Sequence and structural diversity of antibodies are concentrated on six hypervariable loops, also known as the complementarity determining regions (CDRs). Five of six antibody CDR loops presumably adopt a so-called canonical structure out of a limited number of conformations. However, here we show for four antibody CDR-L3 loops differing in length and sequence, that each loop undergoes conformational transitions between different canonical structures. By extensive sampling in combination with Markov-state models we reconstruct the kinetics and probabilities of the transitions between canonical structures. Additionally, for these four CDR-L3 loops, we identify all relevant conformations in solution. Thereby we extend the model of static canonical structures to a dynamic conformational ensemble as a new paradigm in the field of antibody structure design. H) and the light chain (VL) that shape the antigen binding site, the paratope  was performed using the python library PyEMMA 2 employing a lag time of 10 ns . AdditioThe first antibody variable fragment (Fv) studied is the house dust mite allergen binding antibody. Der p 1 and Der f 1 are potent allergens, produced by house dust mites, and cause allergic sensitization and asthma. The PDB structures 3RVW  and 3RVT  were simulated without the antigen present . The CDRAs a second antibody Fv fragment to characterize the CDR-L3 loop ensemble in solution, the antibody binding to lymphocyte function—associated antigen-1 integrin (LFA-1 integrin) was analyzed. LFA-1 integrin plays a vital role in adhesive interactions with both endothelial cells and antigen-presenting cells . Again, The third analyzed antibody Fv fragment is binding interleukin-13 (IL-13), which is a member of the growth-hormone-like cytokine family and plays a central role in the development of asthma , 65. AgaThe last antibody Fv fragment investigated is the anti-hemagglutinin binding influenza antibody . Three cin silico development of antibody biotherapeutics because of their diversity in length, sequence and structure (This present study characterizes the conformational ensemble of the CDR-L3 loop and investigates conformational transitions between different canonical clusters of same length. Structural description of the CDR loops, especially the CDR-H3 and the CDR-L3 loops, are known to be a major challenge for tructure . Anothertructure . The contructure –72.The idea of having ensemble of pre-existing conformations out of which the functional ones are selected was supported by population shift models originating from the Monod-Wyman-Changeux model –77. ThisFor structure design our results imply, that for a given CDR-L3 loop sequence several canonical structures have to be considered. Our results also indicate that there are dominant CDR-L3 loop structures in solution, that are not apparent from X-ray analysis most likely due to crystal packing effects , 32. FurWe characterized the CDR-L3 loop ensemble in solution for different loop lengths and types of light chains. For four antibodies we were able to structurally, thermodynamically and kinetically profile the conformational space of the CDR-L3 loop in solution. Comparison of the resulting the CDR-L3 loop ensemble with the available canonical structures allowed us to calculate transition kinetics between different canonical clusters. Additionally, we identified all relevant conformations in solution. Our results clearly indicate that the static model of canonical structures should be extended to the description of the CDR-L3 loop as conformational ensemble. These findings have broad implications in the field of antibody structure design, antibody docking and might play a key role in the development of biotherapeutics as they provide a new paradigm in the understanding of CDR-L3 loop conformations and their dynamics.All datasets generated for this study are included in the article/All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Many people are accessing digital self-help for mental health problems, often with little evidence of effectiveness. Social anxiety is one of the most common sources of mental distress in the population, and many people with symptoms do not seek help for what represents a significant public health problem.This study aimed to evaluate the effectiveness of a self-guided cognitive behavioral internet intervention for people with social anxiety symptoms in the general population.We conducted a two-group randomized controlled trial in England between May 11, 2016, and June 27, 2018. Adults with social anxiety symptoms who were not receiving treatment for social anxiety were recruited using online advertisements. All participants had unrestricted access to usual care and were randomized in a 1:1 ratio to either a Web-based unguided self-help intervention based on cognitive behavioral principles or a waiting list control group. All outcomes were collected through self-report online questionnaires. The primary outcome was the change in 17-item Social Phobia Inventory (SPIN-17) score from baseline to 6 weeks using a linear mixed-effect model that used data from all time points .P=.001), a standardized mean difference effect size of 0.2. The improvement was maintained at 12 months. Given the high dropout rate, sensitivity analyses explored missing data assumptions, with results that were consistent with those of the primary analysis. The economic evaluation demonstrated cost-effectiveness with a small health status benefit and a reduction in health service utilization.A total of 2122 participants were randomized, and 6 were excluded from analyses because they were ineligible. Of the 2116 eligible randomized participants , 70.13% (1484/2116) had follow-up data available for analysis, and 56.95% (1205/2116) had data on the primary outcome, although attrition was higher in the intervention arm. At 6 weeks, the mean (95% CI) adjusted difference in change in SPIN-17 score in the intervention group compared with control was −1.94 (−3.13 to −0.75; For people with social anxiety symptoms who are not receiving other forms of help, this study suggests that the use of an online self-help tool based on cognitive behavioral principles can provide a small improvement in social anxiety symptoms compared with no intervention, although dropout rates were high.ClinicalTrials.gov NCT02451878; https://clinicaltrials.gov/ct2/show/NCT02451878 Many people are accessing digital tools for self-help for a range of mental health problems . Social Effective psychological and pharmacological treatments exist for social anxiety symptoms, but many people with symptoms do not seek or receive these treatments -7. Self-We, therefore, undertook the first large-scale pragmatic randomized trial of an online self-guided cognitive behavioral intervention for people with self-reported social anxiety symptoms in the general population. Our experimental hypothesis was that participants who received the intervention would have a greater improvement in symptoms of social anxiety compared with participants who did not.A two-arm, parallel-group randomized controlled trial was conducted to compare the effectiveness and cost-effectiveness of a Web-based and mobile-optimized self-guided intervention with a waiting list control condition for treating social anxiety symptoms. The study received ethics approval from the University of Oxford Medical Sciences Inter-Divisional Research Ethics Committee (MS-IDREC-C1-2015-167) and the Australian National University Human Research Ethics Committee (Protocol 2015/229) and is registered on ClinicalTrials.gov (NCT02451878). All participants provided informed consent to take part in the study using a self-completion online form. Outcomes were assessed at baseline, 6 weeks, 3 months, 6 months, and 12 months. The main follow-up time points were chosen to measure immediate effect (6 weeks as the intervention was designed to be used over a 6-week period) and long-term outcomes (12 months), along with interim time points (3 months and 6 months) to strengthen our repeated measures analysis and to support participant engagement. All study administration was conducted using automated online systems. The trial protocol is in Participants were recruited primarily through an online advertisement placed on the UK National Health Service (NHS) website. In addition, study advertisements seeking individuals with social anxiety symptoms were placed on university and charity websites and disseminated via email and social media. We aimed to capture people with a broad range of social anxiety symptoms in the general population, who were likely to be typical of those seeking help from self-directed digital tools. Interested potential participants completed an online screening questionnaire to assess eligibility. We excluded anyone currently receiving therapist-guided treatment for social anxiety disorder or who self-reported a diagnosis of schizophrenia or bipolar affective disorder. Initial inclusion criteria were having access to the internet-based intervention, aged 18 years or older, resident in England, having an email address and mobile telephone number , and an initial criterion of scoring in a subclinical range of 13 to 19 on the 17-item Social Phobia Inventory (SPIN-17). We had initially chosen the 13 to 19 range with expert advice as this would, in theory, capture those scoring above the population mean score (11-12) while excluding those scoring above the commonly used threshold of 19, which indicates further assessment may be warranted . However, early in recruitment, it became apparent that most people in the general population volunteering for this study scored much higher than this, and the distribution of SPIN-17 scores meant that very few scored in the low range. There was clear evidence of a high level of unmet need among individuals living with social anxiety symptoms in the community and not seeking help elsewhere. With advice from our independent Trial Steering Committee, we, therefore, revised and reregistered the protocol  to modify the inclusion criteria to include all individuals scoring 13 or more on SPIN-17, therefore capturing those in our hypothesized subclinical range of 13 to 19, as well as those with a higher score. We continued to exclude anyone receiving professional help, and therefore, the final sample represented adults in the general population who self-reported some level of social anxiety symptoms but who were not receiving treatment for social anxiety. Potentially eligible participants completed consent, and 24 hours later, they were sent an email link to self-complete their baseline measures using online questionnaires.Once baseline measures had been completed, participants were randomized (1:1 ratio) to either the intervention group (E-couch) or the waiting list control group using a computer-generated random number sequence run through an automatic online program using a block size of 2 without stratification. Due to the nature of the intervention, participants were not blind to allocation.Given that this was a general population sample, all participants continued to receive usual care. Participants in the intervention arm were given access to a password-protected website that contained the E-couch social anxiety module. The website was mobile-optimized and could, therefore, be used on a smartphone with the look and feel of a dedicated app, or on a computer browser.The E-couch social anxiety module is a self-directed interactive program based on cognitive behavioral therapy principles. The program is divided into 6 modules: a literacy section, which provides information about the symptoms of social anxiety, types of available help, and effective treatments, and 5 toolkits comprising exposure practice, cognitive restructuring (modifying your thinking), attention practice, social skills training, and relaxation. The content of the toolkits consists of evidence-based information, interactive