkelings Niveau (ESSEON). The Vineland screener 0–6, which is based on the American short version of the Vineland Adaptive Behavior Scales (VABS),26 was used to cover four domains of adaptive behaviour portrayed in the original Expanded Version of the VABS: communication, social, daily and motor skills. The Vineland Screener has been validated for Dutch children aged 0–6 years and for children between 1 and 18 years with a developmental delay.27 Raw scores of each subscale were converted to a DAE. The ESSEON is a Dutch instrument that determines the level of social and emotional development of children from 0 to 14 years of age.28 It is a behaviour rating scale based on interviewing adults with knowledge of the child's behaviour, resulting in a DAE for each subscale. There is some overlap between the Vineland Screener and the ESSEON with respect to aspects of adaptive behaviour-like social and communicative abilities, but these questionnaires are complementary with respect to emotional development.Behaviour, our secondary outcome parameter, was assessed by two questionnaires applied in interview form: the Vineland Screener 0–6 and the 29 and the Child Behavior Checklist (CBCL/1.5-5).30 Methods and results are described in In addition to our primary and secondary objectives, we evaluated behavioural problems, which were assessed using two questionnaires: the BRIEF-P16 It is based on comparing the change in DAE  for each individual subject from the control period, for example, the period of baseline and/or placebo, with the period in which children received intranasal insulin. A P-value<0.05 was considered to be statistically significant. The intention-to-treat analysis was performed with all participants and in all periods. The on-treatment analysis was performed based on all periods in which participants had a >90% treatment adherence, which was evaluated by returned medication and participant diaries. A subgroup analysis was performed for children >36 months who usually show a decrease or stagnation of developmental growth3 to see whether this has implications for the treatment effect.The statistical analysis for treatment effect was conducted using a subject-specific random coefficients (linear mixed) model which has been described previously (SWD-S3).The Institutional Medical Ethical Review Board of the University Medical Centre Groningen approved this study (protocol ID 2012/329) and all parents provided written informed consent. The study was conducted according to the principles of the Declaration of Helsinki  and in accordance with the Medical Research Involving Human Subjects law . The clinical trial  was registered at the national competent authority (NL-41213-042-12), the Netherlands Trial Register (NTR 3758) and the EU Clinical Trial Register (2012-002873-77).3 Nine children were randomised in group A, 8 in group B and 8 in group C.Twenty-five children participated in the trial, 6 males and 19 females, of whom 23 fully completed the treatment period , one child received ~2/3rds of the daily dose in period 3 on the parents' own initiative and one child received ~80% of the medication in period 3 because the parents forgot to administer the medication several times.Two children did not finish the whole-treatment period of the study due to non-SAEs. One child suffered from abdominal pain at the end of period 2 (under placebo) and subsequent paediatric evaluation suggested mild pancreatitis with elevated amylase levels. A relation with the nasal spray was not likely as this child had been using this batch of spray for at least 5 months and none of the other children had similar complaints. Nonetheless, the parents decided to discontinue with the study. Another child used the nasal sprays until the fourth week of period 3, but had suffered from recurrent nosebleeds beginning in period 2 so that the parents decided to discontinue the medication for a while but then forgot to restart treatment.No SAEs or suspected serious adverse reactions have been reported. Expected adverse events such as nosebleeds and irritation of the nasal area were frequently reported (12 of 25 children). These were limited to 1 or 2 days and usually occurred only once per period in both placebo and insulin groups. Most other adverse events reported, like upper airway infections and gastro-enteritis, are common in children and occurred in both placebo and insulin groups.In the pre-treatment period, the mean change in DAE ranged from 0.4 to 1.8 months per 6 months for the five domains of general development (Bayley-III-NL). Intranasal insulin resulted in an additional increase of the DAE for cognition (0.6 months per 6 months), receptive language (0.6 months per 6 months) and fine motor skills (0.4 months per 6 months). Although this additional effect of intranasal insulin for these domains was not significant itself, the total change in DAE with intranasal insulin was statistically significant, as illustrated by the 95% confidence intervals . The meaThere was also a positive effect of intranasal insulin observed on all four domains of adaptive behaviour (Vineland Screener 0-6) and on social development (ESSEON). In the pre-treatment period, the change in DAE ranged from 0.1 to 2.4 months per 6 months, except for social skills of adaptive behaviour. Intranasal insulin resulted in an additional increase of the DAE for all domains except emotional development, including social skills of adaptive behaviour . This adP=0.046) and social skills . The contribution of intranasal insulin to the total change in DAE was 80% for cognition and >100% for social skills because the control rate was negative. Remarkably, the observed additional effect of intranasal insulin on DAE in this subgroup was positive for the domain of expressive language , an effect that was not seen in the total study group (0.0 months per 6 months).The subgroup analysis of children aged >36 months is shown on the right in The aim of this study was to evaluate whether the previously reported positive effect of intranasal insulin on development and