The BDIS estimate was higher in NMP [8.02 (7.40–8.65)] than in SCS  regardless of time. One patient in each group developed ischemic cholangiopathy, with a BDIS of 6 for the NMP-preserved liver. In six other NMP grafts, BDIS ranged 7–12 without development of ischemic cholangiopathy. In conclusion, BDIS increases over time, and the higher BDIS in NMP did not increase ischemic cholangiopathy. Thus, BDIS may overestimate this risk after liver NMP.Static Cold Storage (SCS) injures the bile duct, while the effect of Normothermic Machine Perfusion (NMP) is unknown. In a sub-study of the COPE trial on liver NMP, we investigated the impact of preservation type on histological bile duct injury score (BDIS). Transplants with at least one bile duct biopsy, either at end of preservation or 1 h post-reperfusion, were considered. BDIS was determined by assessing peribiliary glands injury, stromal and mural loss, haemorrhage, and thrombosis. A bivariate linear model compared BDIS  between groups. Sixty-five transplants and 85 biopsies were analysed. Twenty-three grafts were preserved with SCS and 42 with NMP, with comparable baseline characteristics except for a shorter cold ischemic time in NMP. The BDIS increased over time regardless of preservation type (  Biliary complications are a significant cause of morbidity after liver transplantation, particularly in the form of non-anastomotic strictures of the biliary tree, also referred to as ischemic cholangiopathy (IC) .The pathogenesis of IC involves ischemic-reperfusion injury, immune-mediated damage, cytotoxic insults, and defective biliary regeneration . Additioex situ perfusion of the graft with an oxygenated, nutrient-enriched, erythrocytes-based perfusate kept at 37°C  (p = 0.89). The most frequent indication for transplantation was alcoholic liver cirrhosis in both groups, and recipients were transplanted with a median lab MELD of 13 (10–18) points in the NMP group, and 11 (9–16) points in the SCS group (p = 0.44).The included subjects were well matched in terms of donor, recipient, and transplant characteristics. However, there were significant differences in the cause of death and the highest concentration of sodium in the donor, and in cold ischemic time  . In DCD p = 0.01; p < 0.001] and the requirement for vasopressors infusion lower [22/42 (53.66%)] than SCS-preserved livers , as a result of less frequent post-reperfusion syndrome . There was no difference in 1 year patient or graft survival  IU/dL] than in the SCS group  and at 1-h post-reperfusion  (p = 0.25). Representative images of bile ducts with low or high BDIS are provided in post hoc sensitivity analysis excluding the four transplants with BDIS improving after reperfusion confirmed these findings  with a circumferential bile duct biopsy taken 1-h post-reperfusion with a BDIS >6 (range 7–12) that did not develop IC.p = 0.87]. The BDIS estimate in NMP was higher than in SCS in both DBD and DCD transplants (p = 0.989).There was no difference in BDIS estimate between DBD [7.1 ] and DCD liver grafts [7.0 , nsplants . There wTo investigate which factor influences the severity of bile duct injury, we used bivariate linear models with BDIS as endpoint, and tested donor, recipient, transplant, and perfusion characteristics . Donor tIn this study, we provide evidence of the differential impact of different liver preservation modalities on the histological injury to the extrahepatic bile duct. Using 85 biopsies collected during the COPE trial comparing 121 NMP to 101 SCS livers, we showed that the BDIS increases significantly over time, regardless of preservation modality or other donor or transplant characteristics. Interestingly, although continuous liver NMP was associated with significantly higher BDIS, this did not lead to higher incidence of IC in the COPE trial. Next to the effect of time and preservation type, donor blood group emerged as a factor that may influence the severity of histological bile duct injury.Although the results of our study revealed significant differences in BDIS between NMP and SCS groups, the size of this difference  should be placed in context. In this study, BDIS increased significantly over time, independently of the preservation strategy used. As the duration of preservation was longer in the NMP group (>12 h) than in the SCS group 5.75 h, , the bilAdditionally, our study raises concerns about the validity of BDIS as a surrogate endpoint of IC in perfused livers, and about currently defined cholangiocellular viability criteria. Three reports on bile duct histology during liver transplantation highlighted the univariate association between arteriolonecrosis, epithelial loss, stromal necrosis, injury to the peribiliary glands and the risk of IC after transplantation –5. Howevn = 2) and the lack of correction for multiple testing, these results should be interpreted as exploratory.This study also explored the factors influencing the BDIS. Time, preservation type, and donor blood group were found to be independent determinants of the score . In thisThis study has some limitations. Not all centres involved in the COPE trial participated in this substudy, and not all transplants were included, which may limit the generalizability of the results to the entire trial population. Excluded cases had a significantly longer cold ischemic time, a known risk factor for biliary injury . HoweverIn conclusion, histological bile duct injury worsens over time regardless of the preservation method. Continuous NMP is associated with higher BDIS, but the magnitude of this effect remains uncertain due to limitations inherent to machine perfusion trials logistic. However, the more severe histological injury during continuous NMP does not necessarily lead to increased IC. Therefore, BDIS may overestimate this risk in NMP-perfused livers, making it less suitable as a surrogate endpoint for cholangiocellular viability criteria definition. Understanding the biological significance of bile duct injury and cholangiocellular biology during liver NMP is crucial for improving donor liver risk assessment. Further investigations exploring the biological responses of cholangiocytes from different donor types to NMP could shed light on these intricate mechanisms. To this end, the evaluation of bio-banked liver tissue samples from the COPE trial using single-cell -omics studies is being considered, which holds promise for unravelling the complexities of bile duct injury and optimizing liver graft preservation and selection.


Evaluate the feasibility and acceptability of an online guidedself-determination (GSD) program to improve diabetes self-management skillsamong young adults with type 1 diabetes (YAD).An online program comprising seven structured interactive conversations wasdesigned. A pre- and post- interventional study used a sequential, two-phasemultiple method design. Phase one comprised a training program for diabeteseducators (DEs). In Phase two YAD participated in program and completed pre-and post-surveys assessing motivation to self-manage, perceived competencein diabetes and communication with DEs. Both YAD and DEs provided a programevaluation.The online GSD program was acceptable, feasible and effective in improvingautonomous motivation in self-management and communication with DEs. Easyaccess and program flexibility were highly appreciated by both participantgroups and perceived to assist YAD to stay motivated.The program had a significant impact on the diabetes self-management of YADand was a feasible and acceptable way to engage and communicate with DEs.The GSD platform contributes to age appropriate and person-centred diabetesself-management. It can potentially reach geographically distancedpopulations, or with social circumstances or other barriers impedingin-person service provision. Inadequately managed DM resulting in poor glycaemiccontrol is associated with increased risk of developing complications includingvisual loss, amputation, neuropathy, end stage renal disease, cardiovascular disease(CVD), infections and cognitive impairment.3 Living with diabetes requireslifelong self-management to achieve optimal blood glucose levels and well-being andreduce the risk of developing complications. Providing education aboutself-management is critical in diabetes care. Three main pillars of diabetes careand management have been identified: patient empowerment, self-management educationand lifestyle modification. Specific support strategies for young people withdiabetes while recognising their changing life-stage need to be combined with otherbehavioural strategies to motivate them to effectively manage theirdiabetes.4Supporting optimal diabetes mellitus (DM) management in young people, especiallythose aged 18 to 25 years can be challenging for many reasons5 Asself-management of diabetes is ongoing, motivation is a process rather than relatedto a specific goal.6 Self-determination theory, provides a useful, process-basedframework, and addresses the basic human need for competence, while also consideringindividuals’ needs for ownership and internalisation of their behaviour.8Diabetes self-management depends on the individual's motivation and autonomy, asmotivation is an important conceptual variable in diabetes care.10: a motivation seen in people who “experience no meaningfulrelation between what they are doing and themselves” and controlled motivation isidentified in people who wish to obtain a reward or avoid negative consequences areboth connected with poorer health outcomes.10 In contrast, autonomousmotivation, is identified in people whose behaviour is positively endorsed andvalued by them is associated with positive health, behavioural and psychologicaloutcomes, such as to follow medication advice and weight-loss regimens.11Different types of motivation to self-manage diabetes are found to be related todifferent outcomes12 Guided self-determination(GSD) is a person-centred reflection and problem-solving method based onself-determination theory14 has been shown to improve life skills based onself-determination and problem-solving strategies in YAD.15 Life skills are defined as“those personal, social, cognitive and physical skills that enable people to controland direct their lives, and to develop the capacity to live with and produce changein their environment”.12 The GSD method provides educational and behavioural supportfor a person's own motivation and skill development in their diabetesself-management.13 People with diabetes are prompted by health professionalsto systematically explore and express their personal difficulties and experienceswith their condition through