A major reason for poor childhood vaccine coverage in developing countries is the lack of awareness among parents and caregivers regarding the need for immunization and the importance of completing the entire series of vaccines. Short message service (SMS)–based interventions have been quite effective in different programs such as smoking cessation, treatment adherence, health care scheduled appointment attendance, antenatal care attendance, and compliance to immunization. However, there are limited data from low- and middle-income countries on the role of SMS and automated call–based messages and interventions to improve routine immunization (RI) coverage.The primary objective of this study is to evaluate whether automated mobile phone–based personalized messages  can improve RI uptake at 6, 10, and 14 weeks of age per the expanded program immunization schedule, compared with a usual care control group. Secondary objectives include assessing the effects of different types of automated SMS text or calls on RI coverage at 20 weeks of age.This is a mixed methods study using a clustered randomized controlled trial with 4 intervention arms and 1 control arm, augmented by qualitative interviews for personalizing the message. The study is being conducted in Pakistan . In Karachi, 250 administrative structures are taken as 1 cluster, whereas in Matiari, a catchment area of 4 Lady Health Workers is considered as 1 cluster. The intervention targets families to receive weekly 1-way or 2-way (interactive) personalized automated SMS or automated phone call messages regarding vaccination. Possible barriers to vaccination are assessed in each family at the time of inclusion to determine the type of personalized messages that should be sent to the family to increase the chance of a positive response. Finally, in-depth interviews using purposive sampling are conducted before and after the trial to determine the family’s vaccination experience and related factors.All study participants for the cluster randomized trial were enrolled by January 14, 2019. Study exit interviews at 20-weeks follow-up visits will be completed by June 2019.The results of this study will be useful to understand the respective effects of SMS text messages versus automated phone–based communication to improve RI coverage and timelines. Moreover, information regarding families’ perceptions of vaccination and the daily life challenges for timely visits to the vaccine clinic will be used for developing more complex interventions that use mobile phone messages and possibly other approaches to overcome barriers in the uptake of correct and timely immunization practices.ClinicalTrials.gov NCT03341195; https://clinicaltrials.gov/ct2/show/NCT03341195 (Archived by WebCite at http://www.webcitation.org/78EWA56Uo)DERR1-10.2196/12851 Furthermore, 75% and 86% of children had received the third dose of the pentavalent and polio vaccines, respectively, due at 14th week and measles vaccination was 73%, which is due at 9 months. However, these rates are at 1 year of age and much higher than vaccination coverage rate at scheduled time and among conflict hits and displaced populations [Pakistan is one of the countries with the highest rates of child death in the world . It rankAccording to immunization coverage surveys, 1 in 5 children is unimmunized . These bMobile phones offer a new medium to provide education and advocate families or caregivers to enable behavior change so as to improve immunization uptake. Mobile phone use has also increased in countries with low RI coverage and a high risk of VPDs. Good examples are Nigeria and Pakistan, where there were around 170 and 140 million mobile phone subscribers, respectively, in 2014 . There iMobile reminders in the form of phone calls have also proven to be a feasible method of improving RI uptake in resource-limited settings with wide cellphone coverage . A CochrThere are limited data from LMICs set up on the role of SMS-based interventions for improvement of RI coverage, and conventional 1-way reminder SMS text messages were used by most of the studies as the intervention ,21,22. OIn this study, we will identify the factors that affect adherence to RI coverage among Pakistani families and caregivers and will examine an important public health question—do low cost, automated SMS text messages and calls improve RI coverage in resource-constrained settings such as Pakistan? In this study, we will compare the effectiveness of different types of messages: reminder, educational, and interactive SMS text messages and automated calls for improving RI uptake. Different types of messages will be developed to meet the possible RI barriers identified; these messages will be sent specifically to the participants according to the type of RI barriers they faced for immunization. This information will be used to develop strategies to improve vaccine adherence in Pakistan.Our first objective is to evaluate whether personalized automated SMS text messages, 1-way versus 2-way, can improve on-time visits at 6, 10, and 14 weeks of age for RI as compared with standard care. Our second objective is to evaluate whether personalized automated calls, 1-way versus 2-way, can improve on-time visits at 6, 10, and 14 weeks of age for RI as compared with standard care. Finally, our third objective is to compare the efficacy of automated SMS text messages versus automated calls on increasing vaccination uptake.Our secondary objectives are to learn the perception and attitudes of caregivers regarding childhood vaccination and to find out about factors that might influence mobile phone and SMS text–based interventions for vaccination improvement.Our target population are caregivers of newborn (NB) infants younger than 14 days who are due for their RI at age 6, 10, and 14 weeks according to the EPI schedule of Pakistan.Our goal is to identify the barriers faced by families in the uptake of correct immunization practices and to develop adapted messages  to improve vaccine adherence in Pakistan. We will collect information from families regarding perceived sociocultural, technical, and economical barriers that may explain the decrease in vaccine coverage, and possible solutions to overcome these constraints. This information will be used to develop personalized educational messages, reinforced by interactive exchanges  with caregivers in the initial 20 weeks of their child’s life.Personalized weekly message in the form of automated SMS text message or automated calls according to barriers for RI and language preferences can improve RI uptake according to the schedule for vaccine due at age 6, 10, and 14 weeks as compared with standard counseling by health care providers at EPI center and outreach visits for RI. Interactive voice recording (IVR) or 2-way auto calls might be preferred way of communication to caregivers as compared with other intervention and control for possible improvement in RI coverage and timelines.Primary outcomes include: (1) to see a 10% increase in RI through personalized automated mobile phone–based communication  at 6, 10, and 14 weeks of age according to the EPI schedule versus standard care; (2) to see a 10% increase in RI within 1 week of the original timeline at 6, 10, and 14 weeks versus standard care; (3) to compare coverage rates of personalized 1-way versus 2-way SMS text messages on improvement in RI at 20 weeks of age; (4) to compare coverage rates of personalized 1-way versus 2-way automated calls on improvement in RI at 20 weeks of age; and (5) to compare coverage rates of personalized SMS text messages and automated calls on improvement in RI at 20 weeks of age. Secondary outcomes include: (1) to understand the perceptions and barriers of caregivers regarding immunization and (2) to understand the perception of caregivers related to personalized mobile phone–based SMS text messages and automated health messages for vaccination improvement.The study design is a mixed method clustered randomized controlled trial (RCT) augmented by qualitative interviews . The cluThe Department of Paediatrics and Child Health at the Aga Khan University (AKU) conducts active health demographic surveillance systems (HDSS) at several urban sites in Karachi. These sites are (1) Ibrahim Hydri Goth, (2) Ali Akber Shah Goth, (3) Rehri Goth, and (4) Bhains colony, which is part of Bin Qasim Town . Ibrahimstructure is defined as a building with a single entrance and a boundary. These structures can be houses, hospitals, dispensaries, schools, shops, parks, etc. Each structure has a unique number assigned to it. The total population of the active surveillance catchment area is approximately 0.3 million, with around 7115 pregnant women and 6831 NBs being followed annually by the surveillance team. Within 1 block, the 250 administrative structures will be our trial clusters; 4 sites will be participating in the study. NBs will be enrolled from the HDSS, and clusters within the 4 sites will be randomly assigned to the study arms. Hence, participants within each administrative structure  will receive same intervention.The HDSS sites are divided into 195 blocks, each containing 250 structures and mapped using the geographical information system. There are a total of 42,093 structures where 43,098 households are living in areas of Korangi and Bin Qasim towns [Data will also be collected from the rural site of Matiari, which is located in Sindh province 185 km north of Karachi . The depA total of 50 interviews will be conducted from Karachi and Matiari sites . From the HDSS, list of caregivers who have completed the vaccination schedule and those who are dropouts or not vaccinating their child was taken out, and randomly, caregivers will be selected for in-depth interviews. Trained staff will conduct the qualitative interviews. Depending upon the availability, caregivers will be approached at the household, and appointments will be taken from the caregiver so that they can spare enough time for the discussion. Through in-depth interviews, we will explore the perceptions and barriers related to EPI and mobile health from the caregivers. Information collected before the study through qualitative in-depth interviews will help in developing the survey to assess the different categories of barriers. The baseline survey will, overall, collect information on demographics, mobile phone access and usage, possible barriers for RIs, and factors associated with mobile phone–based health messages.Initially, in-depth interviews with a subpopulation in the study catchment area will be conducted to explore RI immunization coverage among Pakistani caregivers and families to assess (1) perceptions regarding risks of infectious diseases preventable by vaccines and vaccine safety and efficacy, (2) barriers to vaccinating children including difficulties in visiting RI centers, and (3) perceptions and barriers that may affect mobile phone–based interventions to improve immunization coverage. The interviews will be conducted across the site regions. We will use purposive sampling to assure that participants represent different