exercises, and workbooks based on cognitive behavioral principles; participants could complete the modules in any order. Participants were advised to access and use the intervention over the initial period of 6 weeks . “Ideal” usage of the intervention would entail engagement with 1 new module each week and ongoing updates to diaries and workbooks based on the user’s real-life experiences. E-couch was developed by the ehub team at the Australian National University National Institute for Mental Health Research. The intervention was adapted for this study to create a “stand-alone” social anxiety intervention that was accessed via a password-protected portal and with the usual E-couch branding removed. The program was adapted for a UK audience by removing Australian-specific terminology and undertaking user testing on the new version. No changes were made to the intervention during the study period.Participants in the control group were informed that they had been put on a waiting list to receive access to the intervention in 12 months. They were asked to complete baseline and follow-up measures at the same time as participants in the intervention group. They received no other intervention.Automated text (SMS) message and email reminders were sent to participants in both groups to reduce attrition. Participants in the intervention condition received 1 text message within 24 hours of randomization to remind them to access the intervention and 3 email reminders during the 6-week intervention period to remind them to log in to access the program. In addition, all participants received email invitations to complete follow-up surveys at each outcome measure time point, with those who failed to complete receiving a reminder email followed by a reminder SMS text message.The primary outcome was the change in SPIN-17 score from baseline to 6 weeks. The SPIN-17 is a 17-item self-rated scale covering the main social anxiety symptoms of fear, avoidance, and physiological disturbance. The responses to 17 statements  are rated on a 5-point scale from “not at all” (score=0) to “extremely” (score=4) to indicate the extent to which each statement reflects how the respondent was feeling in the past week, with higher scores reflecting greater social anxiety symptoms. The SPIN-17 has good test-retest reliability, internal consistency, and convergent and divergent validity . SecondaWe aimed to recruit 2104 participants  to this trial, to detect a small between-group standardized mean difference of 0.2 at 5% two-sided significance level and 90% power, assuming a high level of potential attrition of up to 50% given the fully self-guided nature of the intervention and automated nature of the trial . Although previous studies have suggested a larger treatment effect for internet-delivered interventions, we believed this treatment effect is too optimistic for a pragmatic trial of a self-guided treatment in a general population sample. The target effect size, although small at an individual level, can potentially translate into an important population-level change .The statistical analysis was finalized before unblinding of the data. Primary analysis was modified intention to treat according to allocated group irrespective of adherence and with at least one outcome questionnaire completed post randomization. A linear mixed-effect model was fitted to the primary outcome data, using data collected at 6 weeks, 3 months, 6 months, and 12 months. Participant was included as a random intercept. Randomized group, baseline SPIN-17 score, time, and time by randomized group interaction term were fitted as fixed effects. An unstructured variance covariance matrix was specified between repeated measures on the same individual. Assumptions of normality and constant variance for linear mixed-effects models were assessed by residual plots and other diagnostics plots.Given the high level of attrition expected in online trials of self-guided digital interventions, we also prespecified a Complier Average Causal Effect (CACE) analysis for the primary outcome and 2 other main outcomes, to include only participants who completed at least one module of the intervention and at least one outcome assessment to investigate the effect of the intervention in participants who adhered to the intervention. An instrumental variable approach was adopted to provide the CACE estimate at 6 weeks. This method involved a 2 least squares  estimation from fitting a linear regression model of the primary outcome, adjusting for baseline SPIN-17 and compliance instrumented on randomized group .Similar approaches were undertaken for other outcomes. A CACE analysis at 6 weeks was conducted on fear of negative evaluation (BFNE-S) and mental well-being (SWEMWBS) measures, similarly adjusting for baseline BFNE-S and SWEMWBS in the linear regression models. Safety analyses were not conducted as there were no adverse events reported during the study period.In anticipation of high levels of dropout, we prespecified various sensitivity analyses to explore the impact of assumptions regarding missing data in the primary outcome analysis. These included analyses (1) of participants with complete data at all time periods, (2) adjusting for factors found to be predictive of missingness, (3) fitting a pattern mixture model to assess different degrees of missing not at random, as well as (4) an assessment of missing not at random assumption for the primary outcome by assuming plausible arm-specific differences of missing SPIN-17 score between responders (with SPIN-17 score at 6 weeks) and nonresponders (missing SPIN-17 score at 6 weeks) ,25.Predefined subgroup analyses were conducted on change at 6 weeks for SPIN-17, BFNE-S, CES-D, and SWEMWBS for baseline SPIN-17  to ascertain if the benefit differed between groups scoring above or below the screening threshold and for certain demographic characteristics to determine if the effectiveness of the intervention differed by the individual characteristics we had measured, that is, age , gender , educational level , and ethnicity . Subgroup analyses were conducted by inclusion of an interaction term of baseline subgroup by randomized group by time in the linear mixed model. Descriptive statistics were used to describe usage data, adherence, and self-reports of other help received during the study period. All statistical analyses were performed using STATA SE version 15.1 .A cost utility analysis from an NHS and social care perspective was conducted within this trial to assess the cost-effectiveness of the intervention. The total costs of developing, modifying, delivering, and maintaining the intervention were obtained, and the mean intervention cost was estimated for the participants recruited in the intervention group. Data on health care service utilization (for any reasons) were collected for all participants, including primary care consultations, hospital outpatient appointments, and hospital admissions. Unit costs for these health services were obtained from the Personal Social Services Research Unit (2016-2017) using national average costs . MaximumRecruitment took place between May 11, 2016, and May 9, 2017, when the target sample size was reached. Participants were followed up for 1 year. Final data were locked on June 27, 2018 . P value) in change in SPIN-17 score in E-couch compared with control was −1.94  in change in SPIN-17 score for intervention compared with control was −2.95  adjusted difference in change in SPIN-17 score of −3.07 , and again in this subgroup analysis, the few participants scoring in the lower SPIN-17 range had no benefit on depressive symptoms compared with the control group. There was no evidence of heterogeneity in the effects of intervention for the subgroup analyses involving BFNE-S and SWEMWBS.At 6 weeks, the mean (SD) number of E-couch modules fully completed (out of 6) was 1.87 (1.43), total mean (SD) time in minutes spent on modules was 35.3 (48.1), and total mean (SD) page views was 37.6 (41.3). Greater adherence to the intervention was not associated with baseline SPIN-17 score, age, gender, or ethnicity . At 6 weAt both 6-week and 12-month follow-ups, the waiting list control group, in general, used more health care services than the E-couch group see  and 4. Td=0.2). The study findings provide no evidence as to whether this fully self-guided approach has a role in a clinical setting, where, to date, the evidence base suggests that although unguided self-help has effectiveness, therapist-guided and therapist-led approaches are likely to be superior. The cost-effectiveness analysis demonstrated that the intervention is likely to be cost-effective in both the short and long term, although the gain in general health status and QALY score was very small. The benefit seen in the condition-specific social anxiety outcome measures was greater than the general health status used in the cost utility analysis. Furthermore, the intervention cost could be substantially reduced if the E-couch is used by large numbers at a population level as a public health tool. Given that many people with social anxiety symptoms do not seek help, and that therapist-supported approaches are limited in supply, the findings suggest that unguided digital intervention for social anxiety can be beneficial for some people who do not access professional help and who are increasingly seeking support from apps and other digital tools. The self-help approach tested here might also complement face-to-face therapy, potentially reducing the amount of therapist contact time required and perhaps helping to maintain engagement, although these suggestions need to be empirically tested in future effectiveness and cost-effectiveness work.Our findings showed that this fully self-guided internet intervention gave a small reduction in social anxiety symptoms in participants recruited online from the general population, compared with a usual care waiting list control group, and this small but positive finding was robust to the sensitivity analyses, which explored our missing data assumptions. There was a similarly small but significant improvement in fear of negative evaluation. These improvements were also found in the CACE analyses and maintained at the 12-month follow-up. In the context of a very common mental health problem, this finding suggests that automated self-help delivered via the internet could reduce the overall level of social anxiety symptoms in the population, although at an individual level, the mean symptomatic benefit is small (This study adds to the body of work showing small positive effects for unguided digital self-help for social anxiety ,9-14 andThis study exemplifies both the strengths and weaknesses of undertaking online trials for digital interventions. We were able to recruit large numbers of participants from the general population using digital advertising, and we were able to deliver all measures and the intervention remotely, using little resources and with no requirement for any “real-world” contact between participants and researchers. The flipside of this was that, in common with other fully automated trials of unguided online interventions, there were high levels of dropout from the intervention and attrition from the trial . This isFor people with social anxiety symptoms in the general population who are not receiving other forms of help, an online unguided tool based on cognitive behavioral principles accessed via a computer or mobile phone gave a small but significant improvement in social anxiety symptoms compared with no intervention. As with many online trials of digital interventions, we experienced a “methodological trade-off” between having a cheap, scalable model of intervention delivery versus the statistical challenge of a high degree of missing data. Our findings suggest this intervention could potentially offer the first self-help rung on the ladder of a stepped approach to social anxiety symptoms.