behaviour in children with PMS could be confirmed in a randomized, double-blind and placebo-controlled clinical trial. Our study shows a positive observed effect of intranasal insulin on psychomotor and behavioural development. However, this effect of intranasal insulin was not strong enough to reach statistical significance in our small group of patients, but some statistically significant positive effects were seen in the subgroup of children older than 3 years of age.Obtaining significant results from clinical trials in a small cohort of patients is a challenge, especially in a clinically variable disorder like PMS, and there are a number of factors that need to be considered that may have impacted the size of the signals we measured.16 However, as we recently showed in a study of 34 children, development in PMS is not a linear process and development stagnates after the age of 3 years.3 For most of the developmental domains that were analysed in the present study, the developmental rate under normal circumstances was therefore slower than expected, making an additional 15–45% treatment effect of intranasal insulin based on the previously expected higher basal rate too optimistic, further meaning that smaller effects of intranasal insulin on DAE are hard to detect in this sample size. Nonetheless, we saw an effect of insulin: while the DAE without insulin did not significantly improve over time for most domains, the increase of DAE with intranasal insulin treatment  based on data from four children with PMS.3 Interestingly, subgroup analyses in children older than 36 months suggested a stronger effect of intranasal insulin in this group and showed significant results for the effect of intranasal insulin for the domains of cognition and social skills  and the maximal levels we could obtain in the CSF (based on a proxy for brain volume) because a safety study of intranasal insulin based on brain volume was not available. If we had determined the daily dose based on the maximal safe CSF levels, seven children would have received a higher dose, especially the children in the youngest age group (10–15 kg). Another challenge with intranasal application of insulin is that we cannot determine the exact amount of fluid administered in the nasal cavity nor the actual uptake in the CSF or the distribution and localisation in the brain tissue itself.Finally, even though the treatment adherence appeared to be very high, we relied on patient diaries and returned medication, which are less objective. Still, we think that our estimations of treatment adherence were reasonable since administration of the nose-spray had a relatively low burden, limited adverse reactions and parents were highly motivated to participate in the study.31 The insulin/IGF-1 signalling pathway is involved in dendritic spine and synapse formation upon activation of the PI3K-Akt-mTOR signalling pathway.32 In PMS, dendritic spine formation and synaptic signalling are impaired because of SHANK3 haploinsufficiency as shown in Shank3-deficient animal models.33 Excitingly, IGF-1 was shown to rescue synaptic transmission impairments of neuronal stem-cells of PMS patients by decreasing the number of synapses with SHANK3 and increasing the number of synapses with PSD-95.9 In addition, insulin has been shown to stimulate expression of the postsynaptic scaffolding protein PSD-95 in rat neurons.34 These studies therefore suggest that the effects of SHANK3 haploinsufficiency can be compensated by insulin/IGF-1 signalling via up-regulation of PSD-95 and increasing the number of PSD-95 containing synapses. For future studies, it would also be interesting to investigate the (short-term) effect of intranasal insulin in the brain by measuring event-related potentials or using functional magnetic resonance imaging. Although these techniques have not yet been standardized or validated for testing the effect of medication on development and behaviour in children with developmental delay, they would be very interesting to use in follow-up experiments.Despite our modest results, we do think that intranasal insulin could be a therapeutic strategy in children with PMS, based on the function of insulin in the brain. Insulin and IGF-1 function as neurotransmitters in the brain and both bind cerebral insulin, IGF-1 and insulin/IGF-1 hybrid receptors with different affinity (reviewed by Fernandez and Torres-Alemán.).11 A phase-2 study is currently ongoing in the USA to validate the effect of IGF-1 in patients with PMS . Interestingly, intranasally applied IGF-1 can also be transported to the CSF/brain,35 but no clinical trials have been conducted with intranasal IGF-1 in patients with PMS.A pilot study with daily intraperitoneal IGF-1 injections in patients with PMS showed a beneficial effect on social impairment and restrictive behaviour.In conclusion, the application of intranasal insulin is a non-invasive, safe, easy and interesting therapeutic approach to improve intellectual and behavioural development in children with PMS. Our preliminary results show that it increased the level of developmental functioning, especially in children older than 3 years, who usually show a stagnation of development. Most results were not statistically significant in our small study group but may be considered clinically relevant. Clinical trials in larger study populations are required to prove the true effect of intranasal insulin on development and behaviour, and might be able to determine which individuals would benefit most from it. In addition, future studies should focus on improving the local side-effects by adapting the insulin formulation and by studying the effects of intranasal insulin in the brain by using Shank3-deficient animals, a model for PMS. Still, we think that treatment with medication in children with complex disorders like PMS should only be part of the total and individualised treatment plan. Therapeutic support  with attention to individual needs are as important, if not more important, to optimise health and wellbeing in individuals with PMS.