Deadly Liver Mob (DLM) is a peer-driven, incentivised health promotion program aimed at increasing understanding of hepatitis C, promoting harm reduction in relation to injecting drug use, and linking participants to screening for hepatitis C, other blood borne viruses and sexually transmissible infections among Aboriginal people in Western Sydney, NSW. This paper presents the evaluation of a pilot study examining the acceptability of the program as a first step of a scalability assessment.Deadly Liver Mob operated in co-located needle and syringe programs and sexual health clinics in two sites: . Comparisons were made of the proportion of Aboriginal clients (Site 1) and occasions of service provided to Aboriginal clients (Site 2) in the 12 months prior and post-introduction of DLM. Interviews were conducted with 13 staff involved in delivery of DLM and with 19 clients.A total of 655 and 55 Aboriginal clients, respectively, attended Site 1 and Site 2 for health education. The proportion of Aboriginal clients attending both sites was significantly higher during the DLM compared with prior to its implementation. Of those attending for health education, 79 and 73%, respectively, attended screening following education. DLM clients strongly endorsed the program. Some staff were concerned about workforce capacity to effectively engage Aboriginal clients with multiple and complex needs, managing the differing aims of the participating services involved, and about offering of incentives for attendance at health services.While acceptability was high among staff and clients and preliminary results show high engagement with Aboriginal communities, this evaluation of a pilot program raises some issues to consider in scale up of DLM to other sites. The initiation of additional DLM sites should address issues of alignment with governing strategies and workforce capacity. Globally, widespread adoption of interventions found to be effective in research or pilot programs is required to achieve population health impact and effect . HoweverAboriginal Australians comprise approximately 3% of the population and as a group, experience significant disadvantage including life expectancy of approximately 10 years less than non-Aboriginal Australians . AborigiIt is imperative to engage Aboriginal people in a range of health services to reduce harms associated with BBVs and STIs and prevent further infections. Publicly funded sexual health services are key providers of care for HCV and other BBVs and STIs but the overall participation of Aboriginal people in these services is low. Data arising from mainstream sexual health services, general practitioners, and Aboriginal Community Controlled Health Services (ACCHS) across Australia show that only 4% of all clients are Aboriginal despite the significant burden of disease in this population , 12. EngSince 2013, the Deadly Liver Mob (DLM) program has been running as a pilot program in one Western Sydney publicly funded, co-located sexual health service and needle and syringe program (NSP), and in a second service in Western Sydney since 2015. The program was conceived to address low attendance at the NSP and sexual health clinic by Aboriginal people in a geographical area with recognized high levels of injecting drug use and low STI screening, and very few episodes of care over the immediate preceding years in the adjacent sexual health clinic, despite being one of the most populous areas for Aboriginal people in Australia.DLM uses an incentive-based, peer-driven intervention model. This approach is modelled on The Safe Injecting Cwiz (SIC) project which was conducted in Western Sydney in 1998–2002 and targeted people under 25 years of age who injected drugs . The SICThe peer-driven intervention model offers nominal incentives to encourage participants to attend for education then recruit and educate their peers  for which participants receive an incentive see Fig. . People The DLM education model was developed to meet the needs of the audience and to ensure that messages were culturally appropriate. Aboriginal culture is one of sharing stories (or “yarning”)  hence thAboriginal staff were central to the program design and implementation. A key principle of work with Aboriginal communities is consultation. The development of the DLM program involved consultation with the Aboriginal Programs Advisory Group within the health services and the local ACCHS . It was important that the DLM program and mainstream health services be presented as an additional choice for Aboriginal clients and that consultations with the ACCHS focused on appropriate referral pathways between ACCHS and mainstream services. A key component of the start-up of the DLM program was to invite community Elders to visit the program and experience it even though they may not fall within the target group.This paper uses data collected as part of an evaluation of the DLM pilot program (in relation to participant attendance in various aspects of the program), and qualitative data from interviews with DLM clients and staff to examine the acceptability of the program as the first step of a scalability assessment  . In partSome data have already been presented in relation to preliminary outcomes of the DLM program from one site in the first 12 months of operation . The curThe DLM program operated 2 days per week at Site 1 from 29 April 2013 (2.5 years data available), and 1 day per week at Site 2 from 12 February 2015 (1-year data available). De-identified data on clients participating in the DLM program were obtained. Data included date of initial attendance, demographic characteristics , risk factors (injecting drug use), recruitment of others to the program , and “cascade of care” progression , as identified from incentive payment amounts. The overall numbers of Aboriginal and non-Aboriginal clients attending Site 1 sexual health clinic and numbers of occasions of service provided to Aboriginal and non-Aboriginal clients at Site 2 sexual health clinic were also obtained.Comparisons of the proportion of Aboriginal clients (Site 1) and occasions of service provided to Aboriginal clients (Site 2) in the 12 months prior and 12 months post introduction of the DLM program were conducted using chi-square tests.During 2015 and 2016, semi-structured telephone interviews were used to explore the perceptions and experiences of the DLM program among two key stakeholder groups: (i) health staff ; (ii) Aboriginal DLM participants. Staff were informed of the program evaluation and asked via their professional networks to volunteer for an interview. Interested participants approached a researcher not involved in the running of the DLM program and a time was scheduled for a telephone interview. Aboriginal participants were informed about interviews by the DLM program workers and referred on to a researcher if they were interested in participating. Due to the length of time that had passed between the beginning of the DLM program in Site 1 and the time of the evaluation, many DLM clients could not be reached by the program workers for invitation to an interview. The workers therefore invited clients who had current contact details or who were still engaged with the site.A total of 13 interviews of around 45-min duration each were conducted via telephone with staff involved in the pilot program at these two sites. This sample included the majority of staff engaged with delivery of the DLM program. In addition, a total of 19 semi-structured interviews of around 15–30-min duration were conducted via telephone with Aboriginal clients of the DLM program (10 clients from Site 1 and 9 clients from Site 2). The sample size was sufficient to capture the complexity of program implementation across the two sites. Interviews with staff were conducted by a non-Aboriginal researcher (CT) and focused on: responses from Aboriginal communities and services regarding the program; how the DLM program operated within existing structures; strategies workers used to identify and solve issues; how the DLM program has been/could be embedded in routine policy and practice; and any unanticipated impacts of the program. Interviews with DLM clients were conducted by an Aboriginal researcher (VS) and explored the breadth and relevance of HCV and BBV prevention information, and clients’ level of comfort when attending the program sites.All interviews were audio-recorded and transcribed verbatim. A thematic analysis  was condA total of 677 and 55 Aboriginal clients attended Site 1 and Site 2 for health education, respectively. Demographic characteristics of DLM clients attending both services are presented in Table Figure p < 0.001). This number dropped to 31% in 2016 or year 3 (p < 0.001). For Site 2, the proportion of occasions of service provided to Aboriginal clients between 2013 and 2015 (the DLM program was introduced in 2015) increased from 5.9 to 8.4% (p < 0.001).The overall proportions of Aboriginal clients attending sexual health clinics for Site 1 and occasions of service provided to Aboriginal clients for Site 2 are presented in Fig. DLM was typically well supported by staff and client participants. The DLM program logic identified that involvement of Aboriginal workers was central to the design. The qualitative data supported this assumption but provided a broader lens with which to understand this element of the program design and potential impacts of DLM. The main theme identified in these data was stigma: how stigma related to Aboriginality, HCV, and injecting drug use acts as a barrier to Aboriginal people’s engagement with information and health care services; and, how engagement increased when health-related stigma was minimised via attendance at the DLM pilot program. The qualitative data also supported the assumption that provision of incentives was a key ingredient. However, there were some strong and negative opinions, particularly from staff participants, which would be important to address in scale up plans. For DLM, key elements of context in these two sites were the capacity of the workforce and issues relating to the management of DLM especially in relation to navigating priorities across NSP and sexual health service delivery sectors.‘[I]t’s just nice to have someone on your level that is sort of – I don’t mean to be racist or anything like that, but this is not a white person who talks there with professional talk you know. The people there will have a yarn with you and sort of understand of where you are coming from.’ Analysis of interview data highlighted the constraints that stigma places upon Aboriginal people’s access to health care services, and conversely, how Aboriginal people readily attend services where they do not feel negatively judged. The DLM pilot program was led by Aboriginal workers and provided an innovative model of a one-stop-shop, walk-in clinic for HCV education and harm reduction, BBV and STI screening and care that circumvented the stigma and discrimination experienced by DLM participants (many of whom inject drugs and/or live with HCV). Data from staff and clients demonstrated that a stigma-free, client-centred model of health care and education can significantly increase Aboriginal people’s access to and engagement with health-related services. Aboriginal staff of the DLM program were identified as key to ameliorating the potential impact of stigma in mainstream health services.‘[I]t’s very difficult for them to speak to somebody about it in a non-judgmental way, so they often live in silence in relation to their hepatitis C.’ (Staff #5)‘I think it took off quicker than we imagined … from the kick start, we tapped into a couple of great networks very early and [the Deadly Liver Mob program] was novel and different and everyone was on a high, so our workers were particularly enthusiastic, as were we, and we could not have anticipated the uptake … and we’re still way in front if you look at the sexual health screening numbers, miles in front of where they were prior…’ (Staff #12)‘And the people are so nice here, they make you feel – like if you’re wrong about something, they don’t make you feel stupid, which is what a lot of Indigenous people would have a problem with, because of education and that. I had problems with learning and that, but they don’t make you feel silly here. You feel like you’re equal. They explain it to you in the way that you understand.’ The DLM pilot program’s education and health services were delivered in a friendly, culturally sensitive, non-judgemental environment, which made clients feel comfortable and was described as increasing the likelihood they would return to the service at a later date. In particular, the DLM program was seen as a gateway to sexual health awareness and engagement for younger Aboriginal clients.‘I think that sexual health has such a stigma in that community … I think DLM was able to legitimize [Aboriginal] people getting a sexual health check-up, because I think to actually walk into a sexual health clinic, you are seen to be doing the wrong thing. “Why are you doing that for”, you know? Where it’s attached to some other type of care, or like getting some education … it may assist you to be empowered in other parts of your life and … you know [be seen to be] taking care of yourself …’ (Staff #7)‘I’ve actually found out I can get rid of it [hepatitis C] and I’m going to try and start treatment, yes.’ Overwhelmingly, staff gave a positive appraisal of the DLM pilot program and this was supported by statements from the clients. A large number of Aboriginal clients had been exposed, often for the first time, to health promotion education and BBV/STI screening. The co-location of NSP and sexual health services was said to have substantially improved Aboriginal clients’ access to information about HCV and safer injecting, BBV and STI screening, and referral for HCV treatment.‘I thought it was absolutely amazing, because I knew nothing about what they were talking about and I was very informed. I liked it.’ ‘I thought it was good you know raising awareness of you know hepatitis and you know just harm minimisation and stuff like that you know. I thought it was good information.’ ‘It’s not about the money. It’s about helping the Aboriginal community in my opinion. I mean, I wanted to come in here and just get better educated on it all and it’s been a great experience in my opinion.’ The provision of incentives was a key component of DLM but is not standard practice in health care delivery in Australia. Some staff expressed strong opinions about the use of incentives and questioned the ways in which these were used within DLM, potentially as “bribes” for attendance at a health service which clashed with the ethos of some staff participants. Some operational issues with vouchers were also raised including that storing and activating vouchers was sometimes difficult and required the development of a security process. On the other hand, the incentives were seen to play an important role in attracting clients to the pilot program and for opening discussions about sexually transmissible infections, safer injecting, HCV, and its treatment.Yeah, it’s a funny thing. I’m fairly – I personally don’t agree with [giving incentives], but as far as this group goes, I see that it can be a positive thing, so I don’t know. I’m happy to go with whatever. (Staff #10)I keep on wondering like everybody else, would [the program] work or not, because I for one was not a big fan of incentives. I thought it was not the way to go, because it was just bribing people … but then again after it started and then it just took off, I thought, “Yes this is the way to go”. (Staff #9)‘[I]t seemed that the benefits [of the pilot program] out-weighed the cost, and the cost was not big. You know, think about the money we’ve paid [for incentives] compared to the cost of treating someone if they went into hep C treatment … it feels like a no brainer … but I think for [Aboriginal] people, I think that they’ve got so many other things to contend with’ (Staff #12)‘And you know you can’t help but be touched by the stories and even though I'm experienced in this area I would still feel, at the end of some days, I would just feel wiped out and not from the physical workload it’s the emotional side of it and hearing the stories and the level of disadvantage and the sadness in people’s lives and … the things that have gone on in their lives, and it just makes you realize even more you know, walk a mile in my shoes before you, you can never judge people can you?’ (Staff #8)Staffing composition was said to be important for the success of a scaled-up program. Specifically, it was recommended that workers should comprise a mix of Aboriginal and non-Aboriginal people. Staff were often exposed to clients’ disadvantage and trauma, putting workers at risk of burn-out. The mental and physical health of workers requires monitoring and supporting through debriefing programs and ongoing primary care. The work place in which DLM operates requires mandatory training in Aboriginal cultural awareness. In addition to this, participants suggested that non-Aboriginal staff should be required to attend a pre-employment, awareness raising program to familiarize workers with the social disadvantage faced by Aboriginal communities and the impact that disadvantage has on clients’ health.‘[The staff] are on your level, they talk to you on your level and they don’t make you feel uncomfortable or they don’t get you impatient, you know what I mean? They don’t keep you there … they are very fast and very dedicated to their work, which is good.’ Aboriginal workers helped to recruit clients, greeted clients at reception, conducted the education interventions, provided information and advice to clients, and assisted with follow-up contact and care. Aboriginal workers reinforced an understanding among clients that this was a program administered by Aboriginal people, for Aboriginal people, with information and services relevant to Aboriginal communities. Aboriginal workers were particularly effective at establishing rapport with clients during the pilot program, and they facilitated the building of strong, trusting connections with local Aboriginal communities, which should be a prerequisite for establishing a scaled-up program. Aboriginal communities have diverse cultural conventions, for example, around making eye-contact, so Aboriginal workers are required to consult with communities about their specific needs.‘I think it’s important to talk to the Elders in people’s area to find out what the culture is, whether it’s urbanized, whether it’s traditional … to understand what marks of respect and culturally appropriate steps to take with that client group.’ (Staff #6)Strategies that were thought to increase program adoption and maximise program reach into Aboriginal communities were identified. Several practical suggestions for scale-up included the need for a steering committee and a co-ordinator to oversee the running of the program and to ensure that the DLM program has optimal impact by providing the full range of services. NSPs and sexual health services, although there may be some strategic and operational areas of common interest, are usually discrete entities within the Australian health care system, with different strategic priorities, workforce, operational policies, procedures, and clientele, so it is necessary to develop strategic approaches to managing and promoting innovative co-located services within Aboriginal communities. For example, as a result of logistical barriers and budget restrictions, one sexual health service operating in the DLM pilot program did not provide the full range of tests required to diagnose chronic HCV infection.‘[W]e started off with the optimal kind of site there at [western suburb], we were close to, across the road from the major shopping centre, right next to the transport hub, near the methadone clinic, not far from the drug and alcohol services, right next door to the sexual health service and right in a very, very high density Aboriginal population sort of heartland, so you know, we had a great combination of factors there in setting up and we had some really good community, some great community relationships there.’ (Staff #13)Careful planning and consideration is required to identify geographic areas where there is strong Aboriginal community connections in order to maximise program adoption and reach. The pilot program evaluation indicated that it is useful to locate DLM programs close to where Aboriginal people meet to minimise the effort required for participants to attend DLM and to make it easier for DLM workers to build relationships with the community. Similarly, ongoing community consultation with Aboriginal organisations, such as ACCHS, was recommended as an important management strategy to ensure continuing smooth operation of the program.‘… I think a one stop shop [is the best model], so have the social worker in there, have the sexual health all in the one spot if you can possibly have it … because traditionally … people go to NSP’s, go to methadone clinics, they don’t travel very far if it’s not within that service. Getting them to go to a [mainstream health] service is a lot more difficult.’ (Staff #11)A walk-in, one-stop shop model of care, with flexible appointments, short wait times, and child-friendly waiting areas was described as the most effective model for increasing community adoption and the reach of a scaled-up program. In regard to the make-up of DLM services, ideally, these were thought to include primary health care, HCV PCR testing, HIV and HBV antibody testing, liver disease assessment, access to HCV treatment using the direct acting antivirals (DAAs), pap smear, and a variety of sexual health tests. In the era of DAAs and efforts to increase HCV treatment uptake, a scaled-up DLM program has the potential to give clients direct access to effective HCV treatment. Finally, clients’ low literacy levels need to be considered in the design of the resources they receive in their DLM package and staff should be prepared to assist clients with low literacy to fill out paper work.This evaluation presents evidence to consider in the broader implementation, or scale-up, of the DLM program. Significant numbers of Aboriginal people were engaged in the program, acceptability of the program was high among staff, and clients and additional resources required to deliver the program (relating to incentive payments) were modest. Some issues should be clarified in development of an implementation plan: the relationship of DLM with the relevant strategies for HCV and STIs, better support for the workforce to understand the role of incentive payments, additional resources to induct and support workers, and methods to “manage up” in explaining the impact and role of DLM over time.One purpose of conducting assessments of scalability is to examine the influence of strategy, policy, and operational issues . The comThis slight strategic misalignment also has implications for characterising the DLM target group. Although DLM is framed around engagement of people with, or at risk of, HCV (who may be of any age), sexual health services see people under 35 years as their key target group. There is arguably a third target group for DLM: Aboriginal community members, irrespective of risk factor history or demographics. This third group is important for disseminating information about HCV and reducing stigma associated with it, and in generating community understanding, acceptance, and encouraging participation in DLM. It is recommended that the DLM sites set clear and appropriate goals regarding the extent to which they will engage with the target groups in each component of the program. For example, all Aboriginal community members who seek education will be accommodated at all stages of the program, regardless of history of injecting drug use; all Aboriginal community members agreeing to sexual health screening will be accommodated within the first period of the program (to be determined) to build trust, rapport, and understanding with community and community leaders; after this period, sexual health screening will be targeted to people 35 years and under who are sexually active; and, Aboriginal people who inject (or have injected) drugs remain the priority target population throughout. It is important that the inclusion of other groups does not exclude or dissuade the involvement of the priority group.Financial incentives are increasingly recognized as valid strategies for encouraging the uptake of public health measures. Although financial incentives for community members to improve health-related behaviours have had a limited role in the Australian setting, they have been widely used internationally, particularly in lower and middle income countries , 28, havIn DLM, incentives played a clear role in encouraging decisions to participate. Given the mistrust of mainstream health services that can underpin Aboriginal Australians’ decisions about health care engagement, the provision of an incentive was also a clear signal that the program could offer something of tangible benefit to Aboriginal people. Understanding the perspectives of the Aboriginal community and the evidence-base regarding incentive payments should be key elements of staff training regarding the incentive-based nature of the DLM program.Ways in which DLM staff could be supported were apparent from these data and included, in addition to any Aboriginal cultural sensitivity training offered by the workplace, the need for staff members to understand the role that social disadvantage plays in clients’ lives and how disadvantage affects clients’ health . It was The data from participant attendance draws attention to the need to ensure that all stakeholders understand what constitutes success in the DLM program. These data show very significant increases in attendance of Aboriginal people in the first year of Site 1. Attendance remains high, but lower, in the subsequent years of DLM. DLM sites need to ensure that funders and health managers understand patterns of DLM attendance and have appropriate expectations of community engagement and activity. For example, rates of attendance at DLM may decline over time, but remain significantly higher in relation to baseline. This pattern is expected and appropriate as saturation is reached within the community surrounding the DLM site. Hence, establishment of an appropriate and rigorous baseline measurement is essential.There are limitations in the data collected during this pilot study which effect the generalizability and transferability of findings to other contexts. In particular, the staff interview data was relatively richer than the narratives provided by DLM client participants. This could be due to varying education levels  and issues of stigma and power . Further, there were challenges faced by the DLM program workers in inviting Aboriginal clients to participate in interviews, due to outdated contact details. The interview sample of Aboriginal clients is therefore limited to those who had not changed their contact details or who were still connected to the site, thus may be considered more engaged with the service. The findings are drawn from an exploratory analysis of a pilot program and as such the qualitative data are limited to a descriptive analysis of top-level themes. Despite this, these themes highlight and illustrate the major barriers and facilitators to engagement with the DLM program. These findings, while limited, are important to acknowledge when planning future studies of Aboriginal health care engagement, both in regard to the DLM program, and more generally, and when considering the scale-up and roll out of this innovative model of care for Aboriginal people.Finally, the larger context of this program needs to be considered in the central role of a stigmatized community as well as stigmatised and illegal practices. It was an important aim of this program that DLM did not perpetuate stigma and worked to reduce stigma against Aboriginal people who inject drugs and who live with HCV. Aboriginal people who inject drugs are among the most stigmatised population groups in Australia, and their unequal status often becomes evident when they access health services , 37, 38.Delivering effective and acceptable care for Aboriginal people in Australia is a challenge in all areas of health. This challenge becomes more complicated when sensitive and stigmatized issues of sexual practice and injecting drug use are implicated in the health conditions of interest. While ACCHS remain the cornerstone of delivery of primary care for Aboriginal people, publicly-funded “mainstream” services have a key role to play for BBV and STIs given their specialist services and as an alternative option for service access among this population. However, the capacity of publicly funded services to engage Aboriginal people has been limited over many years, with very low rates of participation in these services. How to effectively engage Aboriginal people remains a complex question that requires an effective and engaged partnership of policy makers, researchers, service managers, and front-line workers to address. The DLM program shows promise in this regard, and the potential impact of this program should be examined when implemented carefully, and at scale.