words and drawings on shared 4–8 worksheets (known as‘conversations’). Discovering and expressing personal challenges and prioritiesrelated to their conditions enables people to discover their potential forchange.14 The GSD method facilitates shared decision-making and improveshealth professionals’ advanced communication strategies.17Current support interventions to achieve optimal diabetes self-management thus tendto support peoples’ autonomous motivation.18 Technologyhas also been found to be successful in supplementing healthcare through theprovision of educational and motivational support.19 Accordingly, the researchteam converted the existing in-person GSD program for online access andfacilitation, so YAD had access regardless of their geographical location.To date, the GSD program delivery with YAD has mainly been facilitated in-person,consequently geographical, travel, cost and time are barriers to in-personparticipation, especially in Australia where YAD may live vast distances fromcities. Most young Australians have internet access and use it daily.21; the online format was a suitable, convenient and improved wayto communicate and engage with YAD managing type 1 diabetes (T1D). In previousstudies, YAD and participating diabetes educators found the online communicationexpedient, flexible and particularly suitable for the YAD who had lostmotivation.21 The online GSD program used in the current study has beenmodified based on feedback from this proof-of-concept study.20The online GSD program has been tested for proof of concept in Australia by membersof the research teamThe main aim of this study was to assess feasibility, acceptability and efficacy ofthe online GSD program to improve self-management of T1D among young adults. Thesecondary aim was to investigate experiences of online GSD from the perspectives ofboth YAD and diabetes educators (DEs).GSD = Guided Self DeterminationYAD = Young adults with DiabetesT1D = Type 1 DiabetesDE = Diabetes EducatorA pre- and post- interventional study using a sequential, two-phase multiplemethod design was applied .25 The firstphase was the delivery of the training program in online GSD for DEs. The secondphase was YAD participation in, and completion of, the GSD online program with aGSD-trained diabetes educator.Multiple-method research focuses on the use of multiple types of data from asingular paradigm to answer the research questions in one study.The DEs were eligible to participate if they had at least 5 years’ experience indiabetes care. Recruitment was through advertisements in social media andnewsletters of the Australian Diabetes Educators Association (ADEA). A plainlanguage statement and consent form was sent by e-mail to each DE who expressedan interest by contacting the research team. Written consent to participate wasprovided by each DE.YAD were eligible to participate if they were aged 18–30 years, diagnosedwithT1D, had access to mobile phone, iPad or computer and ability to provideself-measured blood glucose results.They were recruited from (a) the Young Adult Diabetes Clinic at a metropolitanhealth service in Melbourne, Australia; (b) advertisements in the Victorianconsumer diabetes organisation's social media, newsletters and website; (c)social media advertisements and posters at the university where research teammembers are employed. At the Young Adult Diabetes Clinic, YAD waiting forendocrinologist appointments was approached by a research assistant, whoprovided a flyer about the study and a plain language statement and consentform. YAD was invited to ask questions about the study, and if they wereinterested in participating, they signed their consent and kept their copy ofthe plain language statement and consent form. YAD who saw advertisementsemailed the research team, then received an emailed plain language statement andconsent form that they signed and returned to participate.Training for DEs included online learning materials, and a one-and-a-half day,in-person, introductory workshop. The workshop aimed to provide DEs with anunderstanding of the theories  that underpin the GSD program; the specific behaviouralcharacteristics of older adolescents, in the 18–25-year-old age group; anopportunity to practice the three GSD-informed communication skills of activelistening, mirroring and value clarification; and an introduction to and use ofthe online GSD platform. Completing training and receiving accreditation as aGSD diabetes educator required DEs to complete the online GSD program with twoYADs, while being mentored by members of the research team and experiencedGSD-trained DEs.Indicate significant events/turning points in their lives withdiabetes on a timelineDraw/upload a photo or image to illustrate YAD's feeling of livingwith diabetesFill out unfinished sentences to reflect on how YAD perceivethemselves and why they manage diabetes in the way theydoUse interactive circles to indicate how much diabetes take up time intheir lives and how much they wish diabetes to take time and thedifferences were then discussed with DEsFill out a model that underpins their values and helps to identifythe most pertinent current challenge in their life withdiabetesThe final two conversations focus on decision strategies to solve theidentified challenge and any future steps in managing their life with diabetes.The online GSD consultations between DEs and YAD were conducted via Zoom or bytelephone, according to the preference of the YAD. For each young adult,participation in the GSD online program included seven interactive conversationsover 6 months.The GSD program for YAD consists of synchronous conversations, facilitatedthrough the purpose-built GSD platform, between YAD and DEs at mutuallyconvenient times. The conversations include worksheets which are completed byYAD in discussion with the DE, with some allocated activities completedseparately by each YAD between conversations. The activities encouragereflection by the YAD. For example, the YAD is prompted to: To meet the main study aim, online surveys with validated psychometricinstruments were used before and three months after participation. To meet thesecondary study aim, fixed-response and open-ended questions were included inthe second, post-participation survey for YAD. An online survey includingfixed-response and open-ended questions was used to assess acceptability fromthe perspectives of DEs. A focus group with DEs was facilitated to gatherin-depth experiences of the program (Table 1).26All online surveys were hosted on Qualtrics, an online survey platform.Pre- and post-participation surveys (YAD). YAD was invited toparticipate in surveys before they commenced the GSD program and three monthsafter their last conversation with the DE. By express consent, surveys were notanonymous, as matching of pre- and post-participation surveys was required.The pre-participation surveys assessed YAD demographic and health characteristics.The following validated psychometric instruments were included in the pre- andpost-participation surveys for YAD:Perceived Competence for Diabetes Scale (PCS) (four items):Assesses perceived competence in managing one's diabetes. Competence is assumedto be one of three fundamental psychological needs, so feelings or perceptionsof competence with respect to diabetes are important because they facilitatepeople's goal attainment.27 Responses for each item,assessed on a Likert Scale ranging from 1  to 7 (very true);total scores are presented as the mean of ratings on the four items. Higherscores indicate optimal competence in managing diabetes.Treatment Self-regulation Scale (TSRQ) (19 items): Assesses aperson's motivation for engaging in health behaviours, namely takingmedications, self-checking blood glucose, following an appropriate eatingprogram and exercising regularly.9 Specifically, the scalemeasures the degree to which a person's motivation is autonomous  or the respondent is motivated by expectations ofother significant people in their life . Autonomous motivation is a key principle of self-determination theory,which underpins the GSD approach.12 Responses for each item,assessed on a Likert Scale ranging from 1  to 7 (very true);total scores are calculated as the mean of ratings for each subscale.14 Higherscores indicate optimal motivation for the Autonomous motivation subscale, whilelower scores indicate optimal motivation for the Controlled subscale.Health Care Climate Questionnaire (HCCQ) (15 items): Assessesclients’ perceptions of the degree to which their health care provider, in thiscase, the participating DEs facilitating the online GSD program, is supportiveof their autonomous decision-making.28 Sample items include ‘Ifeel that my DE has provided me choices and options’ and ‘My DE tries tounderstand how I see things before suggesting a new way to do things’. Each itemwas rated on a 7-point Likert scale ranging from 1 (strongly disagree) to 7(strongly agree). Total scores are calculated as the mean of ratings on the 15items. Higher scores indicate optimal healthcare environment.HbA1c: Respondents were asked to provide their most recent bloodHbA1c levelsTo assess acceptability of the program from YAD’ perspectives, thepost-participation survey included 7 Likert-scale questions assessing theirperceptions of the GSD website and Zoom/ telephone communication with DEs, andfour open-ended questions inviting free-text commentsPost-participation surveys (DEs). To assess acceptability of theprogram from DEs’ perspectives, anonymous evaluation surveys were distributed toDEs on completion of the GSD program with at least two YAD. These surveysassessed demographic and work characteristics . To assess acceptability of the program fromDEs’ perspectives, four Likert scale questions assessed DEs’ perspectives on theGSD approach, five Likert-scale questions assess their perspectives of the GSDwebsite and an open-ended question invited any other comments about the GSDprogramFocus group (DEs). Following completion of the GSD program withat least two YAD, DEs were also invited to a focus group hosted on Zoom. Thefocus group was facilitated by BR and KW.p < 0.05. Medians are presented, together withmeans (SD) which are reported for ease of interpretation only.Quantitative data were analysed using IBM SPSS Statistics v25. Scores for thePCS, TSRQ, and HCCQ were calculated according to the instructions of eachinstrument's original authors. Scores on these scales and client-reported bloodglucose levels (pre- and post-GSD program completion) were not normallydistributed; therefore, the non-parametric Wilcoxon Signed Ranks test was usedto test for differences within