ethnic groups. Information gathered through the interviews will help in (1) understanding the types of barriers perceived by caregivers, (2) designing the RCT and, (3) developing content for SMS text messages in several categories of barriers. We expect 20 to 25 parents or caregivers to be interviewed over a period of 3 months, but the number will be guided by saturation of information. Each interview will take 45 to 60 min.The study design is a cluster randomized clinical trial including 4 intervention and 1 control arm .Assumptions used for calculating sample size are as follows: increase in coverage rate from 30% to 40%, having a power of 80% with an alpha error at .05. The sample size per arm would be 615 per arm or 3075 for all 5 arms. Adding a 10% dropout will make it 677 per arm or 3385 recruitment for all 5 study arms. The ICC is taken as .05, with an estimate of 15 NB per cluster see .The inclusion criteria include being a child from the HDSS site, being younger than 14 days with a parent or guardian or at least 1 person in the household, having a working mobile phone connection, and parent or guardian providing consent to participate in the study.The exclusion criteria include a child from outside the HDSS area or if the family plans to stay in the catchment area for less than 20 weeks.The sampling strategy adapted for both sites are as follows .The data for live births and population of each site in Karachi will be obtained from HDSS team. Data will be scrutinized and divided according to 4 sites in the study, with each site consisting of 5 arms (5 clusters). Administrative structures will be assigned to each intervention through stratified  randomization.As Matiari site has not been stratified into clusters according to HDSS, we randomly allocated 30 clusters per arm. The catchment area of 4 LHWs will be considered as 1 cluster. Each intervention arm will then be randomly allocated to 24 administrative structures , a random selection will be made by a program designed in the mobile phone device. After meeting the eligibility criteria, informed consent will be obtained, and the infant will be enrolled in the study and a baseline survey will be conducted.A second survey will be conducted at 20 weeks of child’s age (end of follow-up) to identify vaccination coverage according to the schedule, to be confirmed by physical examination of EPI card see .The intervention arms in addition to the standard counseling will include receiving personalized 1-way or 2- way (interactive) automated SMS or 1-way or 2-way (IVR) automated phone call messages regarding vaccination. In personalized 1-way messages and automated calls, parents will receive educational or reminder or proactive messages related to RI once a week till the child turns 20 weeks. SMS and automated phone calls will contain the same information content and interactive features, only the delivery method will differ. In the interactive arms, in addition to personalized weekly educational or reminder or proactive message, parents will have the option to reply to messages or calls and receive more information related to immunization through SMS text messages or calls see  and 2. TThe control group will receive 1 time standard verbal counseling at the time of initial visit for on-time EPI vaccines at 6, 10, and 14 weeks of age as recommended by EPI, government of Pakistan.Interviews will be conducted after the trial, with a purposive sample of a subpopulation included in the RCT representing all study arms. From these structured in-depth interviews, we expect to further explore participants’ experience of the different study arms having different levels of coverage. We will also assess the factors related to SMS text messages and automated calls associated with vaccination uptake. Sample size will be guided by saturation of the information.failure (conservative approach). Missing the vaccine EPI card to verify vaccine status at home will lead to a deeper investigation in the family and also the vaccine clinic. From previous experience, we expect this process to be successful in identifying the outcome of over 95% (95/100) of the children who remain in the study at 20 weeks. Families that have left the study for any reason  will be interviewed (if possible) and the information gathered used for the outcome classification. If the information is not clear or incomplete, the child’s status regarding vaccination will be failure.Analyses will be conducted according to the intention-to-treat principle. When the information cannot be gathered, the vaccine status of the child will be The main analyses will be comparing our 4 study intervention arms against the control, and with each other. For the purposes of our study, we will compare each of the 4 interventions with each other and with the control arm. For example, 1-way versus 2 SMS, reminder versus educational SMS, 1-way reminders SMS versus control, 1-way educational SMS versus control, 2-way reminders SMS versus control, 2-way educational SMS versus control, 2-way education versus 1-way reminder, and 2-way reminder versus 1-way educational.Analyses will be conducted according to the intention-to-treat principle, and the Bonferroni correction will be used to control for multiple testing. The primary outcome is to assess difference in vaccination status. Chi-square test will first be used to compare the groups’ percentage. Multivariable logistic regression analyses will be conducted to adjust for confounders. As our randomization unit is cluster-based rather than individual-based, generalized linear mixed effects models or generalized estimating equations models will be employed to account for within-cluster correlation, and hierarchal models will be used. The statistical model for the primary and secondary analyses will be developed as explanatory variables. As secondary analyses of the primary outcome, time-to-immunization curves will be constructed using the Kaplan-Meier method and Cox regression for multivariate analysis.In the first section of the semistructured interview guide, we will ask the caregivers regarding common barriers they encounter at the time of RI, whereas the second section comprises perceptions and barriers related to a mobile phone. The data will be transcribed into written form from audio recordings and will be analyzed via qualitative data analysis software NVivo 11 . Written transcripts will then be uploaded into NVivo 11 software to offer easy and organized retrieval of data for analysis. The data analysis will be conducted according to discourse analysis. The interview guides will also be pretested in a similar communityThe audio recording and transcripts will have a unique identifier; original and backup files will be archived in password-protected computer or server at AKU. Only transcriptions and themes will be shared at University of British Columbia (UBC) without nominative information. All study-related data including the recording will be stored in an encrypted server with password protection and having access only to the study specific personnels.Baseline and end line data will be collected on a smartphone device  and 5. TThe study protocol and associated study instruments, including consent forms in English and local language, were approved by UBC’s and AKU’s Ethics Review Committee before commencement of any study activities. The study will be conducted in accordance with the Helsinki declaration and established guidelines. All participants will be administered informed consent before participation. Participants have the right to refuse to participate in the study or leave the study at any time; this will not affect any services provided at the health center. Data confidentiality will be maintained at all times. No personal identifiers will be used in any reports or publication of the study.Initial training of the study staff related to study protocol, SMS text messages and phone calls will be conducted. This will be followed by refresher training in the middle of the study.We recognize that not all barriers identified are modifiable and amenable to phone calls and SMS text messages. However, most of them reflect a priority ranking that does not favor child routine vaccination. One major reservation about SMS-based interventions is the recipient’s level of literacy. However, there has been mixed input related to the preference of phones calls as compared with SMS text messages in populations of low literacy and resource-constrained settings ,27. MobiThe type of intervention will not be blinded, but all families in the same cluster will receive the same intervention. The primary outcome (vaccine completion) is not likely to be influenced by any knowledge of the intervention arm.The baseline survey was conducted from July 2018 to January 2019 where it was found that 97.9% (3797/3877) of care givers either owned or have a sharing access to a working mobile phone. A total of 50 IDIs were conducted before the start of the study, that is, 25 interviews per site, based on which the content of the SMS text messages and automated calls were developed. FGDs with caregivers and stake holders were then conducted to validate the developed content. The RCT enrollment was completed on January 31, 2019, and the participants continued receiving messages till the child was 20-week old. Currently, the study is in its final phase and expecting completion of exit surveys and interviews by June 2019.This study is the first one of this type to assess the efficacy of different types of SMS text messages and automated calls messages, with or without interactive messages in LMICs. Different types of SMS text messages and automated calls messages will also be personalized based on the identification of families’ possible concerns or challenges regarding RI. Each family will receive one weekly message, and we expect families to talk to each other about the messages within the demographic unit, therefore facilitating circulation of information (this aspect will be assessed through the end-of-study interview). This information will be extremely useful to understand the effect of different types of messages in different contexts.The qualitative aspect of our study will generate useful information regarding family’s perception of vaccination and the daily life challenges for timely visits to the vaccine clinic. This information will be used for developing more complex interventions that use mobile phone messages and possibly other approaches to overcome some of the barriers. Finally, conducting this study in Pakistan will generate results that will be useful in most LMICs worldwide.Representatives of public health, Ministry of Health, and community leaders will be part of the study steering committee to ensure transparency, direct communication, and shared decision making as part of on-going knowledge transfer strategy. We will also inform major policy makers, donors, and mobile phone service providers. Study findings will be presented at and published in a scientific journal and national and international scientific and programmatic forums.