A recent major earthquake (M7.8), coupled with appropriate climatic conditions, led to significant destruction in Ecuador. Temperature variations, which may be induced by anthropogenic climate change, are often associated with changes in rainfall, humidity and pressure. Temperature and humidity are associated with ecological modifications that may favour mosquito breeding. We hypothesized that the disruptive ecological changes triggered by the earthquake, in the context of appropriate climatic conditions, led to an upsurge in Zika virus (ZIKV) infections. Here we show that, after controlling for climatic and socioeconomic conditions, earthquake severity was associated with incident ZIKV cases. Pre-earthquake mean maximum monthly temperature and post-earthquake mean monthly pressure were negatively associated with ZIKV incidence rates. These results demonstrate the dynamics of post-disaster vector-borne disease transmission, in the context of conducive/favourable climatic conditions, which are relevant in a climate change-affected world where disasters may occur in largely populated areas. It has previously been shown that environmental characteristics, especially temperature and humidity are associated with mosquito life cycle length3, malaria extrinsic incubation period3, dengue incidence5, emerging infectious diseases6 and other vector-borne disease indicators. Briefly, within an appropriate range, increasing temperatures are associated with shorter mosquito life cycles2 that can lead to increased vector-borne disease transmission. However, excessively high temperatures might actually negatively impact vector-borne disease epidemiology7. In addition, mosquitoes tend to breed in natural or artificial water containers/bodies2, which may abound in environmentally-disrupted post-disaster scenarios8. Therefore, the unfortunate occurrence of disasters in the context of appropriate temperature and/or humidity/rainfall/pressure may represent the “perfect storm” that will lead to an increase in the public health burden of vector-borne diseases. In this study, we evaluated the effect that a recent powerful earthquake, in the context of the local climatic conditions, had on the reported number of ZIKV cases. The tropics of Ecuador and its recent earthquake experience represent a natural experiment to study this phenomenon.Anthropogenic climate change has led to changing global temperatures, which may be associated with changes in rainfall as well as other important climatic variables like humidity and pressure9. It resulted in about 700 deaths and 30,000 injuries, internally displacing an estimated 73,000 people11. Though it was felt nationwide, the earthquake affected two coastal provinces, Esmeraldas and Manabí, the hardest11.In April 2016, Ecuador experienced a massive 7.8 M earthquake along its unstable Nazca and South American plates—the strongest seism in almost four decades15. The 2010 earthquake in Haiti resulted in both immense structural damage and a large cholera outbreak15. The massive damages in the aftermath resulted in a devastated infrastructure15. People were displaced and crowded into unsanitary camps after losing their homes, where they lacked clean water and sanitation16. This allowed for cholera to spread rapidly via contaminated water supplies15. Furthermore, after the earthquake, malaria became an even greater public health concern in Haiti18. Unintentional water collection in building remnants and debris may provide a conducive environment for mosquito breeding19. Similar breeding grounds have been reported in partially built structures in Malaysia20, which may, in this context, fulfil the same vector-breeding function that debris play in post-disaster scenarios. In addition to unintentional water collections, abandoned and unmaintained pools, especially in lower socio-economic communities, can provide breeding grounds for mosquitoes23.Natural disasters, like earthquakes, are often associated with or followed by serious public health consequences such as increased risk for communicable diseases, including waterborne and vector-borne diseases11. Just as in Haiti, post-earthquake scenarios might have led to altered ecological environments conducive to mosquito proliferation, including Aedes spp. (the main vector for Zika virus), causing increased mosquito populations25. Such environments could be associated with rapid and unplanned resettlement, worsening of hygienic and environmental conditions due to significant destruction of water and sanitation infrastructure25. In addition, displaced populations may be at increased risk for vector-borne diseases due to behavioural exposures (i.e. people are forced to spend more time outdoors) as well as housing conditions (i.e. living in temporary shelters without window screens)26. Other post-earthquake/post-disaster conditions that could lead to increased vector-borne disease transmission include landslides, deforestation, river damming, and rerouting of natural water flows26. These populations are also at risk for an increase in psychological disorders and substance abuse as found in post-disaster situations like Hurricane Katrina27.After the earthquake in Ecuador, several health conditions, including acute respiratory infections and diarrhoea, were reported29 is transmitted by Aedes spp31. Aedes aegypti, Zika’s main vector30, is widely distributed in the tropical lowlands (i.e. coast and Amazon basin) of Ecuador. For instance, a recent study reported the presence of Chikungunya virus (CHIKV) and ZIKV from field collected female Ae. aegypti in samples from the cities of Esmeraldas , Portoviejo and Manta (Manabi province) in the coast of Ecuador32. Other cities where Ae. aegypti has been reported include Machala 35 Duran 36, Borbón 37. Indirect evidence of Aedes aegypti distribution is provided by the epidemiological profile of the diseases it transmits. According to the Ministry of Health (MoH) of Ecuador, coastal and Amazon basin provinces reported the highest number of cases for Dengue in year 201638. Finally, a recent study showed that the present-day risk of arbovirus transmission is highest in the coast and Amazon basin39.Zika virus (ZIKV), a flavivirus that was relatively unknown until the past decade29. The spread of the virus has been a cause for concern among international health authorities, as it has been found to be associated with neurological manifestations in both newborns and adults29. These neurological complications include microcephaly in infants and Guillain–Barré syndrome (GBS) in adults29. Responding to these concerns, on February 1, 2016, the World Health Organization declared the Zika-associated neurological complications to be a Public Health Emergency of International Concern (PHEIC)40.Zika virus was first isolated from a rhesus macaque in the Zika forest of Uganda in 1947, but it has made headlines since its rapid spread into the Western hemisphere43. Since then, the number of cases has steadily increased across the nation. In this study, we explored the impact that the 2016 earthquake had on the number of cumulative incident autochthonous ZIKV cases in Ecuador.By January 9, 2016, Ecuador reported its first two laboratory-confirmed ZIKV casesAs shown in Fig. Before the earthquake Fig. , the aveHowever, this relationship was reversed in the post-earthquake period with severely affected cantons having a higher average number of incident autochthonous ZIKV cases (40.19 vs. 4.57)  Fig. .A comparison of epidemiological, socio-economic and climatic characteristics between mildly affected and severely affected cantons is presented in Table When comparing severely affected vs. mildly affected cantons, there were significant differences in all socio-economic variables studied including school years, literacy rate, poverty rate and persons per household. However, all these variables were correlated and only persons-per-household was selected to be included in the regression models since it contained the most normally distributed data. Among climatic variables, only pre-earthquake mean pressure, post-earthquake mean pressure, pre-earthquake mean humidity, pre-earthquake mean cloud coverage, post-earthquake mean cloud coverage, pre-earthquake sun hours, and post-earthquake sun hours showed significant differences between the earthquake impact groups (i.e. earthquake severity groups). In addition, post-earthquake mean maximum temperature and pre-earthquake mean rain days had a marginally non-significant association (p = 0.05 – 0.10). After correlation analysis, only (pre- and post-earthquake) maximum temperature, pressure and sun hours were selected to be loaded onto the regression models.Multivariate analyses were conducted only on post-earthquake cumulative autochthonous ZIKV incidence rates since there were no significant differences on pre-earthquake cumulative ZIKV autochthonous incidence rates Table . Our datA Multiple Ring Buffer Analysis was conducted to visualize pre- and post-earthquake cumulative ZIKV incidence rates in severely and mildly affected cantons and their distance/relationship to 50-, 100- and 300-mile buffers created around the earthquake epicentre and Muisne/Pedernales 46 at any given point after the earthquake in addition to several “unofficial” camps. Although vector-borne strategies were implemented in the official camps, the use of bed-nets  had limited impact in Zika virus transmission given the biting behaviour of aedine mosquitoes. In addition, it is possible that the unofficial camps did not have access to the same levels of vector-control strategies further increasing the risk of Zika transmission in affected populations. Moreover, it is likely that people, especially those living in displaced population camps, spend more time outdoors, which may increase chance of exposure to mosquito bites, especially if mosquitoes are breeding in the camp areas. A recent study showed that time spent outdoors, and the proportion of people spending more time outdoors in a susceptible population, may affect Zika transmission and therefore Zika epidemic size47. Similarly, these camps may have lacked the typical public utilities and sanitation that could have led to conditions like those resulting in the increase in malaria post-earthquake in Haiti17. Altogether, these conditions may lead to an increased likelihood of mosquito-susceptible host interaction  disaster. As ZIKV can be transmitted through sexual contact or blood transfusion in addition to the mosquito vector, population awareness campaigns are needed regarding the different risk factors and transmission prevention strategies1. Moreover, recent studies suggest that displacement of climatic isoclines is faster at higher latitudes, which may lead to new ecological niches and therefore novel species assemblages57. Therefore, this study provides critical information that may help prevent significant public health burden in largely populated areas of North America, Europe and Australia where increasing temperatures may fall within the range for conducive mosquito (or other vector) breeding.This study highlights the importance of appropriate vector-borne disease control following an earthquake, and possibly other post-disaster scenarios like tornado- or hurricane-affected areas, especially in the appropriate climatic context, including changing temperatures. In this natural experiment, conditions in Ecuador created the “perfect storm” for an upsurge of Zika virus cases but it may be possible that, due to changing global temperatures, similar conditions may accrue in northern and further southern latitudes where these diseases currently do not pose a significant threat but which may do so if the “perfect storm” occurs. The Intergovernmental Panel on Climate Change (IPCC) deems very likely that an overall global decrease in the number of cold days and nights with an overall