individuals across time. Significance wasassessed at 29 was conducted with abroad-brush line-by-line coding, and cross checked by two researchers .Responses to open-ended survey questions were managed using an Excel spreadsheetand a manual text analysis30 analysis method: (1) familiarisation with the data, (2)searching for patterns or themes, and (3) reviewing and comparing facilitators’notes and four reporting themes.Focus group data were analysed from notes obtained by the facilitators and amanual thematic analysis was applied, using a modified version of Braun andClarkeThe study was approved by the ethics committee of Western Health (Ethics IDHREC/18/WH/1130) and conducted according to the Declaration of Helsinki. Plainlanguage statements included assurance that if YAD and DEs withdrew from thestudy it would not affect their care, and that confidentiality of information,privacy and dignity would be observed throughout the study.Nine DEs were recruited and trained. Three mentors, one from the research team,experienced in using the GSD program, also attended the training to providesupervision and support. Eight DEs completed the GSD program with at least one youngadult. Of these, four completed it with two YAD and one with three YAD. Six diabeteseducators (with 7–14 years of experience working as a diabetes educator) completedthe post-participation survey. Five DEs responded to email invitation and attendedthe focus group.Forty YAD consented to participate. Of these, 18 (19–30 years) commenced the GSDprogram with a trained DE; the remaining 22 YAD could not be contacted after theyprovided consent, or in discussion with researchers indicated that they did not wishto proceed with the program owing to employment, study or family commitments. Of the18 who commenced the program, 13 (72%) completed the program.Pre- and post-participation survey data were available for eight YAD; the remainingfive participants did not complete the post-participation survey. Of those for whomcomplete data were available, seven were female and one was male. Four respondentsindicated that they lived with their parents, three with their partner and one withother people. Time since diagnosis with T1D ranged from 2.5 to 17 years; five usedan insulin pump. No participants reported any diabetes-related complications.p = 0.028) and the HCCQ scale(p = 0.046). There was no significant difference in PCSscores, TSRQ controlled motivation subscale scores or self-reported HbA1c,following completion of the online GSD program , showed agreementthat the GSD website was easy to use, the website layout was user friendly, andthat the reflection boxes in the GSD conversations were useful. Seven of eightrespondents found navigation between conversations on the GSD website easy; theremaining participant responded, ‘neither agree nor disagree’. All respondentsagreed that the teleconferencing program  utilised for conversationswith DEs was easy to use and adequate for communicating with their DE. Oneparticipant indicated they would have preferred in-person (rather than online)conversations with their DE; three disagreed, one selected ‘neither agree nordisagree’ and one did not respond to this question.Supplement 4.Examples of young adult participants’ responses to open-ended comments areprovided in Diabetes Educators. Responses to Likert-scale items assessingDEs’ perceptions and experiences of the GSD program (post-participation survey)indicated unanimous agreement that the GSD method is an effective approach forworking with YAD to improve their self-management of diabetes. Respondents wouldcontinue to use aspects of the GSD method with young adults as well as otherclients with T1D, and they would recommend the GSD approach to other diabeteseducators. Regarding the online GSD platform, 4 of 6 respondents indicated thatthey found the platform easy to use, and their YADs expressed the same view.Five respondents indicated that if they had open ongoing access to the platform,they would use it with YAD, and four said they would use it with other adultclients with T1D. Five indicated that they would recommend the online GSDprogram to other DEs.Free-text comments by DE respondents indicated they valued the GSD program,including that it was a useful tool to guide conversations. Keeping track ofprogress was a very valuable and rewarding experience. However, DEs highlightedthere were challenges, mainly related to recruitment difficulties.Supplement 5.Examples of free-text comments written by the DE survey respondents are providedin Overall, for the DEs who attended the focus group, their experience offacilitating the GSD program with YAD was positive, particularly as it relatedto ways to engage with young people and their own professional growth. DEshighlighted that they learnt to improve their communication skills, such asusing pauses and active listening skills. Time was an important factor in termsof YAD motivation because YAD needed time to reflect on their issues before theycould decide on solutions.Focus group participants indicated the main challenges related to getting the YADinvolved in the program in the first instance and technological issues, were theuser experience and expecting smoother navigation once on the website.Supplement 6.DEs emphasised that some text in the online activities required high literacyskills of YAD and therefore suggested using tools to improve accessibility forthose with lower health literacy skills. Examples of the DEs responses to thespecific focus group question are illuminated in Young adults. YAD found that GSD online enhanced thecommunication with their DEs as their conversations were more individualised,and solution orientated. These findings echo findings from our previous pilotstudy in which YAD also highlighted that the option of accessing the GSDplatform as required was considered essential to their reflection anddecision-making.2013 As signs of empowerment, YAD and DEs reported shareddecision-making which helped to establish meaningful and effectiverelationships13 enhancing a person-centred approach in self-managementof diabetes.21 Zoffmann and Kirkevold13 highlighted that to keepmotivated and feel empowered, YAD needs to understand their own roles in thesebarriers and enablers.16 In current study, the YAD reported that during the GSDprogram, they talked to their DEs about issues in their lives that were mostpertinent at the time they got involved in the GSD program, not their diabetes.They indicated that because the conversations were centred on their terms andneeds, YAD was able to understand their diabetes in a wider context and takeresponsibility for their own care to keep themselves motivated. Similar findingshave been reported that indicate using GSD seems to positively influencemotivation for self-management.31 The same study reportedthat through self-reflection about how to live with diabetes, participantsreinterpreted their life with diabetes by gradually moving toward acceptance oftheir condition because the dialogue with the DEs was seen to be on a more‘equal footing’ compared to usual consultations, which, in turn, helped supportparticipants to become more self-determined.31 YAD in this studyhighlighted that time to reflect and having open conversations helped them tostay motivated.In the current study, the GSD online program captured YAD information that mayotherwise have not been shared with DEs and was used to inform managementstrategies that helped YAD see patterns of behaviours, recurring issues ordifficulties in diabetes self-management. Zoffmann and Kirkevold reported thatby completing GSD reflections, clients improved their ability to identify,express, and share unique and unexpected difficulties related to living withdiabetes.Diabetes Educators. DEs found that the GSD program had changedtheir practice in the way they communicated with YAD. DEs listened and reflectedmore when using GSD structured conversations. Thus, DEs implemented more of ashared decision-making and person-centred approach with YAD. These changesresulted in a higher level of engagement, motivation and satisfaction in workingwith the YAD as they observed the YAD become more self-sufficient andmotivated.29 Most nurses indicatedthat GSD was a supplement to their practice and professional role because theygained new person-centred knowledge about client challenges. They reported ahigher level of confidence in communicating with YAD and more satisfaction intheir work.29Thus, online GSD provided educational and behavioural support for DEs and YAD. ADanish qualitative study investigated nurses’ experiences of learning and usingthe GSD method in gynaecological settings to determine whether the GSD methodinfluenced nurses’ professional practice.31 especially in the COVID-19 pandemic environment withthe expansion of use of eHealth systems and increased focus on supportingconsumers to directly engage with and use online health care services.32DEs also reported that feeling confident was important in facilitating the onlineGSD method, including their technology skills. Therefore, appropriate trainingand support for DEs are essential in program implementation. Training healthprofessionals in digital technology is well recognised in both international andnational literature,32 Toprovide meaningful communication among health professionals, it is essentialthat the technology meets the end users’ needs.34 Researchers who haveaddressed models of building capabilities using technology indicate that it isintegral for users to believe the technology is beneficial, engaging andmanageable.35 The flexibility of the online GSD and engagementbetween the participants were found to the most important aspects of the onlineexperience of both YAD and DEs, especially it helped YAD to balance competingcommitments between work, studies and families, which otherwise may have beendifficult for example to fit medical appointments into their busy schedules. Itis well established in the literature that online consultations are particularlyattractive to this age group of young adults as they save time, expenses relatedto transport and taking time off work.38 These three issues havebeen established as major barriers preventing young adults with T1D aged 18–30years accessing health services.39 The technology andcommunication flexibility offered by online GSD helped DEs establishrelationships with YADs faster than face-to-face diabetes consultations which,in turn, provided practical solutions and reassurance to YADs in a timely mannerthat kept motivation and confidence high for YAD managing their lives withdiabetes.21 The GSD conversations are