Testing angiogenic potential and function of cells in culture is important for the understanding of the mechanisms that can modulate angiogenesis, especially when discovering novel anti- or pro-angiogenic therapeutics. Commonly used angiogenic assays include tube formation, proliferation, migration, and wound healing, and although well-characterized, it is important that methodology is standardized and reproducible. Human endothelial progenitor cells (EPCs) are critical for post-natal vascular homeostasis and can be isolated from human peripheral blood. Endothelial colony forming cells (ECFCs) are a subset of EPCs and are of interest as a possible therapeutic target for hypoxic diseases such as kidney disease, as they have a high angiogenic potential. However, once ECFCs are identified in culture, the exact timing of passaging has not been well-described and the optimal conditions to perform angiogenic assays such as seeding density, growth media (GM) concentrations and end-points of these assays is widely varied in the literature. Here, we describe the process of isolating, culturing and passaging ECFCs from patients with end-stage renal disease (ESRD), aided by image analysis. We further describe optimal conditions, for human bladder endothelial cells (hBECs), challenged in angiogenic assays and confirm that cell density is a limiting factor in accurately detecting angiogenic parameters. Furthermore, we show that GM along is enough to alter the angiogenic potential of cells, seeded at the same density. Lastly, we report on the success of human ECFCs in angiogenic assays and describe the benefits of live-cell imaging combined with time-lapse microscopy for this type of investigation. Angiogenic assays are commonly used to determine the ability of specific compounds to promote or inhibit angiogenesis. In kidney disease, the discovery of agents that can restore microvascular circulation after injury is highly sought after, as the common end point of all kidney injury is fibrosis resulting in decreased oxygen perfusion and blood flow loss to the kidney. Whilst angiogenesis is stimulated in the early stages of kidney disease, there is a rapid switch to a hypoxic state which denudes the angiogenic processes , which can be achieved via an established culture method  who were dialysis dependent were recruited from Monash Medical Centre  and participated in this study under informed consent. Patients were excluded from the study if their original diagnosis of ESRD was type I/II diabetes, or who were on antibiotics or had a recent infection or inflammatory flare-up.All human studies were approved by the Monash Health Human Research Ethics Committee (CF16/402 - 2016000182), which conforms to the National Statement on 6/well) coated with human fibronectin  and maintained in Endothelial Growth Medium  as previously described . Once a mature phenotype was reached colonies were passaged as previously described  to determine if an ECFC colony had differentiated. This was determined by a change in morphology and rapid proliferation of cells and occurred anywhere between 7 and 10 days  and used in all assays to determine the optimal number of cells and growth media concentration in tube forming assays. These cells were shipped at passage 3 and were negative for viruses and microbes with a doubling time of ~20 h and a 20% seeding efficiency.4, 7.5 × 104, 5 × 104, 2 × 104, 0.5 × 104, and 0.2 × 104 in triplicate in 100 μL of 100% GM. Time-lapse microscopy was utilized on the LX AF6000 microscope (Leica) imaging the cells every 5 min for 6 h. The microscope was enclosed by a custom made perspex housing and contained a custom made lid  in 96-well-plates  as previously described .Additional experiments were conducted in order to determine the optimal GM concentration, which allowed cells to show either an increase or decrease in tube formation ability based on the angiogenic stimulating or inhibiting factors added to the culture media. This was performed as described above with 0.5 × 104, where MSCs were plated with the hBECs at a 1:4 ratio. A macro was written in the langue of FIJI to allow images at specific timepoints to be merged from multiple channels and saved over the whole 7 h experiment. For consistency, the number of cells per well in the hBEC-MSC co-culture experiment was kept at 0.5 × 104, where MSCs were plated with the hBECs at a 1:4 ratio.In order to determine if time-lapse microscopy could capture cell-to-cell interactions using multiple channels over extended time periods, hBECs were placed in tube formation assays with mesenchymal stem cells (MSCs) expressing the enhanced green fluorescent protein (eGFP). MSCs were purchased from the Tulane Centre for Stem Cell Research and Regenerative Medicine  and cultured as described previously  cells were imaged at regular intervals over an extended period of time  and growth media concentration (25%) were determined with hBECS. ECFCs were characterized as mature endothelial cells as according to their cell surface markers by both flow cytometry  were also challenged in tube forming assays using the optimal conditions as determined using hBECs. Patent ECFCs were therefore seeded density of 0.5 × 104 cells per well in a 96 well-plate with 25%GM with or without the addition of MSCs. Time-lapse microscopy was used as described above to capture images, which were analyzed using the macro describe above.Human derived ECFCs . The number of junctions (p < 0.05) and obvious. In a similar fashion, the number of branches (p < 0.05). At 3 h 50% GM did have significantly more mesh formation compared to 25% (p < 0.05) and 1% (p < 0.05) GM however this was reversed at all other subsequent time points Representative images and subsequent quantification of these images using angiogenesis plug-in confirmed that 100%GM significantly increased tube length, and number of, junctions, meshes at all time points analyzed compared to basal media .Results from the analysis of tube formation over 6 h also demonstrated that tubes had a maximal branching time, after which they start to retract away from each other . This waHuman derived ECFCs were confirmed as mature endothelial cells through the localization of specific cell surface proteins by both flow cytometry and immunohistochemical investigation. Patient cells maintained a mature endothelial cell phenotype at passage 2  as demonPatient derived ECFCs were also challenged in tube forming assays at different growth medium concentrations with or without the addition of MSCs and compared to hBECs . Time-laThis study provides an unbiased, imaged-based method to determine the optimal timing of ECFC passaging, when isolated from small volumes of blood from ESRD patients. We furthermore recommend the culture conditions for consistent tube forming assays utilizing continuous live cell imaging combined with time-lapse microscopy. Subsequent analysis of 20 angiogenic parameters was conducted in an unbiased and reproducible way using image analysis software FIJI, an open source image processing package.The starting volume of blood is an important parameter to report, as the number of circulating progenitors will increase when larger volumes of blood are obtained. Previous studies have successfully isolated ECFCs using volumes of blood ranging from 15 to 40 mL and from a variety of sources, including cord blood  with subsequent analysis of colony size easily determined using the macro described.ECFCs are identified in culture according to their cobblestone morphology, high proliferative capacity and ability to be passaged. Optimal ECFC passaging after initial culture from the PBMC fraction of blood has previously been reported as day 14  to patients with renal cell carcinoma (87.8%) (Gu et al., in vitro assays such as tube formation is important for understanding endothelial cell function and mediators of angiogenesis. Currently, there is no consensus or standardization for performing tube forming assays, therefore the optimization of tube forming assays will ensure accurate and reproducible results. By utilizing a microscope that replicates an incubator, with controlled temperature, pH, and humidity, in conjunction with the ability to revisit multiple points and automatically capture images, time-lapse microscopy can be a powerful tool to analyze data at multiple time-points over an extended period of time for numerous assays, including tube formation (Jonkman et al., Measuring and quantifying the angiogenic potential of cells utilizing 4 cells in a 96 well-plate resulted in tube formation that could not be quantified in a consistent manner using the angiogenesis plugin for FIJI. Furthermore, comparison of tube formation at 1 and 6 h demonstrated a significant decrease in all parameters measured, indicating that these assays decline as early as 6 h, which is earlier than the reported 24 h where significant apoptosis occurs disrupting the tubes (Francescone et al., Current reports show the method of performing tube forming assays varies according to plate size, cell number, time of assay and end-point determinants (Choi et al., 2 incubator, overcomes these limitations. Furthermore the use of the angiogenesis analyzer plug-in available for FIJI (Carpentier et al., Most studies that investigate the tube formation ability of endothelial cells reference one time point, usually over extended periods of 12 h (Gulati et al., We were able to show that hBECs and patient-derived ECFCs were able to undergo tube formation, and, contrary to our hypothesis that MSCs would increase the angiogenic potential of cultured cells, MSCs contributed little to angiogenesis. It was noted, however, that MSCs act as an anchor, rather than contribute directly to total tube length, and sit at junction points over the course of the tube forming experiment. This is surprising as the angiogenic properties of MSCs and MSC-derived exosomes are well-documented (Gong et al., ex vivo is not fully elucidated. In summary, we describe analysis macros, in conjunction with time-lapse microscopy as a useful tool that can be used to correctly identify ECFCs ready for passaging to increase culture