increase in the number of warm days and nights has occurred with such changes also likely occurring on a continental scale in North America, Europe and AustraliaThe strengths of this study are multiple and include the fact that the data was nationally collected by Ecuador’s Ministry of Public Health’s Epidemiological Surveillance System, which relied on accurate diagnostic methods (RT-PCR) for Zika infection. In addition, our results were adjusted by socio-economic and climatic variables and used a complex approach to evaluate the impact of earthquake intensity on incident ZIKV rates. The equation for the study of vector-borne disease transmission dynamics in post-disaster scenarios is another important strength and contribution of this study. Our study was limited by the amount of available data. Only canton-level aggregated weekly totals of ZIKV cases  were available. Additional person-level data might have provided further insight on whether there were any other predisposing factors that could have contributed to a rise in ZIKV cases in addition to the earthquake. Natural disasters are unpredictable and potentially disastrous. With the understanding that post-disaster outbreaks of ZIKV, or other vector-borne diseases, are possible, especially in the context of warm(ing) temperatures which are likely to occur in largely populated regions of North America and Europe, it is important to become more vigilant in the prevention of future disaster-induced outbreaks.Ecuador is a democratic republic located in the north-western region of South America Fig. . EcuadorAedes aegypti incubation period) = April 26, 2016 + 12 days (maximum reported ZIKV incubation period) = May 8th. Therefore, all cases occurring up to EW 18 (i.e. up to May 7th) were considered pre-earthquake infections whereas all cases occurring from EW19 (i.e. from May 8th onwards) were designated as post-earthquake infections. In order to control for temporal variations, the study period spans 18 pre-earthquake weeks (i.e. until EW 18 in 2016) and 18 post-earthquake weeks (i.e. from EW 19 until EW 36 in 2016). Climatic data including average rainfall, rain days, temperature, pressure, average wind speed, humidity, cloud coverage, UV index, sun hours, and sun days were collected from an online database (data provided by https://www.worldweatheronline.com). Climatic variables were aggregated as pre- and post-earthquake averages to account for their effect on incident cases (i.e. to assess the effect of average climatic conditions during the pre- and post-earthquake periods on cumulative autochthonous incident ZIKV cases). Canton-level socio-economic data including schooling years, literacy rate, poverty rate and number of persons per household were gathered from Ecuador’s VII Population Census (available at http://www.inec.gob.ec).A natural experiment-based, ecological study was conducted to compare the number of reported ZIKV cases before and after a M7.8 earthquake registered on April 16, 2016 in Ecuador. Nationwide, ZIKV data  were collected from the Weekly Epidemiological Zika Virus Reports published by the Ministry of Health of Ecuador. The total number of cumulative incident ZIKV cases reported by canton and per epidemiological week (EW) was compiled and analysed. May 8, 2016 was used to differentiate pre and post-earthquake cases based on the criteria outlined in Equation (1) .https://earthquake.usgs.gov/earthquakes/eventpage/us20005j32#map). Briefly, MMI scale ranges from I – not felt – to X – extreme. In the aftermath of the April 2016 earthquake in Ecuador, reported intensity ranged from I – not felt – to VIII – severe. Cantons in Ecuador were classified into two groups: 1) mildly affected (n = 17), those with reported intensity between I (not felt) and V (moderately felt); and, 2) severely affected (n = 26), those with reported intensity between VI (strong) and VIII (severe).Cantons in Ecuador were classified according to earthquake impact (i.e. earthquake impact group) into severely affected or mildly affected as per the USGS Modified Mercalli Intensity (MMI) scale-based Community Internet Intensity Map . All statistical analyses were two-tailed. The population of each canton was gathered from Ecuador’s VII Population Census. Cumulative autochthonous ZIKV incidence rates  were calculated. Cumulative autochthonous ZIKV incidence rates at epidemiological week 18 and cumulative ZIKV incidence rates for epidemiological weeks 19–36 were calculated. Pre- and post-earthquake cumulative autochthonous ZIKV incidence rate comparisons by earthquake impact group were performed using the Mann-Whitney test. Similarly, bivariate analyses to compare socio-economic (4 variables) and climatic variables (20 variables) between earthquake impact groups were conducted to identify other significantly associated variables. Pearson’s correlations were utilized to identify correlated variables . Both pre- and post-earthquake measurements were used for selected variables even if only one of those measurements was chosen following bivariate and correlation analyses. Based on these considerations, the regression models included only the following variables: persons per household, pre-earthquake mean maximum temperature, post-earthquake mean maximum temperature, pre-earthquake mean pressure, post-earthquake mean pressure, pre-earthquake mean sun hours, and post-earthquake mean sun hours. In order to control for data over-dispersion, a negative binomial model was fit using SAS 9.4 software to evaluate the association between earthquake impact group and post-earthquake cumulative ZIKV incidence rate.http://www.diva-gis.org/). A vector-based model was created to represent pre- and post-earthquake cumulative ZIKV incidence rates per canton. Finally, a multiple ring buffer analysis was conducted to visually demonstrate the spatial correlation between pre- and post-earthquake cumulative ZIKV incidence rates and areas located within 50, 100, and 300 miles from the earthquake epicentre or from the main two population settlements affected by the earthquake . Ring buffer sizes were determined considering that tertiles were needed to distinguish different distances and that the distance between the Epicenter area to the farthest large city to have felt the earthquake with an intensity of at least IV  is 300 miles.Spatial models were also constructed to visualize the observed differences. Earthquake epicentre coordinates were obtained from the United States Geological Service (available at https://earthquake.usgs.gov/earthquakes/eventpage/us20005j32#origin?source = at&code=at00o5r3xd). In addition, geographic coordinates for the two main population settlements affected by the earthquake  were also obtained. Based on these coordinates, a KMZ file was generated using Google Earth Pro 7.1.8.3036. The KMZ file was imported into ArcMap 10.3 for further spatial analysis. Ecuador shapefiles were downloaded from an online server (DIVA GIS, available at The data that support the findings of this study are available on request from the corresponding author [R.I.]. The data are not publicly available due to third-party restrictions.Supplementary Table S1


Nepal was struck by a massive earthquake on the 25th April 2015 and major aftershock on the 12th of May 2015, resulting in widespread devastation with a death toll in the thousands. The burden of ocular trauma resulting from the recent earthquakes in Nepal has not been described thus far. The aim of this study was to determine the types of ocular injuries sustained in the earthquake in Nepal and its management in Tilganga Institute of Ophthalmology (TIO) in Gaushala, Kathmandu.This is a hospital-based retrospective study of patients presenting to TIO following repeated earthquake. Variables that were recorded included patients’ presenting symptoms and time to presentation, visual acuities at presentation and at follow-up, diagnosis of ocular injury and surgery performed.There were 59 cases of earthquake victims visiting TIO, Gaushala, Kathmandu from April 2015 to July 2015, with 64 affected eyes due to 5 cases of bilateral involvement. The majority of patients were from the district Sindhupalchowk , which was the epicenter of the main earthquake. The average duration between the earthquake and presentation was 13 · 9 days (range 1–120 days). Closed globe injury was most frequent (23 cases), followed by open globe injuries (8 cases). While 24 patients (38%) initially presented with a visual acuity <3/60 in their affected eye, 15 patients (23%) had a visual acuity of <3/60 on follow-up. A variety of surgical treatments were required including anterior and posterior segment repair.Immediate management of ocular trauma is critical in order to prevent blindness. Characterizing the burden of earthquake-related ocular trauma will facilitate planning for service provision in the event of a future earthquake in Nepal, or in countries, which are similarly at risk of having natural disasters.The online version of this article (doi:10.1186/s12886-017-0429-z) contains supplementary material, which is available to authorized users. On the 25th of April, a 7.8 Richter earthquake struck Nepal with its epicenter 77 km from Kathmandu. A 7.3 Richter aftershock subsequently occurred on the 12th of May, equidistant from Kathmandu [Ocular trauma remains an important cause of visual impairment worldwide. Annually, over 2.5 million Americans suffer an eye injury, and more than half a million blinding injuries occur worldwide . In NepaKnowledge of the causes of eye injuries can aid in guiding preventative strategies and optimizing management capacity. For example, implementation of seat-belt legislation and application of occupational eye protection have significantly reduced the number of ocular injuries . In the This is a hospital-based retrospective study, including all patients presenting with ocular trauma to Tilganga Institute of Ophthalmology, a major tertiary eye hospital in Kathmandu, following the major earthquake of April 25th and aftershock on 12th May 2015. This study had ethical approval by Institutional Review Committee of TIO, which is a part of Nepal Health Research Council.For each patient, a brief clinical history regarding the ocular injury and time of injury was obtained. Visual acuity was assessed using an internally illuminated standard Snellen’s chart. All the subjects underwent a detailed eye examination under a slit-lamp bio-microscope (Haag-Striet 900) with intraocular pressure measurement with Goldman’s applanation tonometry in required cases. Fundus evaluation was performed under full mydriasis with 90 Dioptre leans, or binocular indirect ophthalmoscope as appropriate. Relevant investigations such as an Ultrasound B scan, X-ray orbit and skull, Computerized Tomography scan and Magnetic Resonance Imaging were requested whenever indicated. An ophthalmologist examined all the patients and appropriate interventions, whether medical or surgical, were undertaken immediately. Data of each case including demographics, visual acuities and examination findings were recorded and analyzed  followed by Kathmandu , among many other districts . Among the total victims, 12 patients (20%) were children. Males were slightly more affected; 52% patients were male and 48% were female. The average duration between the earthquake and presentation was 13.9 days (range 1–120 days). (Table Uniocular involvement occurred in 54 cases 91.5%), with the right and left eyes being equally involved, and binocular involvement occurred in 5 cases (8.5%) , followed by open globe injuries (8 cases) and corneal ulcer (7 cases)  and surgically . A variety of surgical treatments were required including anterior and posterior segment repair  in their affected eye at follow-up, however no patient developed bilateral blindness. In contrast, previous series in Nepal have