Little empirical evidence is available to support the effectiveness and cost-effectiveness of internet interventions to increase help-seeking behavior for mental health in young adults.Link) in comparison with usual help-seeking strategies.The aim of this study was to evaluate the cost-effectiveness of a Web-based mental health help-seeking navigation tool  or usual care (n=208) with 1- and 3-month follow-ups. The primary outcome of this study was quality-adjusted life years (QALYs) measured by the assessment of quality of life–4D. Costs were calculated based on the self-reported resource use questionnaire and were reported in 2015 Australian dollars. Primary analyses were conducted as intention-to-treat and reported as incremental cost-effectiveness ratios. Completer analyses were conducted in a sensitivity analysis.A cost-utility analysis alongside the main randomized trial of P<.001). The intervention was associated with significantly lower health professional consultation costs at 1-month follow-up . Costs of hospital services were lower at 3 months in the intervention arm (mean costs Aus $47 vs Aus $101); however, there was insufficient sample size to detect a significant difference between the groups. There were no statistically significant differences in the total costs between the 2 arms. Relative to the control group, those who received the intervention experienced 0.01 more QALYs (0.00-0.02) and had lower total health sector costs of Aus −$81 (Aus −$348 to Aus $186) over 3 months. The intervention was found to be more effective and less costly compared with usual help-seeking strategies. The intervention was 100% likely to be cost-effective below a willingness-to-pay value-for-money threshold of Aus $28,033 per QALY. Results were robust in the sensitivity analysis.Significantly more QALYs were gained in the intervention group than the control group (0.15 vs 0.14; Our study found that the online youth mental health help-seeking Web service is a cost-effective intervention for young people aged 18 to 25 years compared with usual search strategies. Further research is required to confirm these results.Australian New Zealand Clinical Trials Registry ACTRN12614001223628; https://www.anzctr.org.au /Trial/Registration/TrialReview.aspx?id=366731 Mental and substance use disorders are a leading cause of disability in children and young adults worldwide , making Barriers to help-seeking and treatment for young people include stigma -7, embarLink compared with usual help-seeking strategies for young adults. The current analysis sought to answer whether an online help-seeking intervention for young adults was cost-effective compared with usual search practices from a health care sector perspective  within a 3-month follow-up.To address these concerns, a randomized controlled trial (RCT) was conducted to evaluate the effectiveness and cost-effectiveness of a brief, internet-based, mental health help-seeking intervention, called Ethics approval was obtained from the University of Melbourne Human Research Ethics Committee, reference #1341063.4, and Deakin University Human Research Ethics Committee, reference #2015-320. All participants consented to take part in this study via an online consent form.This economic evaluation was conducted alongside the RCT. The study adheres to the Consolidated Health Economic Evaluation Reporting Standards Statement (CHEERS) checklist  Multime.Link) or control group  using a random allocation sequence generated internally by the QuON computer software [The study was conducted entirely online. Participants were recruited by electronic direct mail, social media, online advertising, and snowballing, where participants were asked to share the link on the Facebook page with friends and family. Interested participants were directed from a link in the advertisements to the study website where they were provided with more information and a consenting procedure if meeting the eligibility criteria of being aged between 18 and 25 years and residing in Australia. Eligible participants provided informed consent by acknowledging that they had read the information statement by clicking a box, then clicking a separate box to indicate that they consented to participate in the Link Research Project. They then registered for the trial using their email address and a self-generated password. Immediately following registration, all participants completed the baseline survey sent through email including demographic information and the Kessler-10 (K10) measure of psychological distress. Participants were then stratified by responses on gender  and severity of psychological distress (K10>20), then randomized into parallel groups consisting of the intervention group  toward encouraging help-seeking behavior [Link has a 4-step process where (1) users select symptoms they experience, (2) rate how much they are affected by them, (3) choose their preferred way to receive help , and then (4) finally, click on service options presented by the program for more information on how to seek help within that service, including expected costs and website links or online directories. The feasibility of Link was trialed previously and found to be acceptable to young people [The behavior  and the behavior . The funbehavior . In brieg people .The control condition instructed the young adult participants to use their typical strategies to seek help both online and offline such as using internet search engines and face-to-face or phone services.The AQoL-4D was used to measure health-related quality of life . OriginaThis economic evaluation adopted a health sector perspective, which included health care costs paid by the government and out-of-pocket costs paid by patients. All costs were expressed as 2015 Australian Dollars. No discount rate was applied because the time horizon of the study was 3 months.Link platform. The total projected cost for Link was Aus $1.74 million. The maintenance cost of the Link intervention included the time cost of 2 information technology staff (1 senior and 1 junior staff), in addition to the time cost of staff to update content and equipment costs. The total maintenance cost for Link was Aus $29,803 per year . To not overestimate the per-person costs , we estimated the number of people who are likely to receive the intervention when implemented within the Australian population using assumptions based on the published literature. The intervention pathway starts with young adults aged 18 to 25 years in the 2015 Australian population [Link intervention . The RUQ comprised questions on relevant health care services , including the frequency of visits, payment methods , outpatient care services , inpatient admissions, and medications. The different versions of the RUQ have been used in other trials in mental health . The cosThe out-of-pocket costs reported in the RUQ for each service were considered in the health sector perspective. If the reported amount for a community-based health contact was outside of a plausible range, the maximum of out-of-pocket cost of Aus $447 was used based on the recommendation of the Australian Psychological Society . For thoThe primary analysis was performed using an intention-to-treat approach. All participants who were randomized were included in the analysis, and missing data were handled by multiple imputation by chained equations using predictive mean matching. The data were assumed to be missing at random by testing through a series of logistic regression analyses comparing participants’ characteristics for those with and without missing endpoint data. At 1- and 3-month follow-ups, approximately 30% of participants had dropped out or did not complete the survey (29% in the intervention group vs 31% in control group). However, the maximum percentage of missing QALY and cost data was 40%. Thus, to ensure efficient and reproducible estimates, a total of 40 imputations were completed [family, whereas Pregibon link test, Pearson correlation test, and modified Hosmer-Lemeshow test were adopted to identify the link function [General linear models (GLMs) were used to evaluate differences between group on total QALYs and total health sector costs. For the GLMs, a modified Park test was used to identify the appropriate function . GLM witfunction . We firsfunction ,32. GLM function . All regfunction . The regThe bias-corrected CIs around the ICER were reported based on 3000 bootstrap simulations. The bootstrapped data were also plotted on a cost-effectiveness plane . The thrSensitivity analyses included a complete case analysis in which only participants who completed 1- and 3-month follow-ups were included. In addition, the development costs were varied to reflect different proportions of the population receiving the intervention if it was implemented in Australia. In particular, the proportion of people who would receive the intervention was varied from 2% to 17% of the Australian population.All analyses were undertaken using Stata SE version 15.Link and 208 allocated to the control group. Additional details regarding the study flow and Consort diagram are reported elsewhere [Link vs 69% control group). Baseline characteristics were similar between the groups (P<.01).A total of 413 participants were randomized, with 205 allocated to lsewhere . The ovee groups , except Link intervention was associated with a lower number of lengthy health professional consultations; however, this difference did not reach statistical significance. For example, there were less people (2 vs 11) attending extensive GP consultations (duration over 40 min) in the intervention group at the 1-month follow-up compared with those who used usual search strategies.Use of health services is reported for baseline and 1- and 3-month follow-up periods in P<.001). Similarly, there was a statistically significant difference in QALYs at the 3-month follow-up between the groups, which favored the intervention group .The estimated mean AQoL-4D utility values and QALYs for the intervention and control groups over the 3-month follow-up are presented in Link group were statistically significantly higher than those in the control group. No statistically significant differences for other cost categories at any other time points were found. The average total health sector costs for the intervention group were lower than the control group at 1-month and 3-month follow-ups. However, these differences were not statistically significantly different at both follow-up time points. The details of 2-part model results for medication and consultation cost are presented in As shown in Link intervention was associated with significantly higher utility-based quality of life than the control condition . Furthermore, the Link intervention was also associated with lower costs  compared with the control group; however, this difference did not reach statistical significance  compared