success. Additionally, we provide evidence that cell seeding densities and media concentration and length of assay are important considerations when performing tube assays on Matrigel. Further, we demonstrate that these culture conditions can be used to demonstrate tube formation in patient derived cells.EPCs and ECFCs may hold promise as a therapy for vascular disease, however, the optimal culture conditions and measurement of angiogenic potential of these cells This study was carried out in accordance with the recommendations of Monash University Human Research Ethics Committee with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Monash University Human Research Ethics Committee, Project number CF16/402 – 2016000182.SR and CS contributed to intellectual experimental design and funding. BH performed the research and analyzed the data. RD performed and analyzed flow cytometry data. PK coordinated patient sample collection and ethical approval. All authors prepared the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CREBBP, WDR24, ARL8A, PHLDA3, LAD1) that putatively influence quantitative variation in red-brown colouration in chickens. By treating colour as a quantitative rather than qualitative trait, we have identified both QTL and genes of small effect. Such small effect loci are potentially far more prevalent in wild populations, and can therefore potentially be highly relevant to colour evolution.Plumage colouration in birds is important for a plethora of reasons, ranging from camouflage, sexual signalling, and species recognition. The genes underlying colour variation have been vital in understanding how genes can affect a phenotype. Multiple genes have been identified that affect plumage variation, but research has principally focused on major-effect genes , rather than the smaller effect modifier loci that more subtly influence colour. By utilising a domestic × wild advanced intercross with a combination of classical QTL mapping of red colouration as a quantitative trait and a targeted genetical genomics approach, we have identified five separate candidate genes ( The genetics of mammalian coat colour has been studied extensively, with many genes of major effect identified4. Feather colour is more complex than mammalian hair colour, with multiple colours and patterns possible on a single feather (intra-feather patterning)5. Similarly, patterning may also be present more broadly across distinct body regions5. The evolutionary importance of feather colouration can be seen most prominently in their role in signalling a variety of traits relating to sexual selection7. For example, carotenoid pigmentations can reflect increased immunocompetence8, parasite resistance9, and foraging efficiency10, eumelanin pigmentation can signal increased body condition12, testosterone level13, immunocompetence14 and parasite load15, whilst phaeomelanin can signal increased male reproduction16 and survivability17. In this way, understanding the genes underlying colour morphology can have ramifications for a number of evolutionary topics.Plumage colouration in birds is involved in a wide range of functions from camouflage18. In the chicken carotenoid colouration is rare, and has been found to be absent in the feathers of both domestic chicks19 and adults, as well as in wild Red Junglefowl (RJF) adults20. The main form of pigmentation in chickens is via melanin5. Melanocytes are derived from unpigmented melanoblasts, and begin to synthesise melanin around embryonic stage E7-E8. Melanocytes can give rise to either eumelanin (creating black or grey colouration) or phaeomelanin 21. Importantly for understanding their genetic architecture, melanins are not environmentally constrained, unlike carotenoids22.There are three main types of pigmentation in feathers – melanin, carotenoids and porphyrinsMC1R (involved in the melanin pathway where its activation leads to the production of black eumelanin23) and triggers the expression of MITF, which activates the enzyme Tyrosinase24, a rate limiting step in the melanin synthesis pathway. MC1R has been implicated in the control of coat colour in a number of species28. Single mutations in MC1R in the lesser snow goose and arctic skua also explain nearly all the melanin differentiation in these two bird species29. In barn owls a single substitution in MC1R has been shown to explain up to 30% of the variation in three melanin-based colour traits 30. The ASIP gene  is an antagonist of MC1R and acts to nullify it, with loss-of-function of ASIP leading to the production of eumelanin, whilst in the case of MC1R loss of function leads to an increase in phaeomelanin (see review in31). A mutation upstream of ASIP has been implicated in the yellow colouration in Japanese Quail32. Tyrosinase and tyrosine-related proteins also interact with MITF and can lead to hypoalbinism in particular4. Examples of other mutations affecting phaeomelanin also include sut (subtle grey), a mutation in the SLC7a11 gene that reduces phaeomelanin production via lysosome-related organelles33, while the Darkbrown phenotype in chickens reduces black pigmentation and increases red phaeomelanin, and is caused by a deletion upstream of the gene SOX1034.There are numerous genes implicated in plumage colour variation. Examples of causative genes for pigmentation include well studied colour determining genes such as 35). However, to the best of our knowledge, smaller effect loci that have less extreme modifier effects, in essence quantitative trait loci for plumage colour, have yet to be identified . In natural conditions, it may be of greater importance to more subtly modify coat colouration, which is why many of the genes  with more extreme phenotypic effects are identified in laboratory or domestic populations, where natural selection is essentially removed or greatly diminished.The above genetic variants can all be considered of major effect, in that they cause albinism, black plumage, etc in almost a Mendelian fashion. The above polymorphisms are responsible for at least 30% of the variation in the traits in the above examples  or brown , though breeds with more diversity in colouration do exist . Certainly as a comparison, the White Leghorn layer birds are in stark contrast to the colourful Red Junglefowl in that they are completely white and display no sexual dichromatism.Domesticated animals are a classical example of extreme variability in pigmentation within species. A reduction in pigmentation and colouration variability is usually considered as one of the key components of the domestication syndrome2 intercrosses40. The study presented here utilises multiple generations for this analysis. A large-scale QTL scan was performed using the F8 generation, whilst further targeted expression QTL (eQTL) studies were performed using the F10 and F12 generations to assess candidate genes identified in the QTL scan. By using a combination of targeted genetical genomics  to simultaneously map eQTL and correlate gene expression with intensity of red-brown colouration, we identify five putatively causal genes affecting quantitative variation in this plumage colouration trait in the chicken.To identify genes that regulate quantitative variation in the intensity of red-brown colouration, we utilized an advanced intercross between wild Red Junglefowl and domestic White Leghorn (WL) chickens. The Red Junglefowl are elaborately coloured and display some of the widest range of colour variation seen in the chicken . This means that a wide range of red-brown colour variation is seen in these birds, with both light red and dark red plumage present. In contrast the White Leghorn birds are fixed for the dominant white locus and are pure white in colour. The intercross birds display an enormous range of plumage colouration with regards to both hue and intensity, reflecting the large differences seen between the parental types used. Genetically the advanced intercross gives far smaller confidence intervals for detected Quantitative Trait Loci (QTL) than standard F8 birds (n = 380). Three separate QTL were identified for peak intensity on chromosomes 2 at 149 cM, chromosome 10 at 176 cM, and chromosome 14 at 207 cM  and F12 (n = 12) individuals. We correlated the expression levels to the relative intensity of red colouration with all genes  at a genome-wide suggestive level , two for genes associated with overall red intensity and four that are associated with the peak red intensity (see Table (CREBBP and WDR24), and three genes on chromosome 26 , see Fig. 41 located adjacent (~500 kb) to the chromosome 10 eQTL marker and also within the confidence interval for the colour QTL identified in the F8 population.We see a trans regulation of candidate gene expression, with a locus on chromosome 10 controlling multiple target sites. The most significant trans-eQTL effect originates from chromosome 10 and controls two genes on chromosome 14 41. However, whether this sweep contains the causal element responsible for colour variation remains to be validated.The aim of this study was to identify genes and genetic loci that affect red-brown colouration in the chicken. By treating colour as a quantitative trait, as opposed to looking for solely discrete classes, we have identified a total of seven small-effect QTL that until now have been overlooked. Furthermore, by combining this with a targeted genetical genomics approach in a subsequent population we have identified five candidate genes affecting the intensity of red colouration. These genes would be unlikely to have been identified using more classical (presence/ absence of major genes) linkage methods. This is unsurprising given that the average effect size was around 7% in our study, whilst the high degree of epistasis between loci would have meant they would have been extremely hard to pinpoint using single scan approaches. In particular the chromosome 10 locus appears to have a strong effect, controlling loci on chromosomes 14 and 26. It therefore appears that this locus has trans acting effects in combination with epistasis. This locus also contains a selective sweep that is fixed in