therefore in line with theevidence, and the ready accessibility and interactive conversations in theplatform may have supported YAD to stay motivated.The overall experience of undertaking the GSD program using the online platformwas positive from both YAD’ and DEs’ perspectives. Despite a few technicalshortcomings, the interactive conversations encouraged reflections and enhancedmeaningful communication which assisted YAD to self-manage diabetes and theirlives and DEs to apply new communication skill to their practice; essential todeliver effective person-centred self-management support.Strengths of this study include the use of validated psychometric instruments toassess changes in YAD self-management motivation, self-regulation andcommunication with DEs. The mixed-methods approach enabled rich data to begathered regarding acceptability and feasibility of the online GSD program fromthe perspectives of both YAD and DEs to be captured.Limitations include the small sample size. Recruitment of YAD proved verydifficult. YAD cited issues related to time pressures and balancing competingwork, study and family commitments, preventing their participation, althoughtheir reluctance to participate may also indicate a low priority placed on DMmanagement at this stage of their lives, and this was probably reflected in thenumber of YAD who consented but did not engage in the GSD program. Data was notcollected about the quality of internet access for YAD and this may have been anissue in large households and in rural areas with poor internet access at thetime this study was undertaken.It is possible that the sample was not representative of YAD because participantsmay have been more technologically competent and confident and had reasonablehealth literacy skills than those who did not participate.This study confirms that an online GSD program had a statistically significant impacton autonomous motivation in the diabetes self-management of those YAD whoparticipated in this program. The online version is feasible and acceptable inassisting YAD to communicate with DEs about pertinent diabetes concerns and identifyappropriate solutions. With further evaluation of those YAD who did not participatein the program, potentially the program might be more widely applicable. DEsappreciated the flexibility of the online GSD program and reported that it providedpositive opportunities to engage and communicate with YAD, hence enhancing theirpractice. Our evaluation of this online GSD program demonstrated a reproduciblemodel of facilitation of diabetes self-management for YAD. For sustainability, theGSD programs should be integrated as part of treatment-as-usual in health servicesand in DEs’ own practice. Future evaluations should be based on longitudinal andmulticentred programs.Communication technologies such as the online Guided Self-determination platform usedin this study contribute to effective diabetes self-management due to theiraccessibility and relevance to end users. Online GSD also provides healthprofessionals such as DEs opportunities to improve engagement and communication withtheir clients.The online guided self-determination program contributes to person-centred,developmentally age-appropriate care delivery by supporting YAD to be motivated andengaged with managing life with diabetes.The online platform offers potential opportunities to reach populations thatotherwise can be hard to reach due to geographical distances, social circumstancesand other barriers which impede face-to-face health service delivery.Further research about training of health professionals in using technology isessential to improved health care delivery in diabetes care.Click here for additional data file.Supplemental material, sj-docx-1-dhj-10.1177_20552076231167008 for Feasibilityand acceptability of an online guided self-determination program to improvediabetes self-management in young adults by Bodil Rasmussen, Karen Wynter, PeterS Hamblin, Christine Rodda, Cheryl Steele, Sara Holton, Vibeke Zoffmann and JudyCurrey in Digital HealthClick here for additional data file.Supplemental material, sj-docx-2-dhj-10.1177_20552076231167008 for Feasibilityand acceptability of an online guided self-determination program to improvediabetes self-management in young adults by Bodil Rasmussen, Karen Wynter, PeterS Hamblin, Christine Rodda, Cheryl Steele, Sara Holton, Vibeke Zoffmann and JudyCurrey in Digital HealthClick here for additional data file.Supplemental material, sj-docx-3-dhj-10.1177_20552076231167008 for Feasibilityand acceptability of an online guided self-determination program to improvediabetes self-management in young adults by Bodil Rasmussen, Karen Wynter, PeterS Hamblin, Christine Rodda, Cheryl Steele, Sara Holton, Vibeke Zoffmann and JudyCurrey in Digital HealthClick here for additional data file.Supplemental material, sj-docx-4-dhj-10.1177_20552076231167008 for Feasibilityand acceptability of an online guided self-determination program to improvediabetes self-management in young adults by Bodil Rasmussen, Karen Wynter, PeterS Hamblin, Christine Rodda, Cheryl Steele, Sara Holton, Vibeke Zoffmann and JudyCurrey in Digital HealthClick here for additional data file.Supplemental material, sj-docx-5-dhj-10.1177_20552076231167008 for Feasibilityand acceptability of an online guided self-determination program to improvediabetes self-management in young adults by Bodil Rasmussen, Karen Wynter, PeterS Hamblin, Christine Rodda, Cheryl Steele, Sara Holton, Vibeke Zoffmann and JudyCurrey in Digital HealthClick here for additional data file.Supplemental material, sj-docx-6-dhj-10.1177_20552076231167008 for Feasibilityand acceptability of an online guided self-determination program to improvediabetes self-management in young adults by Bodil Rasmussen, Karen Wynter, PeterS Hamblin, Christine Rodda, Cheryl Steele, Sara Holton, Vibeke Zoffmann and JudyCurrey in Digital Health


Of all participants, 186 (39.6%) were injured, with 14 withdrawing from the program due to injury. For those who completed a marathon, 172 (38%) reported 205 musculoskeletal injuries  and 84 boys (48.8%)). More than half  of the reported injuries were soft tissue injuries. Most injuries were localized to the lower leg  and were of a minor nature , requiring only 1–2 treatments. Conclusions: There was a low number of relatively minor injuries for high school participants taking part in a graduated and supervised marathon training program. The injury definition was conservative  and the relative severity of injuries was minor . Overall, these results do not support a need to restrict high school students from taking part in marathon running, though continued emphasis on graduated program development and close supervision of young participants is recommended.Objective: The aim in this study was to quantify the number, nature, and severity of injuries sustained by male and female high school students who took part in a running training program that culminated in the completion of a half or full marathon. Design: This study is a retrospective clinical audit. Methods: Injury reports from high school students (grades 9–12) who participated in a half or full marathon 30-week progressive training program comprising four training days per week (three running days and one cross-training day) were reviewed. The number of runners completing a marathon, together with the number, nature, severity of injuries, and treatment types, as reported to the program physiotherapist, were the main outcome measures. Results: Program completion was 96% ( Physical inactivity is one of the world’s greatest public health challenges . This isMarathon running is perceived as an “extreme” running event that requires a high level of physical and psychological maturity . AdequatIn response to the Rice and Wisniewski statement, an expert panel consisting of an interdisciplinary team of physicians, researchers, and specialists in adolescent physical activity was convened in 2006 to share opinions on safety of adolescents in marathon participation . One theTo address some of these evidence gaps, the primary aim of our study was to identify the number, nature, and severity of injuries sustained by adolescents who took part in an organized high school marathon training program. Additionally, because growth and maturation between female and male adolescents differ, an exploratory examination of differences in outcomes by sex was also undertaken as a secondary aim. With knowledge on the injuries sustained, a better understanding of adolescent marathon runners can be made available to inform future strategies that promote safe participation, and the development of future programs that prevent injury.This is a retrospective clinical audit of prospectively collected data on injury presentations. Ethics approval to collect and report data was obtained through the Boston Children’s Hospital Institutional Review Board. Injury data were collected by licensed physiotherapists at the Advanced Sports Therapy physiotherapy clinic (USA). Treatment decisions were made independently from the organization of the distance running program. The current study was performed prior to the COVID-19 pandemic outbreak.High school students (grades 9–12) volunteered to take part in the marathon training program. All students were welcome to participate if they could commit to attending weekly practices. There were no try-outs or fitness/performance expectations; participants did not need prior running experience; and nobody was excluded from the team due to their performance. Students were required to have a pediatrician’s medical clearance prior to participation. Parental consent was provided for all participants younger than 18 years of age. Individual consent was provided by participants 18 years of age or older.The DREAMFAR High School Marathon program (DREAMFAR) is a noncompetitive marathon-training program available to students from participating high schools in the Greater Boston area . In existence since 2008, over 600 students have successfully trained to complete a half or full marathon. Information sessions are held at participating schools to invite students to participate. All practices are supervised and managed in a noncompetitive manner, with an emphasis placed on fun and participation. The program has a “train-the-trainer” model, whereby high school staff members (adults) volunteer to be a school leader. These leaders take part in an orientation session and receive support and training throughout the marathon season from the program staff. Leaders establish weekly meeting schedules, create safe training routes