with usual search strategies .The results of the incremental analysis suggest that the ificance . An inteLink intervention had a 95% probability of being cost-effective as long as the threshold of willingness-to-pay is over Aus $10,000 per QALY gains  concur with those for the complete case dataset, which show a similar pattern of greater effectiveness and less cost associated with the intervention group compared with the control group . The senLink) compared with usual search strategies. Young people randomized to the Link intervention had significantly higher utility values and QALYs gained at 3 months compared with young people using their usual online search strategies. The online help-seeking intervention was also associated with lower average total costs from a health sector perspective although this did not reach statistical significance. The online help-seeking intervention was found to be a cost-effective treatment option compared with young adults’ current search strategies with a 73% probability that Link would be cost-saving and a 100% probability that it would be cost-effective using a willingness-to-pay threshold of Aus $28,033 per QALY gained. In fact, results suggest that even at a more modest Aus $10,000 per QALY value-for-money threshold, the intervention is still likely to be very cost-effective. The results were robust in the sensitivity analysis when complete case analysis was conducted, or the intervention costs were varied.This study was the first cost-utility analysis of an online intervention to increase mental health help-seeking for young adults . The reason might be that the intervention was associated with a reduction in the quantity of longer health professional  consultations (duration over 40 min) than in the control group. Another important point is that the Link intervention was associated with lower hospitalization costs than the control at the 3-month follow-up, although the number of people who were hospitalized was not different. This might suggest a positive benefit of the Link intervention in reducing severity of mental health problems that require intensive treatments. However, these results did not reach significance, given that the sample sizes of these subgroups were relatively small. As noted above, these results may be explained by the quality of services being accessed via the Link platform. Further research with larger sample sizes and perhaps more evaluation of the type of care being accessed  is needed.Findings from our study showed that although there were no significant differences in terms of health care service use between the 2 groups, the Link platform) demonstrated a high probability of being cost-effective. The initial results from this study are certainly very promising and suggest that if access to the intervention was increased, this could result in significant health impacts and likely cost savings.This study, for the first time, raises the possibility that improving help-seeking not only assists young adults in accessing mental health care services but is also associated with quality of life improvements. More importantly, a Web-based mental health service navigation website  may be associated with even more cost savings. The study was also limited by the relatively short time horizon  and the use of self-reported retrospective utilization of health care services and medication, potentially leading to recall bias. It is not clear whether this may have led to an over- or underestimation of resource use reporting, although any biases are likely to be the same in both groups. Further research using a broader societal perspective and longer follow-up is needed.In terms of limitations, these results do not include any costs beyond the health sector, which may underestimate the cost-effectiveness of the Link, appears to provide a cost-effective and, possibly, cost-saving tool for young adults compared with the usual methods for seeking care. The intervention demonstrated a reduction in health care professional consultation costs at the 1-month follow-up and hospital costs at the 3-month follow-up. These results were robust in the sensitivity analysis. Further research to confirm these results could have important implications for increasing the accessibility of mental health care services for young adults.In conclusion, the online help-seeking navigation website,


Many congenital heart defects and degenerative valve diseases require replacement of heart valves in children and young adults. Transcatheter xenografts degenerate over time. Tissue engineering might help to overcome this limitation by providing valves with ability for self-repair. A transcatheter decellularized tissue-engineered heart valve (dTEHV) was developed using a polyglycolic acid (PGA) scaffold. A first prototype showed progressive regurgitation after 6 months in-vivo due to a suboptimal design and misguided remodeling process. A new geometry was developed accordingly with computational fluid dynamics (CFD) simulations and implemented by adding a polyether-ether-ketone (PEEK) insert to the bioreactor during cultivation. This lead to more belly-shaped leaflets with higher coaptation areas for this second generation dTEHV. Valve functionality assessed via angiography, intracardiac echocardiography, and MRI proved to be much better when compared the first generation dTEHV, with preserved functionality up to 52 weeks after implantation. Macroscopic findings showed no thrombi or signs of acute inflammation. For the second generation dTEHV, belly-shaped leaflets with soft and agile tissue-formation were seen after explantation. No excessive leaflet shortening occurred in the second generation dTEHV. Histological analysis showed complete engraftment of the dTEHV, with endothelialization of the leaflets and the graft wall. Leaflets consisted of collagenous tissue and some elastic fibers. Adaptive leaflet remodeling was visible in all implanted second generation dTEHV, and most importantly no fusion between leaflet and wall was found. Very few remnants of the PGA scaffold were detected even 52 weeks after implantation, with no influence on functionality. By adding a polyether-ether-ketone (PEEK) insert to the bioreactor construct, a new geometry of PGA-scaffold based dTEHV could be implemented. This resulted in very good valve function of the implanted dTEHV over a period of 52 weeks. Approximately 9 out of 1000 children are born with a congenital heart defect . The PulCurrently, there are two generally different types of valve prostheses being used for heart valve replacement: Mechanical and biological valves. However, these prostheses come with certain limitations. Most common are bleeding complications for mechanical heart valves and degeneration for biological valves, leading to the necessity of new prostheses for pulmonary valve replacement ,6. DegenTissue engineering might help to overcome this limitation by providing valves with the ability for self- repair. The concept of scaffold based tissue-engineering on biodegradable synthetic polymers has become widely accepted for heart valve tissue-engineering . PGA  scaffolds promote better tissue formation and higher levels of DNA, GAG, and HYP when compared to PGA-PLA (poly-lactic acid) or PGA-PCL (poly-caprolactone) scaffolds, whereas PGA-PCL scaffolds are more robust under biomechanical conditions when compared to PGA-P4HB and PGA-PLA scaffolds . Fibrin-In a European consortium, a transcatheter decellularized tissue-engineered heart valve (dTEHV) was developed on a PGA-P4HB-scaffold and implanted transvenously using a self-expanding nitinol-stent. A first generation of these valves showed progressive regurgitation after six months in vivo due to a suboptimal geometrical design. An optimized geometry was then developed using computational fluid dynamics (CFD) simulations, leading to more physiological diastolic load on the leaflets and hence better guidance of the remodeling process of the dTEHV ,15,16,17Here we present the role of a P4HB-coated PGA-scaffold combined with a polyether-ether-ketone insert for an improved functionality of decellularized tissue-engineered heart valves.After harvesting the dTEHV from the bioreactor, differences in geometry were clearly visible between the first (dTEHV1) and second generation (dTEHV2) of dTE-valves: Leaflets were much more belly-shaped with higher commissures and larger coaptation area in the second generation when compared to the first generation of dTEHV. Tissue formation was thinner and leaflets were subjectively more mobile in the second generation .With the transvenous implantation of a first generation of dTE-valves, Schmitt et al. proved the general feasibility of this minimally invasive approach to developing and implanting a tissue-engineered heart valve ,16. TheiBasically it can be said that through the newly developed geometry of the dTEHV, valve functionality over the course of 52 weeks was made possible. A direct comparison between the first and second generation dTEHV can be made, especially concerning results of intracardiac echocardiography (ICE), angiography, and macroscopic, as well as histological findings after explantation.Valve functionality directly after implantation as well as after 24 weeks shows substantial improvement from first to second generation dTEHV. Even after 52 weeks, 6 out of 9 second generation dTEHV still remaining in the experiment showed only mild insufficiency (median insufficiency grade: Severe), whereas for the first generation dTEHV 4 out of 5 valves showed severe regurgitation at 24 weeks already (median insufficiency grade: Severe), mainly due to coaptation deficit. .Statistical analysis showed significant improvements in pulmonary regurgitation grades and coaptation length directly after implantation, as well as coaptation length 24 weeks after implantation . Since iDirectly after implantation, 6 out of 15 (40%) dTEHV 1 showed sufficient valve closure during angiography of the pulmonary artery. Eight out 15 (53.3%) showed slight regurgitation of the applied contrast medium into the RVOT. In one case (7.8%), strong regurgitation with contrast medium staining the whole RV was seen.All dTEHV 2 showed sufficient closing function in angiography directly after implantation. No contrast medium regurgitated into the RVOT or right ventricle. No paravalvular leakage was recorded either .After explantation, fusion between the leaflets and the graft wall, as well as leaflet shortening, were macroscopically visible in the first generation of dTEHV. This was more evident after 24 weeks when compared to the explanted specimen after 8 and 16 weeks, leading to the conclusion of a continually progressive process during the recorded follow-up time .Leaflet shortening and fusion between leaflets and graft wall were not present in the second generation dTEHV. Leaflets were still belly-shaped and mobile after explantation. Anatomical