demonstrated monocular blindness in only 5–10% of patients on follow-up , even whNatural disasters such as earthquakes can pose significant challenges for countries’ health systems. Mass casualties often occur and management of patients may be complicated by concurrent damage to hospitals and transport infrastructure which can prevent patients from being able to access healthcare –3. TheseThe spectrum of ocular trauma resulting from the earthquake contrasted markedly with that of general ocular trauma cases seen in Nepal due to agriculture or domestic work , 8–13. TA further explanation for the low initial visual acuity on presentation might be delays in healthcare presentation, as the average duration before presentation was a fortnight. In contrast, 50–70% of patients presented within 1 day of injury in comparable series of ocular trauma patients in Nepal , 13. LatThere have been a few other reports on the management of ocular-related conditions in other natural disasters, which are of interest for comparison. Anecdotal reports following the major earthquake in Haiti reported frequent open globe injuries, crush injuries, ptosis and head injuries associated with cranial nerve defects . HoweverImmediate management of ocular trauma is critical in order to prevent blindness. In this study, delayed presentations were frequent and up to a quarter of patients had sustained severe vision loss at follow-up. The management of earthquake-related trauma is compounded by difficulties in patients accessing specialist healthcare, multiple other injuries and damage to transportation and hospital infrastructure. The institution of a triaging system in the casualty area might help to prioritise injuries requiring urgent attention in the event of a future mass casualty event. Characterizing the burden of earthquake-related ocular trauma will facilitate planning for service provision in the event of a future earthquake in Nepal, or in countries, which are similarly at risk of having natural disasters.


Non-pediatric trainees working in pediatrics in the UK are expected to attend newborn deliveries and provide initial newborn life support if needed. In Swindon, new junior doctors receive a 90-minute teaching session at the start of their pediatrics rotation, but the content has not previously been standardized, and it may be several weeks before a doctor attends a newborn delivery. Thus, the confidence and competence in newborn resuscitation of doctors attending deliveries can vastly vary.A standardized teaching package was developed as part of the pediatrics induction program. This includes an interactive lecture on the physiology of the newborn, skills stations, and mini-simulations to consolidate skills. This is accompanied by a program of regular neonatal mini-simulations as part of the departmental morning teaching program. These sessions allow junior doctors to practice their skills in a safe, simulated environment and reinforce the newborn life support pathway.Qualitative and quantitative feedback was sought following delivery of the induction training session. Junior doctors were asked to rate their confidence before and after the induction session using Likert scales from 1 (least confident) to 5 (most confident). Median confidence in attending term deliveries increased from 2 (range 1 - 4) to 4 (2 - 5), P=0.008. There was evidence that confidence was maintained at one month following induction.A simulation program has been successful at improving confidence among junior doctors in attending newborn deliveries. This has the potential to improve patient care and trainees’ experiences of their pediatrics placement. In the UK, junior doctors rotate through pediatric clinical posts as part of the generic foundation program and general practice (GP) specialty training. These doctors are usually expected to take on the same role as and work alongside pediatric specialist trainees in their first three years of training. Non-pediatric trainees are often required to hold the ‘delivery bleep’ which involves attending medium-to-high risk deliveries and starting emergency resuscitation if needed. Support is available from senior pediatric trainees and consultants, but this may not always be immediately available, especially outside of the normal working day where consultants are non-resident and the senior trainee may be covering multiple clinical areas.The Great Western Hospital, Swindon, is a district general hospital in the UK, with an annual birth rate of approximately 4500. The junior pediatric team currently is comprised of two foundation year-2 (FY2) trainees, eleven GP trainees, and two first-year pediatric trainees. In the past, many of the GP and FY2 trainees rotating through the pediatrics department have reported anxiety around attending newborn deliveries, and the expectation of initiating newborn life support if needed. Resuscitation of the newborn has been covered as part of the local departmental induction, but the session content has previously been variable, and as exposure to clinical situations varies between trainees, there may be several months before the skills learned are needed in real life.Simulation is increasingly being used in pediatrics and neonatology as a way to improve knowledge and skills, and prepare individuals and teams for emergency situations -3. ChallA standardized induction program was developed to help ensure all new trainees are provided the initial knowledge and skills required for competence in newborn resuscitation. The first induction session is 90 minutes in length, with the induction for subsequent groups increased to 120 minutes on the basis of participant feedback. The program includes a brief, interactive lecture on newborn physiology, skills stations using low-fidelity manikins , and mini-simulations to consolidate learning.To help trainees maintain their skills and confidence throughout their placement, a program of regular mini-simulations were developed which are run one or two times per month. Scenarios include basic airway management, meconium at birth, and a baby born with an unexpectedly low Apgar score requiring full resuscitation. Initial scenarios were piloted and adapted on the basis of feedback prior to the formal launch of the program. Ongoing mini-simulation sessions are approximately 20 minutes in length  and have been incorporated into the pre-existing departmental teaching program. This has enabled trainees to attend without an additional pressure on their time. The importance of confidentiality and a safe learning space are emphasized to encourage participant involvement.To measure the quality and impact of the training program, trainees were asked to complete feedback forms prior to induction, following induction, and one month into their placement. Verbal consent was obtained from all participants. Doctors were asked to rate their overall confidence in attending newborn deliveries (primary outcome) as well as in particular aspects of newborn resuscitation (secondary outcomes) using Likert scales from 1 (least confident) to 5 (most confident). There was also the opportunity to provide free-text comments. Participants were asked to include a three-digit identifier known only to themselves to enable matching of pre- and post-induction scoring while maintaining confidentiality. Pre- and post-induction confidence scores were compared using a Wilcoxon signed-rank test, with the threshold for statistical significance set at 0.05 for the primary outcome. For the five secondary outcomes, a Bonferroni correction was applied to account for multiple comparisons  was good to have time to practice individually and ensure we had mastered the appropriate skills" and "[I] feel much more confident but until I've actually done it I think I will still carry a degree of fear."A structured induction program, including manikin-based skills learning and short simulated scenarios, increased confidence in attending newborn deliveries among junior doctors rotating through pediatrics. Embedding regular simulation sessions within a departmental teaching program has been well-received, and there is some evidence that doctors are maintaining their confidence in attending deliveries and performing key techniques. One free-text comment highlighted some remaining anxiety following the induction session; it is hoped the regular simulation sessions help to reinforce the learning and increase confidence, reducing any on-going fear.Less than half of the doctors had attended formal pediatric resuscitation training prior to starting work, and none had received newborn-specific training. The cost of Newborn Life Support courses and the time out-of-work required to attend means it is not possible for many district general hospitals to send new staff on these formal courses. Thus training programs such as this are needed to ensure clinicians have the knowledge and skills necessary to manage emergency situations. The demonstrated increase in confidence and skills in attending deliveries and initiating newborn resuscitation has clear benefits in terms of patient care and will also improve the experience of junior doctors in the department.A strength of the induction program is that the small-group (maximum three per group) skills stations require trainees to demonstrate skills to an experienced faculty member prior to completing the session. Additionally, the regular simulation sessions are run by experienced pediatric clinicians who are able to assess techniques and provide formative feedback to junior doctors.As this is a new project, the number of participants so far has been relatively low, but the results from those who have taken part are encouraging and support the ongoing delivery and development of the simulation program. The results are potentially limited by the use of self-reported measures of confidence; it is possible that participants may over-report their confidence or that confidence may not correlate with clinical competence. However, the small-group teaching and expert feedback from skills practice and simulation scenarios help ensure that trainees are aware of expectations and, therefore, able to assess their confidence and skills appropriately, reducing the potential impact of unconscious incompetence on the results. Anonymous identifiers, where faculty are unable to identify an individual's responses, were used to reduce possible self-report bias.Completion of one-month follow-up questionnaires was 55%, which limited the interpretation of these results. Follow-up questionnaires were sent by email in an effort to improve completion, as shift patterns can make it difficult to contact individuals face-to-face. However, in future work, a dual approach will be considered to try and maximize completion rates.This program has focused on the training needs of junior doctors who are relatively inexperienced in pediatrics. However, there is also a need for other health professionals working in maternity areas to be confident in managing newborn emergencies, and good team working across professions is crucial for high-quality clinical care . It is hOur program of neonatal simulation training has increased confidence among junior doctors in attending deliveries and managing newborn emergencies. This simulation program will continue to be developed, taking into account feedback from those participating. Areas for future development include the use of an objective skills measure to provide feedback for trainees and better measure the impact of the training program. We will also seek to collaborate with other hospitals within our training region to share our good practice and look at ways to standardize training between hospitals. Other simulation programs within the department already have multi-professional involvement, and we are keen to expand this program to neonatal nursing and midwifery staff.