around the school and community, and support the students through prescribed workouts. Separate to the leaders, adult mentors are recruited through various community networks to train with students during longer weekend runs. Mentors are required to undergo criminal background checks and complete a program orientation and training to learn mentorship “best practices” prior to participation in the program. Students and mentors are grouped into “running families” (~1:2 mentor:student ratio) to ensure all students have support on the road. If a student has special circumstances that requires constant supervision, an individual mentor is assigned. The school leaders, together with adult mentors and student participants, also receive education regarding injury recognition, prevention, and management throughout the season by the program’s physiotherapy provider.The marathon training program was modeled on best practice evidence at the time . Three sThe program (“marathon season”) operated for a total of 30 weeks, from October through to May, each school year. Long weekend runs began in November and continued through to the main event—a half or full marathon, held in early May. Three races  were strategically scheduled throughout the training period to maintain participants’ training interests and to provide race experience prior to the main event. All students were expected to attend at least 75% of scheduled training sessions in order to be eligible to run in the main event.The sex and age of all participants were collected from intake forms completed at the beginning of each season. Participant age was calculated based on the date of the marathon for each season as a reference point. Race completion statistics were collected via the marathon organization’s online results database. All information was compiled by the program staff, de-identified, and subsequently shared with the study team.A running-related injury was defined as any self-reported complaint from a participant to the physiotherapist. Initially, notifications of injuries were given to the student mentor or school leader, who, in turn, notified an AST physiotherapist through email to arrange an appointment for injury assessment. An appointment was scheduled within one week of initial notification. Following assessment and diagnosis, a physiotherapist provided treatment or referred the participant on to a local orthopedist if necessary. During runs ≥16 miles, a designated physiotherapist was on-site at the training run to allow students to immediately report a potential injury. Follow-up appointments were scheduled if necessary. Six physiotherapists treated the participants over the course of the study. Their notes and diagnosis were reviewed by the lead physiotherapist and author (MP) at regular weekly intervals. Any concerns were clarified with the consulting physiotherapist, recorded in the main clinical database, and flagged as a DREAMFAR participant. These records were marked for review at the time of the clinical audit. Relevant information was compiled for each participant, de-identified using previously assigned codes, and shared with the study team.Injury types: joint injury ; soft tissue injury ; bone injury .Injured body parts (lower limb only): spine, hip/thigh, knee, lower leg, and ankle/foot.Treatment frequency: number of treatment sessions provided by physiotherapists. This variable was used as an indicator for severity of the injury.Treatment types: consultation, running modification, orthotics/brace/footwear, physiotherapy  and combinations of these treatments.The main variables of interest were the number of participants referred to, and subsequently treated by, the physiotherapy team, together with specific details of the injuries including:t-test was used when this variable was normally distributed. Mann–Whitney U test was used when non-normal data distribution were found. The status of normality was examined using the Shapiro–Wilk test. For categorical variables, the frequency of specific variables, including injury types and injured body parts, were summed and converted to percentages (%). Analysis considered participant sex  using a chi-square (x2) analysis, with comparisons performed to evaluate percentage (%) differences in injury types and injured body parts, between groups, with p ≤ 0.05 used as a critical statistical value. All data were initially provided to the research team as a Microsoft Excel  spreadsheet, and subsequently converted for analysis in SPSS .Descriptive statistics were used to analyze the main outcome measures for the primary aim of the study. Mean, standard deviation, and 95% confidence interval (CI) values were analyzed to describe continuous variables such as number of participants referred to and treated by the physiotherapy team. To compare the number of participants referred to and treated by the physiotherapy team by sex, an independent Over the 5-year period of this study, 469 high school students participated in the DREAMFAR program. Seven participants chose to leave the program due to a change in interest. Fourteen participants dropped out of the program due to injury . In total, 448 participants (253 girls and 195 boys) completed the full training season, which culminated with participation in a half or full marathon .For all registered participants, the proportion with injuries was 39.6% (186/469). Of those who completed the program, 226 musculoskeletal injuries were reported in 172 participants  and 84 boys ). Twenty-nine participants reported multiple injuries during the season. A total of 143 participants reported a single injury: 143/172, 83.1% ; 25 participants reported 2 injuries: 25/172 14.5% ; 4 participants reported 3 injuries: 4/172, 2.3% .n = 154, 68.1%) were soft tissue injuries  than the foot/ankle ; while for male runners, the foot/ankle  had slightly more soft tissue injuries than the lower leg . The types of injuries sustained and the body parts injured did not differ by sex .Two-thirds of injuries ). The most common treatment types reported based on the number of injuries (n = 226) are reported in A mean of 1.8 ± 1.6  treatment sessions were required per participant and 1.9 ± 1.7  sessions per injury. There were no reported differences in the frequency of treatment by sex or for injuries  to as many as 85% of 63 runners in an 18–20-month marathon training program ,12,13. NImportantly, the impact of the reported injuries on participants’ ability to continue training was considered minimal. Four in every five injuries reported in our study required only 1–2 treatment sessions to return to running. When compared with high school runners engaged in a competitive cross-country program, our cohort reported injuries that were less severe in terms of treatment needs . This loAnecdotally, resistance to adolescent marathon participation has stemmed from the supposed vulnerability of growth plate injuries in skeletally immature athletes . This raEngaging adolescents in high school sports is an important goal; yet, many adolescents are not able, or do not wish, to participate in highly competitive settings . The DREA strength of this study was its systematic, consistent data collection structure. The data collection was consistent for the five-year program, with author MP supervising and organizing the collection of information throughout this period. All injury assessments were performed by physiotherapists at the clinic to which author MP belongs. In this clinic, all physiotherapy staff had completed a masters or doctorate level of education. All staff had experience exclusive to sports and orthopedic physiotherapy and utilized their clinical decision making to determine a specific physiotherapy diagnosis, prognosis, and intervention. In cases where diagnostic imaging and/or injury diagnosis confirmation was indicated, each staff member referred the athlete to a local orthopedic specialist, though these referrals were rarely needed.Additionally, as with all descriptive studies of injury, the awareness, recognition, and communication of the injury event is vital to the accuracy of the data collection. In the DREAMFAR program, team leaders, program mentors, and all participants were instructed on several occasions about nutrition and hydration, climate awareness, proper equipment, injury prevention, and symptom recognition and management strategies and techniques by one of the authors (MP) and other professionals specifically trained and experienced in their relevant fields.There are several limitations to this study. First, while free “insurance-blind” injury assessments were available to participants of the DREAMFAR program, some participants might have chosen to seek treatment elsewhere. While there is no guarantee all injuries were captured, we are confident that the most serious were included, with knowledge that only 14 (3%) participants dropped out of the running program due to injury. Second, we were unable to accurately calculate exposure in hours; therefore, injury rates were not calculated, which limits the comparability of results to those of other studies. Third, this study reports on one running program in one region, limiting its generalizability. Replication studies are encouraged to be published as being able to provide adolescent distance runners, as well as coaches and medical staff, with reliable information on injury and injury prevention is important. Fourth, while our study did not find a high proportion of lower limb bone injuries, there are additional factors to be considered before active promotion of marathon running in adolescents. Fifth, we did not have anthropometric data on participants, and participants were of a small age range, between 14 and 18 years. Unlike other studies of injuries in high school aged athletes , we did Our study supports the safety of long-distance running training and the completion of a full or half marathon in adolescent, novice runners. Overall, 40% of marathon completers sustained musculoskeletal injuries. Most of these injuries were minor, requiring 1–2 treatment sessions only. Directly comparing results with those of other studies was limited because of the different definitions used and different age groups included. However, the results suggest that the pattern of injury is similar to that seen in adult populations of novice runners. The results do not support the need to restrict adolescent marathon programs, though a continued emphasis on conservative, graduated, and supervised training programs is recommended. Future studies need to employ a longitudinal study design to monitor the long-term effects.