features of native pulmonary valves, such as a lunula at the upper rim, as well as a small nodulus in the center of each lunula, were seen. Mean distance from upper rim of the leaflet to the hinge region was 21.9 ± 1.46 mm after explantation. Macroscopically, there were no signs of active endocarditis, calcification, or thrombi visible on the explanted valves. Complete engraftment of the stented valve in the surrounding structures was observed. A thin and shiny layer of endocardium covered the stent struts at the proximal and distal part of the valve. .In the histological analysis, the first generation dTEHV showed partial re-endothelialisation after 8 weeks and complete after 16 weeks. Progressive leaflet shortening, that was visible in the macroscopic analysis, was also confirmed in histology of dTEHV 1 as leaflet length continually decreased from implant to explantation after 8, 16, and 24 weeks . In the Complete re-endothelialisation was confirmed in dTEHV 2 as all explanted valves presented themselves with an endothelial layer covering leaflets and the graft wall. Leaflets consisted mostly of collagenous fiber with some elastic fibers. Some remnants of the scaffold were seen in the apical portions of the leaflets, however they did not influence functionality. Foreign body reaction against scaffold remnants in the graft wall was present in the form of foreign-body giant-cells.Chronic foreign body reaction against the stent struts was visible in the form of histiocytic infiltrations surrounding the stent struts. No signs of acute inflammation were found anywhere within the leaflets or graft wall.In most explanted dTEHV 2 specimens, scaffold remnants were found as shown in For second generation dTE-valves, a multi-layered, almost native-like structure of the leaflets was noticeable in histological analyses , implyinWhereas αSMA-positive cells were found to be mainly responsible for an active leaflet retraction in dTEHV1 , differeAs described, functionality improved substantially from first to second generation dTEHV. As there were no MRI scans performed to assess functionality of the first generation, no accurate comparison can be made concerning MRI-data. However, functionality in MRI proved to be generally good over the course of the set follow-up of 52 weeks. As described above, 9 out of the 10 valves reached the set endpoint of 52 weeks, only one sheep had to be euthanized due to regurgitation exceeding 30% in MRI at 24 weeks according to the study protocol.Median regurgitation fraction determined by MRI slightly increased from 9% one week after implantation to 14.7% after 52 weeks, however, all remaining valves stayed under the threshold of 30% at the time of explantation, leading to the conclusion of no hemodynamic relevant insufficiencies 52 weeks after implantation .Over the last years, scaffold-based tissue-engineering has proven to provide a suitable alternative to biological heart valve prostheses made from porcine or bovine pericardium in the future. In contrast to these conventional heart valve prostheses, the biological material that is implanted in tissue-engineered heart valves has not been cross-linked with glutaraldehyde and provides an environment for reendothelialization of autologous cells, furthermore inducing a remodeling process. Decellularization plays an important role in the manufacturing process in order to eradicate foreign antibody structures on the grafts surface, as the dTEHV used in our study were grown from homologous and not autologous stem cells. However, using autologous stem cells would not provide a practical alternative to our homologous approach, as infrastructural challenges would increase substantially.During the in-vivo study of the first generation dTEHV, follow-up of 24 weeks was achieved. However, functionality deteriorated drastically shortly after implantation. Mechanisms for this functional failing of these valves have been identified: Particularly active leaflet shortening conducted through contractile αSMA positive cells and fusion between the leaflets and graft wall due to reduced wash out in the hinge area of the valve were responsible for progressive central regurgitations .In general, it can be said, that by developing a new geometry of these dTEHV, these failing mechanisms could be eradicated in the second generation dTHEV. There were no cell clusters or thrombi found in the hinge areas of the valves. αSMA positive cells were found in our histological analysis. Their functional relevance is at least questionable, though, since Emmert et al. found no αSMA-positive cells on the leaflets surface . HoweverRemodeling seems to be much better guided and controlled in the new generation of dTEHV when compared to the results of Schmitt et al. and Driessen-Mol et al. from the first generation dTEHV ,17. CompAs Emmert et al.  have poiαSMA-positive cells at the leaflets surface as key contractile agents, dTEHV 2 offered a significant step forward as well. Leaflet shortening in previous studies was mainly conducted by contraction of αSMA-positive cells [αSMA-positive cells were nonexistent in the second generation of dTEHV, similar to native pulmonary valves [αSMA-positive, however having no effect on valve functionality.Concerning the presence of ve cells . Emmert y valves . Our hisHence, the newly developed geometry of dTEHV 2 leading to more physiological hemodynamics surrounding the valve itself, poses a big step towards a more native-like pulmonary valve . The useKluin et al. followed a rather different approach in developing a heart valve capable of remodeling with in-situ tissue-engineering on a thermoplastic elastomer PC-BU scaffold that is implanted and then in-vivo recellularized by autologous cells to form a living heart valve, while the scaffold slowly degrades. Their study also shows promising results with good valve functionality after 12 months. However, a polyether-ether-ketone ring supporting the PC-BU scaffold is vital for mechanical support . A minimResults of the present study pose the question of finding the optimal time point for adding the polyether-ether-ketone insert to the bioreactor during incubation of a dTEHV 2. Even though functionality improved quite significantly from dTEHV 1 to dTEHV 2, there were still differences in functional performance of the second generation dTE-valves. Since the PEEK-insert was added to the bioreactor at three different time points (Day 0/10/13 of Incubation), tissue-formation was slightly variable between the production batches. Looking at the functional end-points (regurgitaton fraction (RF) via MRI) of each production batch, it is apparent that the time of adding the insert might influence the functional outcome rather substantially. Median RF at 52 weeks was 14.2%. All four dTEHV 2 of the 13 days batch were above the median RF at that point , whereas the two valves of the 10 days batch presented a rather good functionality, with both valves at a regurgitation fraction of 8.6%. In the group of the start-batch outcome varied the most, with one animal dropping out due to high regurgitation 24 weeks after implantation and three dTEHV 2 with an RF equally (14.7%) or lower than the median RF . In summary, out of nine dTEHV 2 that reached the set follow-up time of 52 weeks, four valves showed RF less than median, one was about median RF, and four valves showed RF higher than the median. The difference of the distance from upper rim of the leaflet to the hinge region before implantation and after explantation provides a suitable macroscopic correlate to the variable functionality. In the group of valves with RF >median, this distance decreased by 3.3 ± 0.94 mm versus 1.1 ± 0.48 mm in the group of valves with RF <median. Due to rather small group size in both cohorts, no statistical significant differences can be identified. However, this result may suggest the importance of finding the right time point of adding the PEEK-insert to the bioreactor. In theory tissue-formation inside the bioreactor is slightly inhibited by the insert, leading to thicker tissue in the leaflets for the 13 days batch when compared to the start-batch. Since radial strain during diastolic loading depends on tissue strength, diastolic pressure, and anisotropy of the tissue fibers , in-vivoValve manufacturing and study protocol of an in-vivo study to test the first generation of decellularized tissue-engineered heart valves have been published before . The folTen dTEHV with an improved geometry were grown in a bioreactor on a biodegradable polyglygolic acid scaffold. Detailed manufacturing process has been described previously . In shor® by Arkema, Colombe, France). At the distal part of these tubes lies the capsule—basically a broadening of the outer sheath—in which the stented valve can be loaded and released. The deployment is performed by pulling back the outer sheath over the inner sheath which functions as a counter bearing for the valve. The process of the deployment can be controlled by the handle which lies on the proximal part of the tube.The dTEHV were implanted minimally invasive into the pulmonary position into sheep, using a custom made delivery system that was introduced through the external jugular vein . This deCT and MRI scans were performed prior to implantation in order to assess proportions and flow dynamics of the native pulmonary valves. The implantations were framed by intracardiac echocardiography, angiography, as well as pressure measurements of the RA, RV, and PA. After implantation, flow measurements in MRI and intracardiac echocardiography were performed monthly in order to gain detailed data of the valves functionality. After 52 weeks, the valves were explanted and examined macroscopically for thrombi, signs of endocarditis and integrity of the leaflets. Histology was performed subsequently. The study protocol determined that valves exceeding a threshold of 30% regurgitation fraction in MRI (marking the border between moderate and severe regurgitation) should be explanted prematurely.Valve insufficiency in ICE was graded according to the recommendations for the echocardiographic assessment of native valvular regurgitation by the European Society of Cardiology .The study was approved by the local authorities . Animals were treated in accordance with the guidelines of the European and German Societies of Laboratory Animal Science.A polyether-ether-ketone insert helps improve the geometry of a transcatheter decellularized tissue-engineered pulmonary heart valve, leading to good functionality over the course of 52 weeks. Failing mechanisms of a previously tested, first generation of dTEHV could be eradicated accordingly. Histological findings show remnants of polyglycolic acid scaffold even after 52 weeks, however without any influence on the functionality of the affected valves. Hence, PGA proves to be a suitable scaffold for heart valve tissue-engineering.