domestic layer birds (the majority of which are white), yet segregating in wild Red Junglefowl. A total of 408 sweeps are present throughout the genome, with five sweeps present on chromosome 10, with each sweep being on average 40 kb in lengthPMEL gene)42, the classic extended phenotype (E) linked to MC1R on chromosome 11 at 19 Mb44, and the Dark Brown (Db) locus on chromosome 1 (linked with SOX10)45 all affect pigmentation , whilst sex-linked barring (on chromosome Z) and the EDNRB2 (panda) locus in quails46 primarily affect patterning. Despite these examples, studies of pigmentation inheritance in crosses imply that many more genes have yet to be identified. Specifically, to examine buff, brown and red colouration, crosses have been made with a variety of breeds, including Junglefowl × Buff Minorca and Rhode Island Red breeds47, Brown and Light Brown Leghorn breeds48, Dark Cornish and Black Breasted Red49, and Villafranquina birds50. These studies reveal a complex pattern of inheritance and multiple modifier loci exist that affect phaeomelanin. For example with the buff phenotype, four autosomal factors were proposed, named ginger, mahogany, dilute and champagne blond47, whilst many studies noted many variations in the intensity of the red colouration present, even when segregating for a known major effect gene50. The wide variety of phenotypes that rapidly emerge indicate that multiple epistatic interactions and numerous genes of small effect is the norm, yet such modifier loci have yet to be identified.Several genes have been identified as affecting colour in the chicken, however these are all major-effect genes that have been identified using a classical linkage/single gene approach. In the chicken, the Dominant White locus  are incompletely dominant to the wild-type and highly influenced by modifying genes45. Our study indicates that by concentrating solely on such major genes, which are easier to identify, much variation is missed.The QTL that we report here overlap none of these previously identified major effect loci. The genes and QTL are all novel loci (as affecting colour), though in the case of the QTL on chromosome 11, this is around 4 Mb downstream of the CREBBP and red intensity. CREBBP is controlled by the trans-acting eQTL locus on chromosome 10 that also contains the selective sweep. CREBBP binds to CREB, whilst CREB itself is a transcription factor strongly involved in melanin synthesis, principally by binding and activating MITF via the cAMP response element51. In turn MITF has a role in activating tyrosinase52, which in turn oxidates tyrosine to DOPA and affects which type of melanin (eu- or phaeo-melanin) is produced53. Given this role of CREB and its close interplay with CREBBP, it therefore represents a strong candidate as a small-effect modifier of red-brown colouration via melanogenesis to alter phaeomelanin colouration. Currently, CREBBP has primarily been found to be involved with neuronal growth and development55, so a role in feather colour expression is relatively novel. Of the other candidate genes identified, WDR24 is a master regulator of lysosome function. Given one function of lysosomes is to regulate and degrade pigment molecules (for example a mutation in the SLC7a11 gene reduces phaeomelanin production via lysosome-related organelles33), this also represents another excellent candidate gene. Similarly, the gene ARL8A, an acetylated Arf-like GTPase, is localized to lysosomes and affects their mobility56. Of the remaining genes, PHLDA3 is implicated in tumour suppressor function, is a target of p53 and also impedes somatic cell reprogramming57, whilst LAD1 encodes ladinin-1, a collagenous anchoring filament protein that is involved with the maintenance of cohesion at the dermal-epidermal boundary58, and defects in this gene are related to linear IgA disease, an autoimmune blistering disease59.Of the candidate genes identified, particular interest comes from the interaction and correlation seen between the gene In summary, by treating the intensity of red colouration as a purely quantitative trait and utilising a variety of advanced intercross generations and targeted gene expression profiling we are able to identify multiple small-effect loci that interact to determine the red colouration present in Red Junglefowl. Using a targeted genetical genomics approach we are able to identify several high quality candidate genes that appear to have direct relevance to the melanogenesis pathway and can thus modify overall red intensity. However, one caveat is that the relatively low number of birds used in the targeted genetical genomics approach means that weaker effect genes will be missed , therefore other candidates may still be present in the regions. Similarly, the genes identified are only putative, and full functional assays are required to verify that these genes are indeed causal to the phenotype. As far as we are aware this is the first study to identify QTL and candidate genes that can more subtly modify the intensity of red colouration in feathers, with such small effect loci being potentially far more prevalent in wild populations.8, F10 and F12) of an intercross between a Red Junglefowl male and three females from a White Leghorn selection line (WL-L13) maintained from the 1960s at first SLU, Uppsala and subsequently in Linköping, Sweden (since 2006). These four parental birds were used to generate 41 F1 individuals that were in turn used to generate 811 F2 individuals. From the F3 generation to the F7 generation, the intercross was maintained at ~100 individuals per generation, until the F8 generation, which consisted of a mapping population of 572 individuals reared in 6 batches, with wings saved from 380 of these birds. The F10 and F12 generations were also of approximately 100 individuals each, with 12 F10 and 12 F12 individuals used in this study. The selection of these birds (from the F10 and F12 generation) was based on those displaying red-brown colouration, with only these 24 birds utilised in this study. Food and water was supplied ad libitum, see60 for further details of housing and the intercross. A flow-chart of the birds used in the experiment, and which generation they are from is provided in Fig. The animals used in this study come from three separate generations  light tent and two fixed 24Watt DULUX L (Osram) lights mounted to brackets to ensure identical lighting conditions were used for each wing. A standardized blue background was used for each shot, whilst an X-rite colour checker chart (with both pure white and red coloured squares) was included in each photograph to normalize colour levels. Wing photos were taken on the blue background, and a corresponding image was taken of the blank background without the wing present. Raw image quality was used for subsequent calculations. Two separate phenotypes were extracted from each wing, peak and overall red intensity. Peak intensity was measured by selecting an area of approximately 1 cm2 (~14000 pixels) in the area with the strongest representative red intensity observed by eye in Adobe Photoshop CS6 and recording the median value from the red RGB channel. This value was then divided by the value measured from the ‘true’ red on the colour chart, to give a normalized peak red intensity for each wing. It is worth noting that since white is composed of full saturation in all channels this red value is negatively correlated with red colour, ie the darker red the feather is the lower the relative colour score will be and vice versa. The second phenotype uses the average red intensity measured across each wing. This was performed using the following steps:Wings were assessed for both the intensity and coverage of red-brown colouration in all the animals tested. The wing was selected for several reasons. We firstly observed that a large degree of colour variation was present on the wing, but importantly it was far easier to accurately assess overall degree of colouration in a far more repeatable fashion and with a high degree of accuracy due to the wing being flat and therefore enabling it to be photographed in a highly standardised fashion (see below). This enabled far more precise inter-individual comparisons to be made, which is essential for genetic mapping experiments. In terms of ecological relevance, the Red Junglefowl more frequently perform wing displays, implying that it is also used for signalling in natural populationshttp://www.guillermoluijk.com/tutorial/dcraw/index_en.htm for a tutorial describing the program). For each of the images the location of the white patch of the colour checker with the highest intensity was automatically detected and the mean RGB vector over all pixels in this patch was computed. The colour vectors of all pixels in the image were then scaled (and converted to 16-bit unsigned integer) such that the resulting pixels in the white patch had all the same grey values. After this step all colour variations due to varying illumination and camera conditions were eliminated and all images could be compared.The raw sensor measurements  were extracted from the image file using the program dcraw  were automatically detected in both images of an image pair. The locations of these registration points were then used to compute the geometric transformation that aligned the images without the wing with the image with the wing. Pixelpairs with blue colour in the background image and non-blue colour in the wing image define a mask indicating the location of the wing.The mask computed in the previous step is used to set all non-wing pixels to a black background colour. Next the colour vectors in the masked image are converted from the RGB colour space to the HSV colour space using the Matlab function rgb2hsv. In this HSV the colour of a pixel is described by three values: H is the hue (described by an angle), S is the saturation described by an interval and finally V is the value, which is a non-negative number describing the lightness. In the application only red/brown pixels are of interest. The corresponding region in colour space was defined as the region where the hue value