The analysis through the HDOCK server showed stable complex formation with a binding energy of -12.3, the lowest among reference drugs. The RetroMAD1-3CLpro complex underwent a 100 ns molecular dynamics simulation (MDS) in an explicit solvation system, generating various trajectories, including RMSD, RMSF, hydrogen bonding, radius of gyration, and ligand binding energy. MDS results confirmed intact interactions within the RetroMAD1-3CLpro complex during simulations. In vitro experiments validated RetroMAD1's ability to inhibit 3CLpro enzyme activity and prevent SARS-CoV-2 infection in human bronchial cells. RetroMAD1 exhibited antiviral efficacy comparable to Remdesivir without cytotoxicity at effective concentrations. These results suggest RetroMAD1 as a potential drug candidate against SARS-CoV-2, warranting further in vivo and clinical studies to assess its efficiency.COVID-19 results from SARS-CoV-2, which mutates frequently, challenging current treatments. Therefore, it is critical to develop new therapeutic drugs against this disease. This study explores the interaction between SARS-CoV-2 3CL As of 6 September 2023, SARS-CoV-2 has caused 770 million confirmed cases and 6.9 million deaths globally according to the World Health Organisation (WHO) . This pandemic has prompted researchers worldwide to join forces, accelerating innovation to combat COVID-19 effectively. Since then, multiple vaccine candidates have entered clinical trials. The large-scale distribution of COVID-19 vaccines that began in early 2021 marked a significant turning point in the global battle against the pandemic. In May 2023, the WHO downgraded the COVID-19 pandemic status and announced that it was no longer a global public health emergency2.In late 2019, an outbreak of pneumonia with an unknown origin emerged in Wuhan, China. Subsequently, it rapidly spread worldwide, placing significant burdens on global public healthcare and economic systems. The outbreak was later attributed to a coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which resulted in the disease being termed coronavirus disease (COVID-19)4. To prepare for future waves of infection, it is crucial to develop vaccines and new drugs using various platform technologies. In the race to combat COVID-19, we introduce our chimeric protein, RetroMAD1 (Patent Number: US 2013/0336955A1). RetroMAD1, when administered orally, has demonstrated effectiveness against the Feline Leukemia Virus (FeLV), a pathogen in cats5. Comprising 41.3 kDa, RetroMAD1 is a combination of three different antimicrobial proteins: Retrocyclin-101 linked to the N-terminus of the 30 kDa momordica anti-human immunodeficiency virus (HIV) protein (MAP-30) via a linker peptide, with Dermaseptin-S1 directly joined to MAP-30 at the C-terminus , MAP-30 exhibits N-glycosidase activity that inactivates ribosomes, thereby inhibiting protein synthesis29. Additionally, MAP-30 can disrupt viral DNA and ribosomal function in virally infected cells, providing novel pathways for its antiviral activity24.MAP-30 is a single stranded protein with 263-residue derived from Phyllomedusa Sauuagii by Mor et al. in 199130. Dermaseptin-S1 has shown potent activity against pathogenic fungi, Gram-positive and Gram-negative bacteria, protozoa, and genital pathogens. Its antimicrobial activity is mediated by the selective interaction of basic and amphipathic α-helix moieties with plasma membrane phospholipids, resulting in membrane permeabilization and lysis33.Dermaseptin-S1, a 34-residue antimicrobial defensin peptide, was initially purified from the skin extract of the South American arboreal frog pro), which plays a critical role in viral replication34. To evaluate RetroMAD1's effectiveness, we conducted molecular docking experiments, comparing it with reference drugs like Andrographolide, Ivermectin, and Remdesivir, all previously shown to inhibit SARS-CoV-2 3CLpro37. To delve deeper into the interaction between RetroMAD1 and 3CLpro, we performed extensive 100 ns molecular dynamics simulations to assess the dynamic stability of the RetroMAD1-3CLpro complex. This computational analysis allowed us to examine the behaviour of the complex over time. Subsequently, we conducted in vitro experiments, including a 3CL protease inhibition assay, to validate our findings from the docking and molecular dynamics simulations. These assays helped confirm RetroMAD1's ability to inhibit 3CLpro enzyme activity effectively. Lastly, we assessed RetroMAD1's capacity to prevent SARS-CoV-2 (Wuhan-Hu-1) infection using an in vitro EpiAirway Model, which consists of healthy human-derived tracheal/bronchial epithelial cells closely resembling the human respiratory tract. Our comprehensive study provides compelling evidence of RetroMAD1's potential as a promising candidate for SARS-CoV-2 treatment, achieved through the inhibition of 3CLpro activity.In this study, we focused on assessing RetroMAD1's potential as a treatment for SARS-CoV-2 by targeting the key non-structural protein, 3-chymotrypsin-like protease (3CL® database (accession number: OQ714810) and served as the template for protein structure prediction using the default parameters of ColabFold in conjunction with Google Colab39. ColabFold replaced the AlphaFold2 homology search with an MMseqs2 search against both the UniRef and Environment sequence databases, using both paired and unpaired sequences41. The models were then visualized using YASARA Structure Version (20.10.4)42 and PyMOLv2.5 43. The predicted structure comprises the complete domains of retrocyclin -101, MAP30, and dermaseptin S-1, and this model was chosen for molecular docking  (PDB ID: 6LU7) of Wuhan-Hu-1 variant are readily available in the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) (https://www.rcsb.org/) and were retrieved in the .pdb format. Swiss Model web server was used for repairing the missing residues within the structures44. All the crystal structures were prepared by removing existing co-crystallised ligands, water molecules and hetero atoms by PyMOLv2.5 43. The 3D structure of reference drugs in this study  were retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/)45. These ligands were converted into the .pdb format using the Open Babel tool46.The crystal structure of SARS-CoV-2 3-chymotrypsin-like protease (3CLhttp://hdock.phys.hust.edu.cn/), an online server which can predict protein–protein interaction through a hybrid algorithm of template-based docking47, was used to carry out protein–protein docking between RetroMAD1 as a ligand and SARS-CoV2 3CLpro as a receptor. His-41 and Cys145 are the catalytic dyad that serve as the active site of 3CLpro48. Therefore, residues that located within 5.5 Å of Cys145 reported from previous study 49 were defined as input active site residues to specify the binding area of the docking complex. Based on the procedure of HDOCK server, the top 100 docking results can be generated from each complex and the top 10 docking results can be visualized on result web page47. The RetroMAD1-3CLpro complex with highest negative docking scores were assessed for the docking poses and visualized using PyMOLv2.5 43. Prodigy website (https://wenmr.science.uu.nl/prodigy/) was used to predict the values of binding affinity  at 25.0 °C.HDOCK server , version 22.9.24. Subsequent to the preliminary structural preparations , the simulation was conducted within YASARA, using the AMBER14 force field integrated into the YASARA structure (macro: md_run.mcr), albeit with slight alterations. To elaborate, the Posisson-Boltzmann approach was the chosen technique for managing the electrostatic force to obtain electrostatic potential with implicit solvent and counter ions. A “simulation cubic cell” was set and inundated with water, maintaining a density of 0.997 g/mL and isobaric pressure at 298 K throughout the 100 ns simulation. To mimic neutral physiological environment, sodium chloride was introduced at a concentration of 0.9%. The distance between the protein and the cell boundary was set to 10 Å. The timestep was designated at 2.5 fs, with simulation snapshots taken at intervals of 250 ps. For a comprehensive analysis of energy and residue alterations throughout the simulation, the md_analyze.mcr and md_analyzebindenergy.mcr macros were employed. The findings, encompassing data on the root mean square deviation (RMSD), the root mean square fluctuation (RMSF), hydrogen bonds, radius of gyration, and the ligand binding energy  stability of the complex binding. In the YASARA binding energy function, the energy is calculated as the difference between