Airborne dust can cause engine wear and contribute to engine gas emission. This study developed a novel submicro-fiber filter medium to provide protection to engines against dust. The wet-laid submicro-fiber medium was prepared by a dual-layer paper machine, and its dust loading performance was compared with other filter media during laboratory and field tests. During the laboratory tests, the dust holding capacity of the wet-laid submicro-fiber medium was 48% and 10% higher than that of the standard heavy-duty medium and electrospun submicro-fiber medium, respectively. During the field tests, the pressure drop of the wet-laid submicro-fiber filter was 45% lower than that of the standard heavy-duty filter after 10,000 km of operation. It was found that there were two crucial ways to design a better filter medium for protection against dust. Firstly, the surface loading rather than the depth loading was preferred for dust filtration. The submicro-fiber layer kept large amounts of dust particles from penetrating into the depth of filter medium. Secondly, particles were captured preferably by fibers rather than pores. The unique fibrous structure of the wet-laid submicro-fiber medium made more particle deposition take place on fibers via interception and inertial impaction. Arid and semi-arid areas, which cover approximately one-third of land surfaces, are a major source of mineral dust . If meteAir filtration is applied to provide protection against the effects of contaminated air. The effects of the engine intake air filter can be both positive and negative . The posIn the combustion engine industry, fibrous filter media are used to prevent airborne particles from entering the engine. Cellulose-type filter media, made of relatively large fibers with fiber diameters, usually larger than 10 microns, are often used in engine air filtration. To improve filtration performance, the application of ultrafine fibers has become of interest in academic research and within the industry ,12,13. MThe advantage of using a submicro-fiber provides a valid case for relatively clean filters. When dust deposits on submicro-fiber, the benefit of a low pressure drop diminishes with increasing amounts of deposited dust. For military vehicles in high dust concentration environments, a self-cleaning method can be used to blow off dust cake using a pulsejet and make the filter medium clean again . HoweverTo develop a new submicro-fiber medium, this study will combine the ideas of submicro-fiber and multilayer papermaking to laminate a submicro-fiber layer on the substrate via the wet-laid process. During the wet-laid process, resin impregnation for a submicro-fiber layer can be easily applied, which will result in a robust submicro-fiber layer and strong adhesion with a substrate. Langner and Greiner  discusseThe preparation of a composite filter medium was conducted on a pilot-scale customized paper machine in Fibrway Co., Ltd. . The experimental results on the paper machine were more applicable for real applications compared to paper preparation in the laboratory . During The main preparation processes included pulp preparation, sheet forming, drying, and coating. There were two sets of the pulp preparation system: Microfiber and submicro-fiber slurry. For the microfiber slurry, the fibers consisted of 20% softwood pulp , 60% flash-dried hardwood pulp , 15% 1.4 D × 5 mm polyethylene terephthalate  fiber, and 5% 3-µm glass fiber was dispersed in clean water and then stored in a tank with continuous agitation to keep fibers away from entanglement. The microfiber slurry with a pulp consistency of 0.1 wt.% was pumped to channel 1 of the headbox, as shown in This dual-channel headbox had been patented by the authors . In the The wet paper sheet was then transferred to the drying and coating section, as shown in 2. The full width of the final paper sheet was 0.7 m after cutting 0.05 m on both edges. Filter paper samples for this study were taken 1 h after stable operation of the paper machine was achieved.The running speed of the pilot-scale paper machine was 5 m/min. The basic weight of the filter paper was 120 ± 3 g/mTM, Phenom-World B.V. Inc., Eindhoven, The Netherland). The pore size was measured by capillary flow porometer .The loading performance of the wet-laid submicro-fiber composite medium was compared to the two other filter media. The standard heavy-duty filter medium  is currently used in many inlet filters of engineering machinery. The electrospun composite medium  is known for its outstanding performance in many studies ,20. The The loading test rig  was used to test the dust holding capacity according to the ISO 5011  standard3. The filter medium clamped in the filter holder with a test area of 100 cm2 was challenged by the dust particles under a constant flow rate controlled by a mass flow controller. The testing face velocity was 11.1 cm/s, which was close to the operating velocity in real applications [The particles used in the test were ISO 12103-1  A2 fine ications . The tesM0) and fully loaded (M1). The final dust mass deposit, finalM, was calculated by Equation (1).S is the test area of filter medium.The filter medium was weighed when it was clean .totalt is the total loading time (min).Because the gravimetric efficiency for all filter media was close to 100%, as shown in The curves of pressure drop versus dust mass deposit per unit area were used to analyze the loading performance of the different media. Two parameters obtained from the pressure drop curves were considered for discussion: Dust holding capacity and energy consumption. Dust holding capacity was defined as the dust mass deposit per unit area when the pressure drop reached 2 kPa. The energy consumption of different filter media under a fixed amount of dust mass deposit can be compared by calculation methods described in Eurovent 4/21 .The loading performance of the wet-laid submicro-fiber composite medium and two commercial filter media was evaluated in the laboratory. Because the wet-laid submicro-fiber composite medium was a dual-layer structure, loading tests using the microfiber or submicro-fiber layer at the inlet were conducted to analyze the difference in filtration performance, as shown in The loading performance of the wet-laid submicro-fiber composite medium and the standard heavy-duty filter medium were evaluated in the field test. Because one sample of the electrospun composite medium was not big enough to make a full-size filter, no field test was conducted for the electrospun medium. During the field test, two heavy-duty trucks  with close mileage were used. The original equipment manufacturer (OEM) air filter  for this truck was made by the standard heavy-duty filter medium. In comparison, a filter based on identical filter geometry was made using the wet-laid submicro-fiber medium, as shown in 3/h. After running 10,000 km, these two filters were taken out of the trucks and put into an ISO 5011 [3/h. The pressure drop results were used to evaluate the loading performance of the wet-laid submicro-fiber medium and the standard heavy-duty filter medium.The two trucks equipped with the OEM filter and submicro-fiber filter operated at the same coal mine  under the same tasks. The surrounding region of this coal mine was semi-arid, and many roads are unpaved and dusty, as shown in ISO 5011  test rigThe properties of the wet-laid submicro-fiber medium are in 2.5), and it must be noted that the loading behavior of coarse particles was very different. When a dual-layer filter medium was used for filtration, it was important to determine which media was the inlet. Several researchers have studied the loading difference of the microfiber and submicro-fiber layers. Leung and Hung , Leung e2. When the submicro-fiber layer was the inlet, the dust holding capacity was 95 ± 1 g/m2, which was 13% higher than that of the microfiber layer as the inlet. This meant that when the filter medium was challenged by dust particles, the lifetime was 13% longer if the submicro-fiber layer was placed in the upstream. This conclusion was contradictory to the conclusion of Leung et al. [The loading results demonstrated that when the submicro-fiber layer was placed as the inlet, the pressure drop increased at a lower rate. When the microfiber layer was the inlet, the dust holding capacity was 84 ± 2 g/mg et al. , which iThe pressure drop of the loaded filter medium can be considered as the sum of the pressure drop across the loaded filter medium and the pressure drop of the dust cake, as shown in cakeP is pressure drop across the dust cake; μ is gas viscosity; su is face velocity; H is cake height; ε is cake porosity; κ is dynamic shape factor of A2 dust; vgd is geometric mean of volume equivalent diameter; gσ is geometric standard deviation.According to Endo et al. ,44, the To illustrate the loading behavior of the particles, SEM images of dust deposition on the filter medium were obtained when the pressure drop increased 10, 50, and 100 Pa, respectively, as shown in 2, which meant that the dust holding capacity of the wet-laid submicro-fiber medium was 48% higher than that of the standard heavy-duty medium.The loading results of the wet-laid submicro-fiber medium and the standard heavy-duty medium are shown in p(iM) was the pressure drop when dust mass deposit was iM. The average pressure drop in a complete loading test can be calculated by Equation (4). The polynomial of fourth order Δp(m) = nMia4 + nMib3 + nMic2 + nMid + ΔIp and parameters na, nb, nc and nd were used to curve fit the pressure drop data as a function of loading time. ΔIp is the initial pressure drop of filter media.The energy consumption of air filter media was proportional to average pressure drop in loading process . Δp(Mi) 2, the estimated energy consumption of the wet-laid submicro-fiber medium was 36% lower than that of standard heavy-duty medium based on calculation of average pressure drop via Equation (4). The laboratory results showed that the wet-laid submicro-fiber medium had a longer lifetime and lower energy consumption