was between 0.6 and 0.7 and the saturation value was greater than 0.75. Both the hue and the saturation values are located in the  interval. This procedure thus isolates all the red/brown patterned feathers from the rest of the wing. After the pattern isolation, the red channel of the entire resulting trimmed image was measured in Photoshop. Therefore this second phenotype takes the median red intensity of every red/brown area of the wing into consideration. The average value for this second phenotype is 184.4 with a standard deviation of 42.4 compared to the average value of 0.71 with a standard deviation of 0.18 for the single feather analysis used for the eQTL analysis (see below).8 generation. These birds were individuals from both the F10 and F12 generations from the advanced intercross (n = 12 F10 and 12 F12 birds). Multiple generations were required to obtain a broad spectrum of red colour phenotypes, see Fig. 8 birds, with only the peak intensity measured. The developing basal root of the feather follicle  was then used for RNA extraction and microarray analysis for eventual eQTL analysis.A separate eQTL fine mapping population was used for expression analysis of budding feather tissue for candidate gene assessment in the previously detected QTL regions identified in the F62). Microarrays were first preprocessed using DEVA software to normalize the data and all arrays were processed together.5–6 growing feathers of 2–3 cm length were collected from each individual. The calamus was immediately cut from each feather and snap frozen in liquid nitrogen for RNA extraction, the rest of the feather was kept at −30 and used for colour measurements. RNA was extracted from a pooled sample of three to four calami per individual which had been homogenized in Lysing matrix D tubes in a FastPrep using TRIzol (Sigma Aldrich) reagent and following the manufacturers instructions. After quality checking the RNA with RNA Nano Chips(Agilent Technologies) in an Agilent 2100 Bioanalyzer to ensure that RNA was not degraded, the RNA was treated with DNAse I (Thermo scientific) followed by cDNA synthesis using a RevertAID KIT from Life technologies, and following the instructions provided with the kit. The cDNA was again quality checked using a Bioanalyzer 2100 and RNA Nano chips (Agilent Technologies) and subsequently labeled with a NimbleGen One colour labeling kit(Roche) according to the manufacturer’s instructions. Finally, the cDNA was hybridized to NimbleGen 12 × 135 k custom gene expression microarrays (Roche) and scanned using a NimbleGen MS200 Microarray scanner (Roche). The custom Microarrays have been used in previous publications by the group and contain all known Ensembl and RefSeq genes as well as EST probes  of the genotype data was performed, and the first ten PCs fitted as covariates in the QTL model, with all significant PCs retained in the final model (see62 for further details). A sex interaction was also added, where significant. Digenic epistatic analysis was performed as per the guidelines provided in67. Initially a global model was used that incorporated standard main effects and sex interactions. Epistasis was then built on this model, starting with the most significant loci and working down for each trait. Significance thresholds were calculated by permutation69, with thresholds of 20% and 5% genome wide significance being the cut-offs for suggestive and significant loci respectively. A suggestive threshold was used as per70, though we used a 20% genome wide threshold, which is slightly more stringent than 1 false positive per scan. These thresholds corresponded to approximately LOD cut offs of 3.6 and 4.4. Confidence intervals were calculated using a 1.8 LOD drop method71. Epistasis thresholds were calculated in a similar manner, with 20% and 5% genome-wide thresholds used. If two loci were significant by main effects alone then an epistatic effect for this locus was included if the interaction passed the nominal 5% threshold in the same manner as a sex interaction. Note that the sample sizes required to detect epistatic interactions can be much larger than a standard QTL analysis , so certain interactions will most likely be missed, though ones that are detected will still be valid.DNA preparation was performed by Agowa GmbH , using standard salt extraction. A total of 652 SNP markers were genotyped using an Illumina GoldenGate system, and gave a map of length ~92675 cM, with an average marker spacing of ~16 cM. Of these markers, 551 were fully informative , with markers selected to be evenly spread throughout the genome (as reflected in the average marker spacing) and based around the markers generated for the F10 and F12 fine mapping eQTL population were genotyped for sixteen SNP markers which had been identified in the F8 mapping population as being flanking markers for the QTL with the two colour phenotypes . These probes were then correlated with the specific red colour phenotype obtained for each feather sample (see above), using a linear model to fit gene expression levels and colour score using the lmFit function in the limma R package73. All probes that had a significant association between gene expression and colour score at a FDR significance level of 0.05 were retained  included as fixed factors. All the genes from second step were tested against the regions genotyped using the 16 SNP markers mentioned above . Permutation testing was used to set an experiment-wide threshold for each gene (1000 permutations per gene), with a 5% significance and a 20% suggestive threshold obtained . To control for the total number of genes (n = 76), a principal component analysis was first performed on these 76 genes. 7 significant eigenvalues accounted for 97% of the total variation in the data. Thus, as it is only required to control for the number of independent tests, a multiple testing correction of 7 was added to each LOD threshold. Using a log10 transformation to convert this to a LOD score, this gives an increase of 0.85 LOD to each threshold, meaning an eQTL was suggestive with a LOD score of ~3.05, and significant with a LOD score of ~3.85.Analysis was performed using a three-step procedure. Initially, all genes that were present in the colour QTL regions detected in the FSupplementary Information.


The use of readily accessible ammonium ylides for (asymmetric) transformations, especially cyclization reactions, has received considerable attention over the past two decades. A variety of highly enantioselective protocols to facilitate annulation reactions have recently been introduced as an alternative to other common methods including S-ylide-mediated strategies. It is the intention of this short review to provide an introduction to this field by highlighting the potential of ammonium ylides for (asymmetric) cyclization reactions as well as to present the limitations and challenges of these methods. These 22.13)-based ammonium ylides for cyclopropanation reactions was reported by Bhattacharjee et al. in 1982. cyclizations was reported by Gaunt’s group in a series of four publications that appeared between 2003–2006. Alt Alt22 an29 for cyclopropanation reactions  gives, upon its addition to benzaldehyde (43a) and acidic workup, the corresponding β-hydroxyammonium salts 44  was generally higher yielding for several substrates relative to quinuclidine (55) but with lower diastereoselectivities.  as a suitable chiral amine for the synthesis of chiral stilbene oxides 53 through an asymmetric ammonium ylide mediated epoxidation. They succeeded in carrying out the in situ formation of the chiral ammonium ylide by treating chloride 58 with a slight excess amount of amine 57. The subsequent addition of base in this one-pot approach in the presence of an aldehyde gave epoxide 53 with moderate to good ee values for a range of different aldehydes and activated (electron poor) benzylic ylides  allowedtereomer . We alsoidations , we recem ylides . This prm ylides  and 5.2.3Aggarwal’s group has shown aziridinations are faster than epoxidations for S-ylides, and ammo69 is possible to give the aziridine 70 as a mixture of diastereomers (The earliest example was reported by David’s group in 2007. Upon theereomers .11 can be used to access aziridines 71 in high yields with good trans selectivities (61 but found the reaction to occur with N-Boc-imines 72 (Boc=tert-butoxycarbonyl) and not with N-Tos-imines 69  can have a pronounced effect on the outcome.Very recently, Namboothiri and co-workers investigated the reaction of cyano-stabilized ylides derived from ammonium salts ptors 78 .54] Her Her11 wiN-oxides 81 in an efficient manner.[82 as ylide precursors that can undergo highly enantio- and diastereoselective formal [4+1] cyclizations upon addition to nitroolefins 83  side products. In contrast, some of the first reports that describe the use of ammonium ylides suggest that cyclization reactions do not readily occur with these systems compared with those of S-ylides. Over the last two decades, however, a variety of different approaches clearly show these initial assumptions to be wrong, and the use of in situ generated ammonium ylides has emerged as a powerful complementary strategy for cyclization reactions. Since the pioneering work by Gaunt’s group, which describes highly enantioselective catalytic cyclopropanation reactions that proceed by using chiral ammonium ylides, the field has received considerably more attention. Recent years have witnessed an increasing interest in such transformations, which has resulted in the development of protocols that render reactions enantioselective where other methods fail. There are of course several limitations, particularly when it comes to the development of catalytic approaches, as quite a few recent reports are still chiral auxiliary based methods. It is therefore without doubt that future research should focus on the development of catalytic and more atom efficient methods. In our opinion, recent results are rather encouraging, and we are convinced that a variety of strategies and synthetic methods that use ammonium ylides will be reported in the near future.