the sum of potential and solvation energies of the separated compounds and the sum of potential and solvation energies of the complex in the AMBER14 force field. Thus, more positive energy (difference) means higher affinity.The molecular dynamics of the RetroMAD1-3CLpro inhibitor, feline antiviral drug (GC376) provided by BPS Bioscience, was employed as an inhibitor control. The effect of RetroMAD1 on inhibiting the activity of the 3CLpro was assessed using the Untagged 3CL Protease Assay Kit  in accordance with the manufacturer’s instructions.Test concentrations of RetroMAD1, ranging from 400 to 33.55 µg/mL, were prepared in the Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham (DMEM/F12) non-phenol red cell culture media . The 3CLpro was diluted in assay buffer (containing 1 mM DTT) to achieve a concentration of 0.5 ng/µl (15 ng per reaction). Subsequently, 30 µl of the diluted 3CL Protease enzyme solution was introduced into wells designated as “Positive Control,” “Inhibitor Control,” and “Test Sample.” In parallel, 30 µl of the Assay Buffer (containing 1 mM DTT) was added to the “Blank” well. Additionally, 10 µl of GC376 (500 µM) was pipetted into the wells labelled as the "Inhibitor Control." Furthermore, 10 µl of RetroMAD1 at specific diluted concentrations was added to each well marked as the "Test Sample." Finally, 10 µl of Assay Buffer with DTT was introduced into both the "Blank" and "Positive Control" wells.In brief, 3CLThe enzyme and inhibitors were subjected to preincubation for 30 min at room temperature with slow shaking. The reaction was initiated by the addition of 10 µl of the 3CL Protease substrate solution (200 µM) to each well. Subsequently, the sealed plate was incubated for 4 h at room temperature with slow shaking. Finally, fluorescence intensity was measured using an M3 multi-mode microplate reader (SpectraMax Molecular Devices) at an excitation wavelength of 360 nm and detection of emission at a wavelength of 460 nm.RetroMAD1 was diluted to test concentrations as follows: 400, 300, 200, 150, 100, 75, 50, and 37.5 µg/mL in the MatTek culture medium (AIR-100-MM). Remdesivir  was tested in singlet wells at concentrations of 1, 0.1, 0.01, and 0.001 µg/mL as the positive control.The USA-WA1/2020 strain of SARS-CoV-2 was passaged three times in Vero 76 cells to create the virus stock. The virus was diluted in AIR-100-MM medium before infection, resulting in a multiplicity of infection (MOI) of approximately 0.03 CCID50 per cell.Chlorocebus aethiops) kidney; ATCC CRL-1587) were seeded in 96-well plates and grown overnight  to 90% confluence. Samples containing the virus were diluted in tenfold increments in the infection medium, and 200 µL of each dilution was transferred into respective wells of a 96-well microtiter plate. Four microwells were used for each dilution to determine 50% viral endpoints. After 7 days of incubation, each well was scored positive for the virus if any cytopathic effect (CPE) was observed compared to the uninfected control, and counts were confirmed for the endpoint on day 10. The virus dose that infected 50% of the cell cultures (CCID50 per 0.1 mL) was calculated using the Reed-Muench method52. The day 7 values are reported. Untreated, uninfected cells were used as cell controls.Vero 76 cells (African green monkey (https://www.mattek.com)  and arrived in kits with either 12- or 24-well inserts each. The TBE cells were grown on 6 mm mesh disks in transwell inserts. During transportation, the tissues were stabilized on a sheet of agarose, which was removed upon receipt. One insert was estimated to consist of approximately 1.2 × 106 cells. Kits of cell inserts  originated from a single donor, # 9831, a 23-year-old, healthy, non-smoking, Caucasian male. The cells have unique properties in forming layers, with the apical side exposed only to air, creating a mucin layer. Upon arrival, the cell transwell inserts were immediately transferred to individual wells of a 6-well plate according to the manufacturer's instructions, and 1 mL of MatTek’s proprietary culture medium (AIR-100- MM) was added to the basolateral side, while the apical side was exposed to a humidified 5% CO2 environment. The TBE cells were cultured at 37 °C for one day before the start of the experiment. After the 24-h equilibration period, the mucin layer, secreted from the apical side of the cells, was removed by washing with 400 µL pre-warmed 30 mM HEPES buffered saline solution 3X. Culture medium was replenished on the basal side following the wash steps.The EpiAirway Model consists of normal, human-derived tracheal/bronchial epithelial (TBE) cells that have been cultured to form a multi-layered, highly differentiated model resembling the epithelial tissue of the respiratory tract. The cell cultures were custom-made by MatTek Life Sciences (Each compound treatment (120 µL) and virus (120 µL) were applied to the apical side, and compound treatment only was applied to the basal side (1 mL) for a 2-h incubation. As a virus control, three of the cell wells were treated with placebo (cell culture medium only). Following the 2-h infection, the apical medium was removed, and the basal side was replaced with fresh compound or medium. The cells were maintained at the air–liquid interface. On day 6, the medium was removed and discarded from the basal side. Virus released into the apical compartment of the tissues was harvested by the addition of 400 µL of culture medium pre-warmed at 37 °C. The contents were incubated for 30 min, mixed well, collected, thoroughly vortexed, and plated on Vero 76 cells for the virus yield reduction (VYR) titration assay. Triplicate and singlet wells were used for virus control and cell control, respectively.Uninfected cells treated with each concentration of the test compound were run in parallel with the infected, treated wells. The cell cytotoxicity was determined under the microscope to check for any changes in cell appearance compared to the normal control cells and toxicity control cells that were run on the same plate. These changes may include enlargement, granularity, cells with ragged edges, a filmy appearance, rounding, detachment from the surface of the well, or other changes. These changes were designated as T (100% toxic), PVH , PH , P , Ps , or 0 (no toxicity–0%), depending on the degree of observed cytotoxicity.50) and half maximal inhibitory concentration (IC50) of RetroMAD1 were determined through non-linear regression of the inhibitor's concentration–response curve fit model. The EC90 was calculated using the GraphPad Prism 9 calculator, based on the EC50 value and hill slope obtained from non-linear regression analysis. Statistical analysis employed an unpaired t-test with Welch's correction, and the results are presented as the mean ± SD. P values less than 0.05 were considered statistically significant.Data and statistical analysis were conducted using GraphPad Prism 9 software . The half maximal effective concentration  and II (residues 102–184) feature a six-stranded antiparallel β-sheet structure, while domain III (residues 201–303) comprises five α-helices and is responsible for enzyme dimerization54 , we assessed the potential of RetroMAD1 to inhibit 3CLpro by conducting molecular docking of the RetroMAD1-3CLpro complex. We utilized the SARS-CoV-2 3CLpro model retrieved from the Protein Data Bank (PDB ID: 6LU7). Among the top 10 results, we selected the one with the highest negative scores for interaction with the catalytic site (His41-Cys145) and the other 25 important catalytic pocket residues 49. Andrographolide, Ivermectin and Remdesivir, which have previously shown efficacy against 3CLpro37, were used as reference drugs. It was found that RetroMAD1 exhibits the strongest interaction with 3CLpro (− 219.74), followed by Remdesivir (− 182.36), Ivermectin (-142.85) and Andrographolide (− 117.10)  underwent further evaluation for their binding affinities and dissociation constants using the PRODIGY server. Table pro complexes with RetroMAD1, Andrographolide, Ivermectin and Remdesivir were − 12.3,  − 5.43, − 5.42 and − 5.53 kcal mol−1, respectively. RetroMAD1 exhibited a relatively higher binding affinity and bound to the His41-Cys145 catalytic dyad and other catalytic pocket residues.Using the HDOCK server . The complex’s interaction was later computed using RMSD, RMSF, MM/PBSA, Hydrogen Bond, and Radius of Gyration values. The snapshots of the interaction of the complex were illustrated in Fig. To investigate the dynamic stability of the RetroMAD1-3CLpro. Fortunately, these regions were not implicated in