than the standard heavy-duty medium when challenged by dust particles.Under a dust mass deposit of 64 ± 1 g/mThe SEM images of the clean standard heavy-duty medium and the loaded medium when the pressure drop increased 10, 50, and 100 Pa were obtained, respectively, as shown in 3/h, the pressure drop of the standard heavy-duty filter was higher than 2 kPa and the filter service life ended. The pressure drop of the used wet-laid submicro-fiber filter was approximately 45% lower than that of the standard heavy-duty filter, which demonstrated the advantage of the novel filter medium in the field test. During the laboratory tests, when the pressure drop of the standard heavy-duty medium reached 2 kPa, the pressure drop of the wet-laid submicro-fiber medium under the same dust mass deposit was 38% lower than that of the standard heavy-duty medium, which was close to the field test results. The field test results showed that the wet-laid submicro-fiber medium had a longer lifetime when challenged by dust particles.2, which meant that the dust holding capacity of the wet-laid submicro-fiber medium was 10% higher than that of the electrospun medium. Under a dust mass deposit of 86 g/m2, the estimated energy consumption of the wet-laid submicro-fiber medium was 10% lower than that of the electrospun medium based on calculation of average pressure drop via Equation (4). The laboratory results showed that the wet-laid submicro-fiber medium had a longer lifetime and lower energy consumption than the electrospun medium when challenged by dust particles.The loading results of the wet-laid submicro-fiber medium and an electrospun composite medium are shown in The SEM images of the clean electrospun composite medium and loaded filter medium when the pressure drop increased 10, 50, and 100 Pa were obtained, respectively, as shown in The governing mechanisms of capturing coarse particles were interception, inertial impaction, and sieving . ParticlThe current study is limited to room temperature and controlled RH (50 ± 10%). In the future, the effect of excess RH and temperature on dust loading performance of wet-laid submicro-fiber media should be studied.In this study, a novel wet-laid submicro-fiber composite medium was prepared by a dual-layer paper machine, and its loading performance and dust deposition behavior was compared with a standard heavy-duty medium and electrospun medium in laboratory and field tests. It was found that:(1) The submicro-fiber layer, laminated by dual-channel headbox technology and reinforced by acrylic resin, was highly porous without pore clogging. The loading results demonstrated that when the submicro-fiber layer was placed as the inlet, the pressure drop increased at a slower rate than that when the microfiber layer was the inlet. It was concluded that it was preferable for surface loading to happen earlier for dust particles.(2) During the laboratory test, the dust holding capacity of the wet-laid submicro-fiber medium was 48% higher than that of the standard heavy-duty medium, and the estimated energy consumption was 36% lower. In the field test, the pressure drop of the wet-laid submicro-fiber filter was approximately 45% lower than that of the standard heavy-duty filter after 10,000 km of operation. This showed that the wet-laid submicro-fiber medium had a longer lifetime and lower energy consumption than the standard heavy-duty medium when challenged by dust particles.(3) The dust holding capacity of the wet-laid submicro-fiber medium was 10% higher than that of the electrospun medium, and the estimated energy consumption was 10% lower. The key point of submicro-fiber medium design was that the particles were preferable to be captured by fibers rather than pores. The fibrous structure of the wet-laid submicro-fiber medium made more particle deposition take place on fibers via interception and inertial impaction. Thus, in this work, the submicro-fiber medium showed a lower pressure drop than the electrospun medium in the loading process.


Human genome harbors 55 receptor tyrosine kinases (RTK). At least half of the RTKs have been reported to be cleaved by gamma-secretase-mediated regulated intramembrane proteolysis. The two-step process involves releasing the RTK ectodomain to the extracellular space by proteolytic cleavage called shedding, followed by cleavage in the RTK transmembrane domain by the gamma-secretase complex resulting in release of a soluble RTK intracellular domain. This intracellular domain, including the tyrosine kinase domain, can in turn translocate to various cellular compartments, such as the nucleus or proteasome. The soluble intracellular domain may interact with transcriptional regulators and other proteins to induce specific effects on cell survival, proliferation, and differentiation, establishing an additional signaling mode for the cleavable RTKs. On the other hand, the same process can facilitate RTK turnover and proteasomal degradation. In this review we focus on the regulation of RTK shedding and gamma-secretase cleavage, as well as signaling promoted by the soluble RTK ICDs. In addition, therapeutic implications of increased knowledge on RTK cleavage on cancer drug development are discussed. Human genome harbors genes encoding 55 receptor tyrosine kinases (RTK) . RTKs caFull-length RTKs and RTK fragments have been observed in the intracellular cell compartments such as nucleus and mitochondria . Active ERBB4 was recognized as the first RTK cleavable by gamma-secretase almost 20 years ago . Since tMost of the work addressing the molecular mechanisms by which the gamma-secretase complex recognizes and interacts with its substrates has been carried out with Alzheimer’s disease-related amyloid precursor protein (APP) and to some extent with the notch receptors. Only relatively little research has been conducted on the intracellular cleavage of RTKs and, so far, practically no work has addressed aspects of substrate recognition, substrate binding, and actual cleavage catalysis in the context of RTKs. Thus, it is plausible that gamma-secretase-mediated cleavage is regulated by mechanisms resembling those exploited by both APP and notch, but this remains to be confirmed.The formation of a soluble ICD requires the full-length receptor to be cleaved by two sequential proteolytic cleavage events in a process called regulated intramembrane proteolysis , anterior pharynx defective-1 (APH-1), and nicastrin . Human pThe requirement for substantial conformational changes in the gamma-secretase complex during the substrate binding and catalysis of the cleavage have been reported . The subA conserved structural motif in the transmembrane domain of substrates is probably needed for the recognition by gamma-secretase complex. So far, no conserved amino acid consensus sequence has been identified serving either as the substrate recognition sequence or as the actual substrate cleavage site. The recognition of substrates is thought to be based on a combination of the length of the remaining ectodomain after shedding , the strWhile most of the substrate interactions with gamma-secretase complex have been indicated to take place within N- and C-terminal presenilin sequences, interactions of the substrates with PEN-2 and nicastrin subunits have also been observed . NicastrGamma-secretase has over 100 identified substrates , 17, 18 The main factors that regulate RTK RIP are (i) ectodomain shedding that is required before gamma-secretase cleavage and is thus critical for the initial selection of substrates, and (ii) subcellular localization of substrates and gamma-secretase complex in relation to each other.While gamma-secretase cleavage of cell surface proteins could be considered a relatively passive event taking place almost automatically after exposure of the substrate by prior ectodomain shedding, it is not promiscuous. In agreement, not all RTKs that have been reported to be shed undergo cleavage by gamma-secretase. RTKs such as ERBB2 and TIE2 have been found to undergo ectodomain shedding, but no indications for gamma-secretase cleavage has been reported so far , 23. ThiIn any case, as shedding is typically a prerequisite for subsequent processing, RTK cleavage by gamma-secretase is in principle subject to control by any circumstances that affect the expression or activation of the sheddase. For example, it has been shown that ERBB4 ectodomain is present in the serum of the breast cancer patients at higher concentrations than in the serum of healthy individuals, and that the enhanced shedding associates with more ADAM17 protein present in breast cancer tissue as compared to paired samples representing histologically normal adjacent breast tissue . UpregulWhile basal RTK cleavage has been reported, quite often activation by ligand is needed for RTKs to be cleaved by gamma-secretase. For example, ligand-induced cleavage has been observed for CSF1R, EPHB2, ERBB4, FGFR3, RYK, VEGFR1, and VEGFR2 , 29–34. In addition to ligand activation, activation of other cellular receptors and changes in ion concentrations across the cellular membranes have been associated with induction of gamma-secretase cleavage. Examples include cleavage of CSF1R, EPHB2, and ERBB4 RTKs , 29, 37.It has further been indicated that shedding of ectodomains requires phosphatidylserine exposure at the outer layer of the cell surface that interacts with ADAM17 membrane-proximal-domain leading to a conformational change in the ADAM17 ectodomain enabling its catalytic site to interact with cleavage sites of its transmembrane substrates , 39 Fig. OverallO-glycosylation of the substrates [O-glycosylation frequently takes place. Glycosylation has been found to predominantly reduce ADAM17-mediated shedding, with some reports indicating alterations in cleavage sites after glycosylation as well [O-glycosylation are controlled by only one or few GalNac-transferase isoforms indicating that shedding could be regulated to some extent by expression of specific GalNac-transferase isoforms [O-glycosylation [O-linked glycosylation of RTKs influences the