A first generation of CRISPR-based gene drives has now been tested in the laboratory in a number of organisms, including malaria vector mosquitoes. Challenges for their use in the area-wide genetic control of vector-borne disease have been identified, including the development of target site resistance, their long-term efficacy in the field, their molecular complexity, and practical and legal limitations for field testing of both gene drive and coupled anti-pathogen traits. We have evaluated theoretically the concept of integral gene drive (IGD) as an alternative paradigm for population replacement. IGDs incorporate a minimal set of molecular components, including drive and anti-pathogen effector elements directly embedded within endogenous genes – an arrangement that in theory allows targeting functionally conserved coding sequences without disrupting their function. Autonomous and non-autonomous IGD strains could be generated, optimized, regulated and imported independently. We performed quantitative modeling comparing IGDs with classical replacement drives and show that selection for the function of the hijacked host gene can significantly reduce the establishment of resistant alleles in the population, while drive occurring at multiple genomic loci prolongs the duration of transmission blockage in the face of pre-existing target site variation. IGD thus has potential as a more durable and flexible population replacement strategy. Summary: Here, we describe and model a novel gene drive strategy – integral gene drive – for population replacement based on the hijacking of endogenous genes. Homing gene drives were first proposed 15 years ago as potential tools for enabling the genetic engineering of natural populations , in partAlthough gene drive research is now mainly centered on drives built using the CRISPR genome-editing toolset, proposed strategies adhere to the classic transgene paradigm, namely the use of pre-characterized promoter and terminator elements, each driving tissue-specific transgene expression as required for different functions in the germline (drive) and various somatic tissues (anti-pathogen effect), and with the resulting constructs inserted at arbitrarily chosen genomic sites, e.g. genes that are presumed to be neutral A. It hasPlasmodium transmission. A growing set of anti-pathogen effectors now exists . Each IGD effector transgene also carries a ubiquitously expressed U6-driven intronic gRNA for triggering homing in the germline. Unlike the drive component, however, the effector component does not encode an endonuclease and thus is not able to initiate gene drive on its own. Indeed, targeted cleavage mediated by the gRNA and Cas9 complex can only occur when the latter is provided by the drive component in the germline. Cleavage in the germline triggers homing of the effector component, which is a non-autonomously homing allele of an endogenous gene and is designed to spread in a population, thereby increasing the allele frequency of the coupled effector trait. We define non-autonomous gene drive as the ability of a certain allele, encoding a gRNA that renders itself capable of homing, to only show gene drive, provided that a Cas9 endonuclease is supplied by another genetic element.For simplicity, we consider here the effector to be an exogenous polypeptide that when expressed in the target tissue reduces or abolishes parasite development in the mosquito. Various mosquito tissues – such as the midgut, the hemocytes and the salivary glands – are at the interface of the vector-parasite interactions and are thus ideal candidates for hosting the expression of effectors. The fat body is another good candidate tissue, as its secretions into the hemocoel can directly interact with  parasiteDrive and effector traits inserted at various suitable loci throughout the genome could be generated and tested independently from each other, including by different research teams. On the one hand, the drive component can be specifically optimized in the laboratory for its propensity to induce homing, the faithfulness of Cas9 expression and to minimize the rate at which cleavage triggers the undesirable non-homologous end joining (NHEJ) or microhomology-mediated repair (MHMR) pathways. In addition, one would seek to reduce the fitness cost incurred by the expression of Cas9 itself or by its integration interfering with the function of the hijacked host gene. On the other hand, the effector component can be optimized in the laboratory for its efficacy in reducing or blocking parasite/pathogen development and transmission, its own intrinsic fitness cost and any negative effect it may have on the expression of the hijacked host gene. In addition, the rates of non-autonomous homing of the effector component, assisted by the presence of the IGD drive component or another source of Cas9, can be measured under laboratory containment. Laboratory crossing of the transgenic strains harboring the IGD drive and effector components would allow assessing the likely performance of these trait combinations in the field, including the spread of each modified allele and their resulting frequency in cage populations. It should be noted that the existence of independent IGD strains requires them to be appropriately isolated to minimize the risk of them combining before it is desired.To predict the behavior of one or multiple interacting IGD traits at the population level, we used a discrete-generation (non-overlapping) model, comparing the dynamics of a classic replacement drive  to the dn) of expression due to the nuclease is less than the cost (se2) of expression for the effector (sn=0.05 versus se2=0.1), resistance replaces the transgene faster at the effector locus, while the nuclease persists for longer in the population, whereas in the conventional strategy, the compound cost for expressing both causes the construct to be lost rapidly. We find that at the same rate of formation of resistant alleles, their impact is reduced in the IGD strategy since mutations that result in a loss of function of the hijacked endogenous target gene and are selected against – unlike the conventional strategy, which assumes the target to be neutral.A comparison of a conventional replacement strategy and the IGD two-loci model is summarized in Fig. S2). We find similar dependencies as for the classical model; however, for a number of parameters IGD appears more robust with minor or no effects on the duration of protection evident. For example, increasing the proportion of resistance and loss-of-function alleles formed by either NHEJ or aberrant HDR at locus 1 within a biologically sensible range does not affect the duration of protection. This can be explained by the main effect the drive component (locus 1) has on locus 2, which is to convert wild-type alleles to effector alleles in heterozygous individuals, allowing the effector to rapidly propagate in the population and to establish protection before resistance at locus 1 takes over. Protection starts decreasing only when resistant alleles start forming at locus 2 and eventually replace effector alleles in the population. The eventual subsequent loss of the drive allele at locus 1 and its replacement by resistant alleles is no longer of any consequence to the duration of protection because the conversion of wild-type alleles at locus 2 into effector has already taken place.In order to determine which parameters have the strongest effect on the duration of protection, we varied each while retaining all others at baseline values . By contrast, levels of protection begin to crash only when initial resistance at locus 1 approaches 80% i.e. that target sequence represents a minor allele. Should pre-existing resistance alleles occur with a frequency of 10% at both loci, 95% protection is reduced from 81 generations to 15 generations. Pre-existing resistance must be assumed to be present in significant proportions in many target species including mosquitoes , the longer it will be present in the population and the longer protection can be extended. The condition for full protection to be achieved after release of the second effector at locus 3 is that the allelic frequency of the nuclease is greater than ≅55% at the time of release. If the second effector is released before protection starts significantly decreasing, it can optimally prolong the duration of maximal protection. We find that the ideal time for secondary effector release at locus 3 is when protection drops below 95%.A second effector component allele at a separate locus could also be introduced after a given time to extend the duration of protection. The second effector is driven through the population when in the presence of the nuclease allele at locus 1, extending the duration of protection until the time when the second effector is in its turn replaced by resistant alleles. The IGD paradigm reflects our view that a successful gene drive intervention will have to involve the use of multiple interacting traits rather than attempting to make singular constructs evolution safe. It consequently should allow (informed by continuous monitoring) the ability to flexibly react to predictable as well as unexpected developments in a target vector or parasite population by adapting the release strategy in a context-dependent manner. Control would require the constant development and refinement of engineered genetic traits rather than the one-off or continued application of a static product.Modeling of allele frequency dynamics suggests that for the parameters investigated, the IGD strategy could confer significant advantages over conventional replacement drive designs. By integrating components into endogenous loci, undesired repair outcomes following DNA cleavage are predicted to result in loss of function of the hijacked gene. Selection, in turn, reduces the rate at which such resistant alleles accrue. Whilst IGD does not prevent the onset of resistance, it could significantly extend the duration and level of protection available to the human population. This approach also could resolve the longstanding issue of the arbitrary selection of a target sequence for population replacement where neutral sites show poor conservation, whereas conserved sites are likely to be functionally constrained and thus costly to disrupt. For example, trying to tackle this conundrum, Gantz and colleagues disrupt the kynurenine hydroxylase gene in their replacement approach with unclear consequences for the behavior of these drives in the field . The IGDns=0.05) is lower than the cost