RetroMAD1-3CLpro contact sites thus might suggest structural integrity or flexibility that was not attributed directly from the RetroMAD1-3CLpro complex formation  was calculated to be at -110.460 kJ/mol, with the best interaction during 43.25 ns at 604.556 kJ/mol. Additionally, the hydrogen bond count remains proximate to 1100  against GC376 at a concentration of 500 μM . We also generated a concentration–response curve for RetroMAD1's inhibition of 3CLpro activity, which revealed an IC50 of 86.89 μg/mL .To assess RetroMAD1's antiviral properties in a model that closely resembles human cell infection, we utilized the EpiAirway Model. This model involves the cultivation of normal human-derived tracheal/bronchial epithelial (TBE) cells to create a multi-layered, highly differentiated structure that closely mimics respiratory epithelial tissue10 CCID50 values of 2.67 and 2.5, respectively . In the absence of any antiviral compound, cells exposed to the infection media produced a log10 CCID50 value of 4.4.To assess the effectiveness of RetroMAD1 in preventing SARS-CoV-2 infection in human-derived TBE cells, we conducted an evaluation of its inhibitory impact on SARS-CoV-2 at various concentrations, comparing it to the efficacy of Remdesivir 5. Drawing on the structural congruence between FCoV and SARS-CoV-261, our study delves into the prospective efficacy of RetroMAD1 against the 3CLpro of SARS-CoV-2. We embarked on this exploration using molecular docking and advanced it with rigorous evaluations, including 100 ns molecular dynamics simulations to discern the nuanced interplay between RetroMAD1 and 3CLpro. Additionally, a series of in vitro inhibitory assays further illuminated the potential of RetroMAD1 in countering COVID.RetroMAD1, designed to express three anti-viral peptide fusion proteins, offers a compelling direction for research. Since 2020, RetroMAD1 has been globally available for therapeutic intervention against FeLV and FCoV infections in pro is a non-structural protein responsible for cleaving at least 11 sites on large viral polyproteins that are essential for replication and transcription63. Consequently, inhibiting the activity of 3CLpro could disrupt the SARS-CoV-2 viral replication, making it a crucial target for COVID-19 treatment. In line with this, numerous research groups have focused on 3CLpro as they seek effective treatment for SARS-CoV-2 infection, leveraging repurposed antiviral drug such as Ribavirin, Remdesivir, Favipiravir, and Lopinavir, which are designed to impede viral replication66. Our docking study indicates that RetroMAD1 achieves a better docking score against 3CLpro compared to Andrographolide, Ivermectin and Remdesivir, employing similar docking parameters  analysis provides insights into binding drift70. Our RMSD trajectory results revealed an overall stability of the protein complex, especially within the first 80 ns. More pronounced conformational shifts arose after this period and later stabilized until the end of the simulation. Intriguingly, the RMSD superimposition assessment of RetroMAD1 illustrates that the ligand's conformation exhibits considerable constancy throughout the investigation, as visualized in Fig. pro complex  trajectory analysis simulations were completed using Yasara Structure version 23.5.19.W.64 over a 100 ns with 400 snapshots, using AMBER14 force field. A steady-state NpT molecular dynamics simulation was utilized to validate the predicted binding between the RetroMAD1 and 3CLpro enzyme activity at concentrations ranging from 200 to 400 μg/mL. This inhibition is equivalent to the inhibitory activity of GC376 at 500 μM, resulting in 101.35% inhibition , where the EC50 of Remdesivir had to be increased from 0.01 to 0.06 μM in evolved coronavirus cultures carrying mutations within non-structural proteins 12 (nsp12) of the viral RNA-dependent RNA polymerase (RdRp)76. The constant evolution of SARS-CoV-2 may necessitate frequent increases in dosage or prolonged treatment with Remdesivir. Additionally, exposure to Remdesivir in concentrations ranging from 1 to 8 μM has been shown to lead to alterations in gene expression profiles and morphology77.It's worth noting that Remdesivir has shown limited effectiveness in treating hospitalized COVID-19 patients, as evidenced by its impact on overall mortality, the need for ventilation, and the duration of hospitalizationpro78. By combining Remdesivir with RetroMAD1, our aim is to potentially enhance their antiviral effectiveness. RetroMAD1's unique mechanism of action, attributed to the co-expression of three different peptides within RetroMAD1, equips it with the ability to combat a wide range of viruses. This distinctive feature makes RetroMAD1 less susceptible to the development of viral resistance, positioning it as a promising candidate for combination therapy alongside Remdesivir and other antiviral drugs. This strategy has the potential to reduce the risk of drug resistance while enhancing therapeutic efficacy. It should be further explored through in-vivo studies, followed by clinical trials79. It's worth noting that RetroMAD1 can be administered orally, which presents a significant advantage. This attribute could potentially make RetroMAD1 a preferred treatment option for mild-to-moderate COVID-19 cases in adults at high risk for severe diseases, including hospitalization and mortality.With this in mind, we propose a strategy for future studies involving the combination of repurposed drugs such as RetroMAD1 with Remdesivir. This strategy leverages Remdesivir's dual antiviral action, targeting both the RNA-dependent RNA polymerase  and 3CLpro mutations that confer resistance to RetroMAD1. This hypothesis is grounded in the limited reports of resistance to 3CLpro inhibitors80. Furthermore, recent research81 has shown that in vitro results indicate that GS-441524, Remdesivir, EIDD-1931, Molnupiravir, and Nirmatrelvir retain their activity against all Variants of Concern (VoCs), including Omicron. This suggests that RetroMAD1 may also have the capability to maintain its activity against future subvariants of Omicron. These findings provide a promising outlook for the potential effectiveness of RetroMAD1 in addressing emerging Omicron subvariants, although further research and testing are required to confirm this hypothesis.While this study did not demonstrate RetroMAD1's effectiveness against any Omicron subvariants, which is a limitation of this study, it is hypothesized that there may be no 3CLConsequently, it is advisable for RetroMAD1 to undergo in vivo non-human primate challenge studies, followed by clinical trials, as a promising antiviral candidate for addressing infections caused by various SAR-CoV-2 variants, including Omicron subvariants. In-vivo studies are necessary to explore the limitations and strengths of RetroMAD1 in combating current SARS-CoV-2 variants before progressing to human clinical trials. The use of RetroMAD1, either alone or in combination with FDA-approved repurposed drugs, requires further validation through high-quality evidence and additional validation studies.pro represents a crucial target for COVID-19 treatment, and our study underwent rigorous validation, encompassing docking, molecular dynamics simulations, enzyme inhibitory assays, and EpiAirway Model testing. During the docking analysis, RetroMAD1 was compared to reference drugs , and RetroMAD1 exhibited a strong interaction with 3CLpro. Its binding affinity had the lowest negative binding energy , exceeding that of Remdesivir, Ivermectin, and Andrographolide . Exploring the dynamic stability of the RetroMAD1-3CLpro complex through 100 ns molecular dynamics simulations, the RMSD trajectory showed an initial destabilization phase followed by stable structural behavior. Minor fluctuations in root mean square fluctuation (RMSF) were observed in specific residue regions rather than between the contact points of the complex. Additionally, the MM/PBSA analysis indicated favorable interaction energies during the simulation, consistently accompanied by hydrogen bond formation. Radius of gyration data further suggested limited structural movement, confirming overall complex stability. In vitro tests on human-derived TBE cells using the EpiAirway Model and 3CL protease inhibition activity supported the potential of RetroMAD1 as an antiviral therapy for COVID-19. However, further clinical research and rigorous trials are necessary to confirm its efficacy and safety for clinical application.SARS-CoV-2 3CLSupplementary Information 1.Supplementary Information 2.Supplementary Information 3.