intracellular cleavage and signaling by reducing ectodomain shedding.The regulation of gamma-secretase cleavage at the level of shedding may also be modified by bstrates  Sheddase as well . The sitisoforms . Interesisoforms  and is sisoforms  was showsylation . Hence, It has been reported that most of the mature ADAM17 is localized intracellularly as a result of constitutive internalization, with only a small fraction residing on the cell surface . FurtherChen et al. showed that ADAM10 and gamma-secretase complex reside in the same multiprotease complex . While tDifferent gamma-secretase complexes containing either presenilin-1 or presenilin-2 have been reported to direct their gamma-secretase complexes to distinct subcellular localizations , 14. PreCleaved ICDs of RTKs have been detected in various cellular subcompartments. Also in this respect, the most-studied RTK is ERBB4. Soluble ERBB4 ICD, as detected by overexpressing constructs encoding solely the ICD domain, or by analyzing endogenously produced ICD fragments using subcellular fractionation analyses and chemical inhibitors of nuclear export, has been detected in nucleus by several models both in vitro and in vivo , 61–63. In addition to ERBB4, soluble ICDs of endogenously expressed AXL, RYK, and TRKA have been reported to translocate to the cell nucleus while those of FGFR3 and PTK7 have only been observed in nucleus when overexpressed , 66–68. Soluble ERBB4 ICD has also been reported to translocate into the mitochondria , 70 whilAs discussed below, the soluble ICD of several RTKs may also rapidly translocate to proteasomes for degradation.After the release from the membrane, the RTK ICDs can act as signaling molecules with various functions Fig. . The ICDVarious processes in development and differentiation of different organs have been reported to be at least partially dependent on the functionality of nuclear ERBB4 ICD. The ICD has been shown to localize into the nuclei in mammary epithelial cells and found to induce differentiation of the mammary gland in a mouse model , 75, 76.ERBB4 is expressed in estrogen receptor-positive breast cancers and ERBB4 ICD has been found to increase estrogen response element-mediated transcriptional activity [The molecular mechanisms relaying signaling from the nuclearly localized ERBB4 ICDs have reported to include nuclear interactions with several transcriptional activators or repressors. For example, it has been proposed that STAT5 is escorted to nucleus by ERBB4 ICD in mammary epithelial cells where the complex associates with beta-casein gene promoter inducing its activity and mammary differentiation . During activity . The cleactivity , 83, 85 activity , and an activity .Other transcriptional factors that have been reported to directly associate with and be modulated by ERBB4 ICD include ETO2, Krab-associated protein 1 (Kap1), and AP-2 , 88, 89.ERBB4 and RYK ICDs have been associated with functions in neural development. In the developing mouse, the generation of ERBB4 ICD has been identified to be required for control of astrogenic differentiation . The ERBDLX1/2 intergenic regulation element and are involved in regulation of its transcription and neuronal differentiation of mouse primary cortical neural stem cells.The cleavage of RYK has been reported to be required for Wnt3-dependent neuronal differentiation and nuclear localization of RYK ICD requires Wnt activation in neural progenitor cell cultures . RYK ICDNuclear localization of PTK7 has been found to affect the proliferation of colon cancer cell lines . This obNuclear localization has also been observed for the ICD of TRKA by immunostaining human liver tissue samples with antibodies recognizing intracellular epitopes of TRKA, but not with antibodies recognizing the receptor ectodomain . TRKA ICStimulation with FGF1 has also been reported to induce gamma-secretase cleavage of FGFR3 in COS7 and T-Rex 293 cells. This was found to result in nuclear localization of the released ICD . In contIn addition to promoting differentiation and growth, the ERBB4 ICD has been demonstrated to induce apoptosis in various breast cancer cell lines. In SKBr3 cells ERBB4 ICD has been found to localize into mitochondria and has been proposed to directly function as a proapoptotic protein, as the ICD contains a BH3 domain similar to the BH3-only type of BCL2 family members . ConsistN-methyl-d-aspartate receptor (NMDAR) in cultured rat primary neurons [The cleavage of EPHB2 by gamma-secretase has been suggested to result in the formation of ICD residing in the cytoplasm where EPHB2 phosphorylates another receptor,  neurons . The pho neurons .Taken together, the soluble ICDs of RTKs have been observed to act as active signaling molecules with distinct functional properties. In the case of ERBB4 ICD, multiple functions have been reported in various cellular and organelle contexts. This growing body of evidence indicates that ICDs released from the cell membrane by gamma-secretase cleavage can have an active signaling role in various biological contexts.While ICDs released by gamma-secretase cleavage can add diversity to intracellular signaling by creating active signaling fragments, the cleavage also represents a mechanism of membrane receptor turn-over and degradation. The two steps of cleavage, generating the ectodomains and ICDs could participate together in downregulating RTK signaling through releasing the extracellular domain with the ligand-binding capacity to the extracellular space, and degradation of an active kinase domain, respectively. While there are a few examples indicating roles for RTK ectodomains in neutralizing the respective ligands  , 94, theAs the ICDs released from the cell surface include the functional kinase domain, and a truncated ICD fragment anchored to the cell membrane prior to gamma-secretase cleavage may even possess enhanced kinase activity , the kinCSF1R is quickly downregulated by gamma-secretase cleavage in macrophages during the activation of toll-like receptors by lipopolysaccharide, the major component of the cell wall of Gram-negative bacteria . The resIt has also been reported that proteolytic cleavage TIE1 in endothelial cells leading to ectodomain shedding and rapid ICD degradation may result in activation of TIE2 signaling . As TIE1A few articles have addressed the role of RIP in regulating signaling of the VEGFR subfamily of RTKs. Pigment epithelium-derived factor (PEDF) has been reported to induce shedding and gamma-secretase cleavage of VEGFR1 resulting in inhibition of VEGF-induced endothelial tube formation  and endoIn addition to VEGFR1, VEGFR2 has been reported to be cleaved by gamma-secretase in retinal pigment epithelium cells following induction by PEDF, a VEGFR1 agonist, leading to reduced VEGFR2 agonist-induced cellular permeability . In thisThe third member of the VEGFR subfamily of RTKs, VEGFR3, was also found to be cleaved in a systematic screen for gamma-secretase-sensitive RTKs . While iRTK signaling is frequently altered during carcinogenesis, and tumor cells may become addicted to specific activating variations in RTK sequence or copy number . TherefoMost of the human RTKs are targeted by one or several of the currently approved cancer drugs. These drugs belong to two major drug classes: Tyrosine kinase inhibitors that block RTK signaling by competing for ATP to block the intrinsic RTK kinase activity, or monoclonal antibodies that block ligand interaction or RTK dimerization, facilitate RTK endocytosis and degradation, or augment antibody-dependent immunological mechanisms , 105. SiWhile the relevance of RTK cleavage as a resistance mechanism has not been extensively studied, one can speculate on several possible mechanisms by which RTK processing could affect the sensitivity of cells to currently existing RTK-targeted therapies. Indeed, the fact that gamma-secretase-mediated RTK cleavage releases receptors from the membrane alters the well-characterized RTK signaling mode that has also been the primary target for drug development.The release of ectodomain by shedding does not only deplete the cells from structures available for interaction with therapeutic antibodies binding to the ectodomains but can also create a decoy receptor that binds the antibody within the extracellular space further neutralizing the effect on cells that would still express an intact receptor. Small molecule inhibitors against RTK kinase domains, on the other hand, could still be active therapeutic agents even after the release of a soluble ICD domain into intracellular compartments. However, in spite of attempts to develop ADAM inhibitors as cancer drugs , recent Most of the cleavable RTKs have no identified cellular function for their gamma-secretase released ICDs, and only a handful of RTK ICDs have any identified function. Future research will need to expand our view of ICD signaling beyond current understanding that is to a large extent based on reports focusing on one RTK, ERBB4. As most of our understanding about the biological relevance of gamma-secretase-mediated processing of RTKs is based on in vitro observations, there is also need for more thorough in vivo validation of the findings. This goal would greatly benefit from the characterization of cleavage sites and regulatory mechanisms critical for the process, thereby enabling specific molecular and genetic interference with the events.Development of new and more specific inhibitors targeting RTK cleavage needs better understanding of the biological significance, as well as about the mechanisms by which gamma-secretase substrates are distinguished from nonsubstrates. In addition, almost all the knowledge about molecular mechanisms and signaling events associated with regulation and substrate recognition by gamma-secretase is based on the research focusing on APP cleavage and, to some extent, on notch cleavage as well. Validation of these findings among RTKs would provide broader spectrum of tools for development of therapeutic agents specifically interfering with gamma-secretase-mediated cleavage of RTKs.