The benefits of mentorship on residents are well established. The current state of mentorship in General Surgery (GS) residency programs in Canada is unknown. The objectives of this study were to obtain GS residents’ and program directors’ (PD) perspectives on resident mentorship.An electronic survey was developed and distributed to all 601 GS residents in Canada. All 17 PDs were invited for telephone interviews.A total of 179 of the 601 residents responded. Ninety-seven percent (n=173) felt mentorship was important. Only 67% (n=116) identified a mentor and only 53% (n=62) reported a mentorship program. Most who identified a mentor  were satisfied with the mentorship received. Significant variations in mentorship existed between demographic subgroups and mentorship program types. Overall, residents  favoured having a required mentorship program.A total of 11 out of 17 PDs participated in the telephone interviews. The majority of PDs  were satisfied with current resident mentorship but most acknowledged that barriers exist .GS programs in Canada should ensure they are providing equal opportunities for mentorship across demographic subgroups. Programs are encouraged to examine both their program’s and their residents’ needs as well as local barriers to improve mentorship. Over the last decade, the number of applicants to surgical residencies has declined and one out of five surgical residents do not complete their training.27Our hypothesis is there is a lack of emphasis and resources placed on the development and maintenance of mentorship relationships among General Surgery residents in Canada. Thus, the objectives of our study were to obtain Canadian General Surgery residents’ and program directors’ critical perspectives on current resident mentorship with the goal of implementing or strengthening mentorship programs across the country.Ethics approval was obtained from the University of Manitoba Research Ethics Board.We adopted the most common definition of mentorship in the surgical literature, consistent with vertical mentorship: “the process whereby a more experienced, usually senior, individual (the mentor) guides the personal and professional development of someone more junior (the mentee).”30An e-mail invitation requesting participation in an online, anonymous survey on mentorship was sent to all General Surgery residents in Canada (n=601). The first invitation was sent in September 2015 followed by a two-week reminder. The survey was open for one month. Two $50 Starbucks gift cards were raffled to increase participation. An invitation to participate was included in the September and October 2015 Canadian Association of General Surgeons monthly newsletter.An e-mail invitation requesting participation in a semi-structured telephone interview was sent to all General Surgery program directors (n=17) in April 2016 with one two-week reminder. No response after the two-week reminder was interpreted as a decline to participate. Questions were not provided to program directors prior to the telephone interview. Telephone interviews were conducted by the primary investigator (M.D.) and were standardized using three questions. Interviews were recorded and transcribed. A semi-structured telephone interview was chosen to improve participation and to obtain richer data than possible with a survey.®. Test-retest and inter-observer reliability were deemed unnecessary.31An electronic survey  was creaThe Mentorship Effectiveness Scale was included in the survey.Program size was collected as a categorical variable based on number of residents  and was dichotomized using the median. Resident training level was collected as a continuous variable and dichotomized using commonly accepted definitions of a junior resident (≤2 years of training) and a senior resident (≥ 3 years of training).Statistical data analysis was performed using SPSS . Dichotomous outcomes and dichotomous/categorical predictors were compared using chi-square analyses. A factorial ANOVA with 95% confidence intervals (CI) and standard error of the means (SEM) were calculated to determine if mentorship program type influenced residents’ Mentorship Effectiveness Scale scores. Chi-square analyses were performed to determine if: 1) resident satisfaction was associated with type of mentorship program or how mentor was obtained and 2) resident preference of mentorship program type was associated with year of training or residency program size. A two-sided p-value equal to or less than 0.05 was considered significant.A total of 179 General Surgery residents (30%) completed the questionnaire .Ninety-seven percent (n=173) of General Surgery residents in Canada who answered the questionnaire felt that mentorship was important to their training, but only 67% (n=116) could identify a mentor. Significant differences in reported prevalence of mentorship among demographic subgroups existed between males and females .Of the 116 residents who identified a mentor, 115 responded to the question regarding how they obtained their mentor. Of these, 79% (n=91) obtained a mentor on their own. Men more commonly obtained a mentor because the mentor was approachable or easy to work with compared to women  . ResidenSignificant differences in reasons for not obtaining a mentor existed among juniors and seniors . CompareSixty-seven percent (n=76) of the 115 residents who identified a mentor had more than one mentor. Mentors were most frequently obtained in residents’ junior years  compared to their senior years  and clerkship . Mentors included an attending , a more senior resident , the program director  or a fellow .Of the residents who identified a mentor, 53% (n=62) stated that a formal or informal mentorship program existed in their residency program . SignifiSeventy-nine percent (n=87) of residents reported being satisfied with their mentors and 21% (n=23) felt neutral. Resident satisfaction with mentoring was not significantly associated with type of mentorship program (p=0.51) or if the mentor was assigned versus obtained by the resident (p=0.7).Significant differences in mentorship satisfaction existed between demographic subgroups. Ninety-five percent of residents with mentorship programs (n=56) felt their mentors were accessible compared to only 83% with no program . Junior residents were less satisfied with mentors’ guidance on professional and personal issues  compared to senior residents .Average Mentorship Effectiveness Scale scores ranged from 46  for informal programs compared to 48  for formal programs, with 55 representing the highest attainable score. The average score for mentors that were assigned was similar to scores if mentors were self-obtained  versus 47 , respectively). Mentorship Effectiveness Scale scores were not significantly associated with type of mentorship program or how residents obtained their mentors . The observed power for measuring Mentorship Effectiveness Scale scores by type of mentorship program was β=0.13. The observed power for measuring Mentorship Effectiveness Scale score by how mentor was obtained was p=0.05.The most common difficulties identified with mentor-mentee relationships included time constraints  and scarcity of qualified mentors . Residents with no mentorship programs felt that scarcity of qualified mentors  was more of a difficulty compared to residents with a mentorship program .Residents felt that the most important attributes of an ideal mentor include:Someone who works in General Surgery or a General Surgery subspecialty Someone who is chosen by them Someone who does not have an influence on their academic standing .Gender , age , and ethnicity/religion  were less important attributes. However, 19% of women (n=18) valued having a mentor of the same gender compared to only 7% of men .Significant differences in desired subjects for mentorship existed between demographic subgroups. Women reported wanting mentorship on professional development  and clinical confidence  significantly more commonly than men  . ResidenResidents from larger programs favored an interest based matching process compared to smaller programs . Men  more commonly favored having topics to stimulate discussion with their mentors compared to women .The majority of residents  felt that a mentorship program should be required. Mentorship program preference did not vary significantly with year of residency training (p=0.5) or residency program size (p=0.63).Eleven program directors (65%) completed the telephone interview.Only one program reported having a formal mentorship program. Four reported assigning mentors only to residents needing remediation. The remaining six programs had no mentorship program and these program directors reported being primarily responsible for facilitating resident mentorship. These same program directors felt that they were the primary mentor for most of their residents.Eighty-two percent of program directors (n=9) felt that residents were satisfied with the current mentorship situation. One program reported having a formal mentorship program in the past but discontinued it due to lack of success with assigned mentors. In particular, this program director felt mentorship was more successful if self-initiated and was contemplating starting an informal mentorship program where mentors were self-selected. The program directors felt that some residents were “falling through the cracks” since the elimination of the program. Three program directors contemplated starting a formal mentorship program, but felt that mentorship is most meaningful when it is organic.Two program directors stated that it was possible some residents were not finding mentors due to lack of a mentorship program. Another stated that it was challenging because of generational gaps and professionalism issues. Two felt overwhelmed by having to take on the role of a mentor for all their residents. One program director felt that the main challenge with mentorship is time constraints. Two program directors felt that there was a lack of resources, including dedicated time off, faculty development and financial compensation.Our study found that only two-thirds of respondents currently identify a mentor, despite almost all understanding its importance. The prevalence of mentorship in Canadian General Surgery programs appears to be higher than that reported in other studies 28% – 65%).