of expressing an effector gene from an effector component (es2=es3=0.1). This is justifiable when considering that germline genes tend to have lower expression levels than those present in the soma and that expression of antimalarial effectors needs to be sufficiently strong to ensure effective concentrations. Probabilistic models of finite populations (versus our deterministic model for an effectively infinite population) predict that resistant alleles generated at low rates suffer early stochastic loss , the cost of disrupting the drive component locus (d1s), the cost of expressing the effector (es) and the cost of disrupting its corresponding locus (d2s). Modeling shows that changes in the cost of expressing the nuclease and the cost of disrupting the drive component locus are robustly tolerated. By contrast, the IGD strategy is sensitive to changes in the cost for disruption of the effector component locus, and the cost of expression of the effector. This is due to the efficiency with which the drive component, even if not present at high allele frequencies, is able to convert wild-type alleles into effector alleles. Thus, even if the drive component carries significant costs of expression and disruption, there will not be a sizable reduction in the duration of protection. These findings may be useful in the design of IGD traits, particularly with respect to the effector components. Minimizing the cost of effector expression and avoiding disruption of the hijacked gene are critical to the successful implementation of an IGD-based release. Previous work has highlighted the difficulty with which these parameters can be evaluated in a field setting , including an independent ecological risk assessment. It is expected that gene drive strains will face a significantly tougher and prolonged regulatory pathway compared to strains harboring such a conservative genetic modification. These foreseeable regulatory and operational challenges must inform the design of gene drives, and the potential theoretical properties of IGDs should thus be considered in conjunction with the practical aspects of generation, testing, optimization, regulation and deployment of gene drives for population replacement.Recently, the first transgenic mosquito strain, carrying a dominant male-sterilizing transgene , was impField testing of the effector component would be aided by the simplicity of the genetic modification, i.e. insertion of a single effector coding sequence containing the intronic gRNA, with all other functions provided by the hijacked host gene. Given the inability of the effector component to spread autonomously, the regulatory threshold for such strains is expected to be lower than that of conventional fully driving transgenes harboring a population replacement payload A. This wThe scientific working group on a pathway to deployment of gene drive mosquitoes for elimination of Malaria in Sub-Saharan Africa  recommenThe IGD strategy offers an increased degree of versatility by allowing independent testing and release of overall less complex components. The scientific working group on gene drives recommends a stepwise pathway for the deployment of gene drive mosquitoes i.e. to progress testing from laboratory studies, possibly involving large indoor and outdoor cage trials, to small-scale isolated and open releases to full-scale open releases . HoweverThe modularity and interdependence of IGD components does provide a straightforward path for moving testing from self-limited to self-sustaining traits in the field by modulating the propensity to spread in the population . First, https://github.com/genome-traffic/igd. The classical model for a drive+effector construct  considei . Locus 1 corresponds to the drive component, and loci 2 and 3 correspond to effector components. At each locus i, there are four possible alleles: a wild-type allele (iw), a transgene it , and two types of alleles at each locus that are resistant to the drive and do not have an intact nuclease or effector, ir and im. The first type of resistant allele, ir, arises from incomplete homing or mutations that are in-frame and do not cause loss of the function of the host gene, and therefore are not considered to carry any fitness cost (similarly to the wild type). The second type of resistant allele, im, corresponds to a mutation that results in frameshift of the host gene, disrupting the endogenous locus. If the host gene is an essential gene, im alleles are considered to convey lethality when homozygous. Resistance can be pre-existing or occur either by NHEJ or by incomplete HDR at either locus. We neglect resistant alleles created from spontaneous mutations, as rates are likely to be low, compared to generation of resistance during homing.While the IGD model may involve multiple nuclease genes and effectors on many different loci, here we consider a simplified version with transgenes on either two or three independent loci An individual is considered to have IGD drive if it carries at least one functional drive component at locus 1 and at least one functional effector component at locus 2 . This is a necessary condition for the effector component to home and propagate in the population at a super-Mendelian rate. For both models, we assume that the initial field population consists entirely of the wild-type allele, and there may be pre-existing resistance due to standing genetic variation at each locus . Individuals homozygous for different IGD drive components are subsequently released as a relative proportion of the field population.w1/n1 at the first locus). Cleavage and homing at effector locus 2  can occur only in those genotypes if there is at least one nuclease allele at locus 1 and a wild-type and effector allele at locus 2 (w2/e2 at locus 2). Transmission of the nuclease at locus 1 occurs with probability 1d. Transmission of the effector transgene at locus 2 occurs with probability 2ad when the drive component is heterozygous, and probability 2bd when homozygous, and similarly for an effector at locus 3 if included (3a d3bd). Resistance to the drive, sometimes accompanied by loss of gene function, is considered to occur during homing. We conservatively consider mutations to produce resistance (ir) in 1/3 of cases, and loss of gene function (im) in 2/3, here predominantly caused by frameshift mutations. Resistant alleles (ir) arise at loci 1, 2 and 3 from incomplete HDR with probabilities 1l, 2l and 3l, and by NHEJ with probabilities 1u, 2u and u3, respectively. Resistant alleles (im) that cause loss of the endogenous gene function occur via incomplete HDR at loci 1, 2 and 3 with probabilities 1L, 2L and 3L, and by NHEJ with probabilities 1U, 2U and U3, respectively. Due to germline cleavage, homing, incomplete HDR and repair events, individuals that are heterozygous for the nuclease at locus 1, i.e. w1/n1, contribute alleles 1:n1:r1:m1w in proportions (1−1d) (1−1u−1U):1d (1−1l−1L):(1−1d) 1u+1 d1l:(1−1d) 1U+1 d1L. Allele contributions from individuals with the wild-type and effector allele (w2/e2) at locus 2, if there are one or two nuclease alleles at locus 1, are according to w2:e2:r2:m2 in proportions (1−2kd)(1−2u−2U):2kd (1−2l−2L):(1−2kd) 2u+2 d2kl:(1−2kd) 2U+2 d2L, where k=a, and b corresponds to locus 1 heterozygous or homozygous for the nuclease. If no nuclease allele is present at locus 1, gene transmission at locus 2 is Mendelian, as is inheritance in all other individuals. Similar expression can be written for an additional effector at locus 3.Cleavage and homing can occur only in the germline of genotypes with a wild-type and nuclease allele at locus 1 , which has a fitness of one. Fitnesses are modeled using ten parameters for transgenes at two loci and 14 parameters for three loci. We consider the following homozygous fitness costs: the cost of in-frame disruption  at locus 1, 2 and 3 , the cost of disruption (by mutations of type im) that lead to loss of function at each locus , the cost of expressing the nuclease from the drive component (ns) and the cost of expressing the effector from the effector components . The corresponding dominance coefficients are d1h, d2h, d3h, mh, nh, e2h and e3.h The range of fitness costs is from 0 (no cost) to 1  and dominance coefficients range from 0 (completely recessive) to 1 (completely dominant). The fitness of each genotype is derived as the product of costs at each locus associated with site disruption, number of nuclease components and number of effector components. For example, for a two-loci model (drive at locus 1 and effector at locus 2), the fitness of a genotype that is heterozygous for the transgene at both loci  is given by (1−d1 sd1h) (1−d2 sd2h) (1−n snh) (1−e2 se2h) to reflect costs of host gene disruption by transgenes  at both loci as well as the cost of expressing the nuclease (at locus 1) and the effector (locus 2).With four possible alleles at two independent loci, there are 16 gamete types and 100 diploid genotypes; for three independent loci, there are 64 gamete types and 1000 genotypes. The fitness of each genotype is relative to the wild-type homozygote (Allele frequencies and genotype abundances are modeled using deterministic discrete-generation recursion equations. We assume a one-life-stage model (adults) with a field population composed of equal numbers of male and females with the same genetic and fitness parameters, such that allelic and genotypic frequencies are equal between them. Mating is random, with unsuccessful mating events not considered. We assume the population to be sufficiently large to ignore stochastic effects. The system of equations is solved numerically using Wolfram Mathematica .rc1rch if the effector component is present at locus 2 in one copy (heterozygous) and by cr if two copies (homozygous) are present. For the three-locus model, we assume that the reduction in vector competence depends on the total number of effector alleles, giving rci rch for i=1, 2 or 3 effector alleles in total over loci 2 and 3, and cr for i=4 alleles in total (i.e. homozygous for the effector element in both loci). Values of cr range from 0 (no effect) to 1 , and the dominance coefficient for refractoriness rc1h (and rc2h and rc3h for the three-locus model) ranges from 0 (completely recessive) to 1 (completely dominant). We quantify the effect in terms of the reduction in vectorial capacity at time t as 1−CV[t], where CV[t] is the vectorial capacity. CV[t] is calculated as the sum over the genotype frequencies multiplied by their individual vector competence. For the two-locus model, this yields:As in