% – 65%.2The first comparison is between men and women. Women more often reported not having a mentor compared to men. A mentorship gap among women in medicine has been previously identified.The second interesting comparison is between junior and senior residents. Our survey showed junior residents more commonly reported not obtaining a mentor because they were unfamiliar with potential mentors compared to senior residents. Mentorship is most critical during junior years as these are when burnout and attrition rates are the highest.23Another important comparison can be made between formal, informal and no mentorship programs. The importance of structured mentorship programs is highlighted in our data. Residency programs with formal and informal programs had significantly more resources for mentorship and had more active mentorship relationships compared to those with no program. Only 53% of respondents in our study reported a formal or informal mentorship program.a team of mentors who work well together and have complimentary skill sets.”45A reassuring finding in our study is that General Surgery residents’ satisfaction with current mentorship is considerably higher than all other Canadian residency programs .Our data were obtained via self-reporting and are therefore subject to bias. Residents who did not respond may be systematically different than those who responded. For example, the most common respondents may have been those who were particularly satisfied or unsatisfied with their mentor, a phenomenon known as the social desirability bias.32A response rate of 30% represents a favorable number for an online, voluntary, anonymous survey, but it may not be representative of all 601 General Surgery residents in Canada. Although our response rate is low, it is on par with or higher than similar studies conducted among residents in North America.There are variations in mentorship among General Surgery residents in Canada. Efforts need to be made to improve available resources while respecting that each resident’s mentorship requirements are unique. One possible strategy may involve standardizing mentorship through accreditation to help level the disparities that exist among residents. Stakeholders need to understand that investing the resources in mentorship today is important for training the mentors of tomorrow and will strengthen the postgraduate medical education system.25


Relaxin family peptide receptor 2 (RXFP2) is a GPCR known for its role in reproductive function. It is structurally related to the human relaxin receptor RXFP1 and can be activated by human gene-2 (H2) relaxin as well as its cognate ligand insulin-like peptide 3 (INSL3). Both receptors possess an N-terminal low-density lipoprotein type a (LDLa) module that is necessary for activation and is joined to a leucine-rich repeat domain by a linker. This linker has been shown to be important for H2 relaxin binding and activation of RXFP1 and herein we investigate the role of the equivalent region of RXFP2. We demonstrate that the linker’s highly-conserved N-terminal region is essential for activation of RXFP2 in response to both ligands. In contrast, the linker is necessary for H2 relaxin, but not INSL3, binding. Our results highlight the distinct mechanism by which INSL3 activates RXFP2 whereby ligand binding mediates reorientation of the LDLa module by the linker region to activate the RXFP2 transmembrane domains in conjunction with the INSL3 A-chain. In contrast, relaxin activation of RXFP2 involves a more RXFP1-like mechanism involving binding to the LDLa-linker, reorientation of the LDLa module and activation of the transmembrane domains by the LDLa alone. Relaxin is a pregnancy hormone with roles that differ between species, but common mechanistic actions include tissue remodelling as well as having cardiovascular effects such as vasodilation and organ protection3. The human relaxin peptide, human gene-2 (H2) relaxin, has attracted recent attention due to its successful passage through three phases of clinical trials for acute heart failure where the drug significantly reduced mortality in groups receiving recombinant H2 relaxin (serelaxin) compared to placebo4. The precise mechanism by which relaxin functions in heart failure is not known but is thought to involve numerous positive actions on vessels and organs5. INSL3 is predominantly produced in testicular Leydig cells6 and plays a vital role in testicular descent in many species7. In females INSL3 is found at lower levels than in males, but it is detectable in serum and is largely produced in the theca interna cells of ovarian antral follicles in humans8. The role of ovarian INSL3 in humans is currently unknown however animal studies have demonstrated putative roles for thecal derived INSL3 in germ cell survival9 and follicular androgen production10. In addition to its reproductive roles, INSL3 has been implicated in bone metabolism in males where low levels are associated with an increased risk of osteopenia and osteoporosis11. Both hormones are also overexpressed in a number of human cancers16.Relaxin and insulin-like peptide 3 (INSL3) are two members of the insulin superfamily of peptides that are best known for their roles in the reproductive tract of females and males respectively18. They act via their respective receptors, the relaxin family peptide receptors RXFP1 and RXFP2, which are G-protein coupled receptors (GPCR) and contain the seven transmembrane alpha-helices typical of this group. However, RXFP1 and RXFP2 are unique in that they also contain a large ectodomain consisting of a series of 10 leucine-rich repeats (LRRs) and an N-terminal low-density lipoprotein type a (LDLa) module  of the transmembrane domain (TMD) of RXFP124, thus making the LDLa module the true ligand and uncovering an interesting archetype in GPCR signalling. Other examples of GPCRs making use of tethered ligands include the protease-activated receptors (PARs), which are first cleaved to expose the N-terminal agonist that interacts with the receptor ELs26. In the case of RXFP1 and RXFP2 however, it is the binding of the peptide hormone that allows the LDLa to come into contact with the ELs and bring about the final active receptor conformation. The LRRs and LDLa module are connected to each other by a stretch of linking residues termed the linker, and this region differs in length and sequence between the two receptors  studies on a recombinant protein consisting of the RXFP1 LDLa module and linker in titration experiments with a protein displaying RXFP1 ELs revealed that they may be the point of interaction between the linker and EL2. Our current model of ligand-mediated activation therefore suggests that binding of H2 relaxin to the linker stabilizes a helix which then positions residues of the LDLa module and linker to interact with the TMD to activate the receptor.We recently showed that the RXFP1 linker is involved in ligand binding and contains a transient helical structure that is stabilized in the presence of relaxin28. Mutants were made in the linker region of full-length RXFP2 and tested for INSL3 and H2 relaxin binding and activation. Parallel studies assessed ligand interaction with a soluble RXFP2 LDLa-linker protein using NMR. Our results demonstrate that INSL3 does not bind to the RXFP2 linker but the GDxxGW is absolutely required for activation. In contrast H2 relaxin weakly interacts with the linker and also requires the GDxxGW motif for activation.In this study, we use similar methods to establish the role of the RXFP2 linker, which contains the conserved GDxxGW (where x is any residue) found in RXFP1, but lacks the proposed relaxin binding site  would be conveniently removed by chemical cleavage at the carboxyl side of the unique Met residue by CNBr. After overexpression, purification, and in vitro maturation truncated relaxin was obtained with correct molecular mass  and high yield (~5 mg mature peptide from one litre of culture broth). Truncated relaxin demonstrated equivalent potency to native H2 relaxin in cAMP activity assays in HEK-293T cells expressing RXFP1 or RXFP2 , which acts as a solubility and stability enhancement tag34. This construct is designated RXFP2(1–65) and it was recombinantly expressed and purified. 2D 1H-15N HSQC spectra of this construct, as well as the equivalent construct lacking the GB1 domain, show that the RXFP2 portion behaves identically both in the absence and presence of GB1. Nevertheless, a distinct heterogeneity was seen in the spectra, most easily observed for the indole resonance of W47  P4F with increasing concentrations of truncated relaxin. Significant chemical shift changes were observed throughout both the linker and LDLa module .A 2D ker Fig. , it stan µM Fig. . For com36. In order to explore the interaction of the RXFP2 LDLa module and the LDLa-LRR linker with the TMD extracellular loops (EL) we used the same approach that we employed within the RXFP1 system27. We designed a soluble scaffold based on GB1 onto which was grafted the partial 1st and entire 2nd exoloops of RXFP1 or RXFP2. Designated ssRXFP1 and ssRXFP2, the constructs contain a disulphide bridge between loops 1 and 2 that is essential for correct receptor structure and function37. Characterisation of the presence of structure in ssRXFP2 was done using circular dichroism (CD) spectrophotometry, which showed 23.4% helix, 34.5% strand, 10.7% turns and 21.8% unordered  and a greater percentage of strand  according to our analysis.We have previously demonstrated that chimeric RXFP1 receptors with an RXFP2 LDLa module can signal in response to relaxin, although the presence of the native linker seems essential to this activity15N-labelled RXFP2(1–65) P4F with increasing concentrations of both ssRXFP1 and ssRXFP2. Interestingly, in response to both scaffold proteins an interaction was seen most strongly around the N-terminal of the LDLa module. While small chemical shift changes occurred in the linker, residues Cys13 and Cys26 moved significantly in both experiments, as did Ser5 and Gln7 in response to ssRXFP1 , contrary to what was reported for RXFP1.The NMR titration studies for the RXFP2 linker interaction with truncated relaxin highlighted a specific interaction which involved the C-terminus of the LDLa module along with the first 8 residues of linker (Gly42 to Ile50)  as well as the observed heterogeneity in glycine resonances, suggests that the linker residues C-terminal to the LDLa module (GDxxGWxxxF) may in fact fold around to come into close contact with the N-terminus of the receptor. The structures of the LDLa module of RXFP1 (pdb: 2jm4)35 and RXFP2 (pdb: 2m96)47 have been solved in the absence of the linker. However, inspection of the structures, especially the LDLa module of RXFP2 , consisting of the LDLa module and the 32-residue linker, with a soluble scaffold displaying partial EL1 and full-length EL2 revealed a potential binding site between residues within or near the GDxxGW motif and Phe564 and Pro565 of EL227. In order to explore whether the same motif in the RXFP2 linker was also interacting with the extracellular loops a titration was performed with RXFP2(1–65)-P4F and the same RXFP1 soluble scaffold protein displaying EL2 and a shortened EL1 (ssRXFP1) as well as an equivalent RXFP2 scaffold protein (ssRXFP2). As the LDLa modules can be interchanged without a dramatic loss of signal we expected RXFP2(1–65)–P4F to interact with both scaffolds36. Indeed, titrations with RXFP2(1–65)-P4F showed interactions with the scaffold constructs, displaying two potential interaction sites, one clustered around the N terminus of the LDLa module and the other within the linker (Gly42 –His55)  Fig. . Interes48. Additionally, truncation of the relaxin A-chain leads to peptides that have concurrent drops in both binding and activation of RXFP1 and RXFP249, whereas similar truncations on INSL3 maintain RXFP2 binding but lose their ability to signal50. Furthermore, a truncated INSL3 that is missing the first 9 residues of the A-chain acts as an antagonist to INSL3 activity50, as do some B-chain only variants51. The different roles of the peptide A-chains are consistent with our previous study where we have demonstrated that the peptide B-chains have distinct binding orientations to the LRRs of RXFP1 and RXFP2 respectively41. Relaxin interacts with RXFP1 by binding of the relaxin B chain α-helix across the face of the LRR at an angle of 45° whereas the binding mode of INSL3 to the RXFP2 LRRs requires a 90° binding angle between the LRR β-strands and the B chain α-helix. This study also highlighted that relaxin is likely to bind to RXFP2 in a manner similar to the way it binds to RXFP1.The data from our study are consistent with previous studies on relaxin and INSL3 peptide analogues and RXFP1/2 chimeric receptors. Replacement of either the INSL3 A-chain or the B-chain individually with that of relaxin gives rise to poor activators of RXFP2These previous results and data from our current study lead to a proposed model of activation of RXFP2 by relaxin and INSL3 Fig. . For the2 and 85% humidity.Receptors were assayed after transient transfection into human embryonic kidney 293 T (HEK293T) cells  using lipofectAMINE 2000 (Invitrogen) per manufacturer’s instructions. Cells were grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 1% l-glutamine and 1% penicillin/streptomycin (referred to as complete DMEM) in 37 °C incubators with 5% COTM/Zeo + AmpR expression vector (Thermo Fisher Scientific) using BamHI and XhoI restriction sites. GB1-RXFP2(1–65) and ssRXFP1 were both inserted into pET15b expression vectors (Novagen Inc.) using NcoI and BamHI restriction sites and NdeI and PstI restriction sites respectively. ssRXFP2 was inserted into a pET28a vector (Novagen Inc.) between NcoI and BamHI sites. GB1-RXFP2(1–65) and ssRXFP2 were produced commercially at Genscript, NJ, USA. Single point-mutations for all mutants except RXFP2-G42A, RXFP2-G46A and RXFP2-F51A were introduced into wild-type receptor using PrimeSTAR DNA Taq polymerase (Takara Clontech) according to manufacturer’s instructions. Forward and reverse strands for mutants G42A and G46A were amplified separately and resulting single-stranded plasmids were annealed together in a further reaction (1 min at 95 °C followed by cooling to room temperature). RXFP2-F51A was made according to the protocol outlined in52. Mutant Primers are in Supplementary Table E. coli cells for subsequent DNA extraction using a Bioline Isolate II or a Promega Wizard Plus SV miniprep kit. All mutations were verified by sequencing and entire inserts were sequenced to ensure no accidental mutations occurred during the process.Wild-type RXFP2 cDNA with a bovine prolactin signal sequence was inserted in a pcDNA3.13+)-labelled INSL3 (Eu-INSL3)53 or H2 relaxin (Eu-H2)54 at increasing concentrations in the presence or absence of 1 µM unlabeled ligand. Following a 1 h incubation, media was removed and 100 µl Delfia Enhancement solution (PerkinElmer) was added to each well. Plates were incubated in low light for 20–30 mins with shaking and then read on an Omega POLARstar plate reader using a time-resolved fluorescence protocol with excitation at 340 nm and emission at 614 nm. Data from at least three independent experiments, all performed in triplicate were pooled and presented as mean fluorescent specific binding ± SEM using GraphPad PRISM 6. Experiments were further analyzed by nonlinear regression one-site binding curves and Kd values were subjected to one-way analysis of variance (ANOVA) and uncorrected Fisher’s least square difference comparison test.Comparison of ligand affinity for mutant receptors with wild-type RXFP2 was assessed using Europium  at 570 nm. All experiments were performed in triplicate a minimum of three times and data were pooled and presented as percentages of the maximum response induced by wild-type RXFP2 ± SEM. GraphPad PRISM was used to fit a nonlinear regression sigmoidal dose-response curve and resulting pEC50 and maximum response (Emax) values were subjected to one-way ANOVA and uncorrected Fisher’s least square difference comparison test using only wild-type RXFP2 that was on the same plate as any particular mutant for comparison.cAMP activation in response to ligand stimulation in HEK293T cells was measured using a colorimetric assay(1–65) was designed with a GB1 solubility tag on both the N- and C-termini as well as an N-terminal His6 tag which was removable along with the N-terminal GB1 by thrombin cleavage. The construct was expressed in BL21 (DE3) trxB (Novagen) cells using autoinduction56. N5052 medium was used for uniform 15N-labelling using 15NH4Cl (Sigma-Aldrich)56 and the method described in57 was employed for 13C, 15N labelling, with cells grown in a 1 L Braun Biostat fermenter, and 15NH4Cl and D-[13C] glucose being the only nitrogen and carbon sources. Following harvesting and pelleting cells were stored at −20 °C.RXFP2g for 40 mins and then loaded onto approximately 10 ml pre-equilibrated (with 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM imidazole) Talon Superflow resin (Takara Clontech) to be purified by affinity chromatography. Elution was done with 400 mM imidazole in 20 mM Tris-HCl (pH 7.4), 150 mM NaCl and the resulting protein refolded overnight in a mixed redox reaction with stirring , 150 mM NaCl, 10 mM CaCl2), such that the three disulfide bonds of the LDLa module should form correctly. The His6-GB1 tags were removed by overnight incubation with 5 units of thrombin (Sigma-Aldrich) per mg of protein, and a second pass over Talon Superflow resin. Flowthrough and wash fractions were collected and further purified by reversed-phase high-performance liquid chromatography (RP-HPLC)  using an Agilent Zorbax 300SB-C18 column. Purity and molecular weight of collected fractions were assessed by mass spectrometry before they were lyophilized and stored at −20 °C.Cells were resuspended in 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM Imidazole and lysed with an Avestin EmulsiFlex C3 cell crusher. The soluble fraction was separated from insoluble debris by centrifugation at 4 °C, 13,000 × 27. Briefly, after 16 h of expression at 16 °C via isopropyl –D-1-thiogalactopyranoside (IPTG) induction in LB medium after cells reached an OD600 of 0.6, cells were harvested, pelleted and purified by affinity chromatography over Talon Superflow resin (Takara Clontech). The His6 affinity tag was removed by thrombin cleavage and the proteins further purified with a HiLoad 16/60 Superdex 75 prep grade column  in 20 mM Tris HCl (pH 7.4), 150 mM NaCl. Purity and molecular mass was assessed by polyacrylamide gel electrophoresis and mass spectrometry.The scaffold proteins were expressed in BL21 (DE3) cells and purified as described for the RXFP1 scaffold protein previouslyobs)/(l.c.n/M) where θobs = observed elipticity (mdeg), l = path length (cm), n = number of amino acid residues, M = the molecular weight and c = concentration (mg/mL). Data were analyzed using the online Dichroweb server with the Selcon3 method and reference dataset 758, and compared to data previously acquired for the tGB1 protein for estimates of expected structure24.The ssRXFP2 construct had not previously undergone characterization or quality control, so it was submitted to CD measurements to ensure secondary structure was present and refolding had occurred. An AVIV Model 410SF spectropolarimeter (Biomed) was used and samples at 0.15 mg/ml were placed in a cell with a 1 mm path length for wavelength scans to be performed from 260–190 nm at 1 nm intervals. Buffer used was 20 mM Tris HCl (pH 7.4), 100 mM NaCl. Background (buffer only) readings were subtracted and data were expressed as mean residue elipticity  according to the equation [θ] =  and the N-terminally shortened precursor purified by high performance liquid chromatography (HPLC) using a C18 reverse-phase column . Finally, the shortened precursor was sequentially treated with endoproteinase Lys-C, papaya glutaminyl cyclase, and carboxypeptidase B and the mature two-chain relaxin purified HPLC using a C18 reverse-phase column . After lyophilisation, its purity and molecular mass was confirmed by mass spectrometry. The peptide was tested for its activity at both RXFP1 and RXFP2 in HEK-293T cells overexpressing the receptors and a pCRE reporter gene as described above23.A B-chain truncated analog of H2 relaxin with lysine residues at position 9 on the B-chain and positions 9 and 17 on the A-chain replaced by arginine  was produced in 2 at pH 6.8. Proteins for titrations were dialyzed in the same buffer and same vessel overnight. Backbone resonances  of residues were assigned from 3D HNCACB, HN(CO)CACB, HNCO and HN(CA)CO experiments using non-uniform sampling (NUS) on a Bruker Avance II HD 800 MHz spectrometer equipped with TCI cryoprobe at 298 K. For NUS, sampling schedules were generated using poisson gap sampler with 10% of the total number of points collected for all the 3D NMR experiments60. Spectra were reconstructed with compressed sensing algorithm using qMDD61 and processed using NMRPipe62 as described in27 and data analyzed in SPARKY . Samples for titrations of truncated H2 relaxin, ssRXFP1 and ssRXFP2 to RXFP2(1–65) were dialyzed in the same buffer as RXFP2(1–65). Chemical shift and intensity changes were monitored via the acquisition of 2D 1H-15N Heteronuclear Single Quantum Coherence  as described for RXFP1(1–72) in27, using increasing concentrations of each titrant against 50 µM RXFP2(1–65). 2D 1H-1H NOESY and TOCSY of H2 relaxin and truncated relaxin were acquired on a 600-MHz Bruker Avance HDIII spectrometer at 25 °C. Samples were 500 µM, pH 6.8.NMR experiments were all performed at 25 °C on a 700-MHz Bruker Avance HDIII spectrometer equipped with triple resonance cryoprobe, with proteins suspended in 50 mM imidazole, 10 mM CaClSupplementary information