Patient engagement in research agenda setting is increasingly being seen as a strategy to improve the responsiveness of healthcare to patient priorities. Implementation of low‐dose computed tomography (LDCT) screening for lung cancer is suboptimal, suggesting that research is needed.This study aimed to describe an approach by which a Veteran patient group worked with other stakeholders to develop a research agenda for LDCT screening and to describe the research questions that they prioritized.We worked with Veterans organizations to identify 12 Veterans or family members at risk for or having experience with lung cancer to form a Patient Advisory Council (PAC). The PAC met repeatedly from June 2018 to December 2020, both independently and jointly, with stakeholders representing clinicians, health administrators and researchers to identify relevant research topics. The PAC prioritized these topics and then identified questions within these areas where research was needed using an iterative process. Finally, they ranked the importance of obtaining answers to these questions.PAC members valued the co‐learning generated by interactions with stakeholders, but emphasized the importance of facilitation to avoid stakeholders dominating the discussion. The PAC prioritized three broad research areas—(1) the impact of insurance on uptake of LDCT; (2) how best to inform Veterans about LDCT; and (3) follow‐up and impact of screening results. Using these areas as guides, PAC members identified 20 specific questions, ranking as most important (1) innovative outreach methods, (2) the impact of screening on psychological health, and (3) the impact of outsourcing scans from VA to non‐VA providers on completion of recommended follow‐up of screening results. The latter two were not identified as high priority by the stakeholder group.We present an approach that facilitates co‐learning between Veteran patients and providers, researchers and health system administrators to increase patient confidence in their ability to contribute important information to a research agenda. The research questions prioritized by the Veterans who participated in this project illustrate that for this new screening technology, patients are concerned about the practical details of implementation  and the technology's impact on quality of life.Veterans and Veteran advocates contributed to our research team throughout the entire research process, including conceiving and co‐authoring this manuscript. Historically, research priority setting has been controlled by scientistsOne area where patient input has been limited is lung cancer screening research. The National Lung Screening Trial (NLST) and other studies have demonstrated that screening with low‐dose computed tomography (LDCT) reduces lung cancer mortality among adults at high risk of lung cancer.Lung cancer is of particular importance to US military Veterans as they are often at increased risk for lung cancer due to high rates of smoking and exposure to carcinogens . Higher rates of lung cancer have been observed in Gulf War VeteransIn this paper, we describe an approach by which a Veteran patient group developed a research agenda for LDCT screening in collaboration with other stakeholders. We then present the patient‐centred research priorities around optimizing LDCT implementation; this was generated through this collaboration.22.1This study reports an approach for identifying patient‐centred research questions around optimizing LDCT implementation through facilitating a process of two‐way co‐learning between two groups of stakeholders: the Patient Advisory Council (PAC) and Stakeholder Group (SG). Co‐learning is one of the Patient‐Centered Outcome Research Institute (PCORI) principles of patient and stakeholder engagement. The project focused on Southeast Wisconsin, where the research team has deep connections to Veteran groups. Figure 2.2We recruited 12 PAC members from various racial backgrounds based on (1) fitting the NLST entry criteria ,We recruited 10 SG members to represent lung cancer providers , researchers, health system administrators and patient advocates. Our patient advocates included senior Wisconsin members of the American Cancer Society, American Legion and Vietnam Veterans of America organizations. Their involvement was designed to both facilitate dissemination of project products back to the Veteran community and to include a Veteran voice in discussions by the SG. These Veterans were not involved in PAC activities.PAC and SG members were paid based on meeting attendance in accordance with PCORI's best practice.2.3Meetings were based on an evidence‐based design called the ‘building block approach’ for consensus building.We arranged two joint meetings for PAC and SG members in year two, adhering to the PCORI engagement principle of co‐learning. During the meetings, we used moderation techniques to ensure that there were opportunities for both groups to speak. Despite these efforts, after each joint meeting, we discussed how to improve the dialogue with both groups (PAC and SG), so that our moderation strategy evolved over time. Additionally, PAC feedback caused us to orient PAC members more fully about the planned joint meeting activities before that meeting. A designated staff took minutes for all meetings. The project staff also summarized each meeting immediately after the meetings and sent the meeting recap follow‐up emails to all PAC and SG members.2.3.1PAC members met quarterly, and each meeting lasted approximately 2 h, with lunch provided. Between June 2018 and December 2020, the PAC had seven meetings: one kick‐off, two joint meetings with SG and five PAC meetings. PAC meetings had two major objectives: (1) share personal experiences with lung cancer screening, diagnosis and/or treatment and (2) develop a Veteran‐centred research agenda in collaboration with the SG. Responding to PAC requests, learning sessions to address questions related to LDCT from the patients' perspective were incorporated into meetings.The SG met less regularly, and their meetings were scheduled to coordinate with PAC needs. The SG met three times per year (the last meeting was cancelled due to COVID‐19) for a total of five meetings, including one kick‐off, two joint meetings with PAC and two SG meetings. The main objectives of the SG meetings were to: (1) translate the PAC's ideas into research questions and (2) generate a sustainability plan to maintain the PAC for future research engagement.2.3.2The PAC and SG convened as a single group two times (joint meetings). These learning collaborative 2‐h sessions were designed for co‐learning. The third joint meeting, a project celebration, was cancelled due to the COVID‐19 pandemic. The goals of the first two joint meetings were to develop a research agenda and identify research questions. The project team had a synthesis responsibility during the ‘reaching consensus’ section on the research agenda, combining the agendas developed by the two groups. The two sets of agendas were revised and negotiated at each joint meeting and presented back to members at the individual meetings (of SG and PAC) in between those two joint meetings.2.3.3The research agenda development process involved several steps, including PAC and SG independent meetings, PAC and SG joint meetings and a final voting. The voting process was anonymous following the REPRISE priority setting reporting checklist formulated by Tong et al.33.1Among 12 PAC members we recruited, one‐third were women, one was African American and one was Hispanic. The 10 SG members included 3 VA and 3 MCW healthcare providers, an MCW population health scientist and representatives from the American Legion, the Vietnam Veterans of America (VVA) and the American Cancer Society.3.2The agenda reflects Veterans' prioritized research topics influenced by their life experiences. These topics were different from and sometimes viewed as less significant than the topics identified by researchers.3.2.1The PAC prioritized their top three topics of interest by their third meeting Table . The firThe second topic focused on the best way to do outreach to encourage screening. Much of the concern on outreach revolved around those Veterans who were ‘falling through the cracks’: the young, uninsured, isolated and marginalized . Notably, PAC members provided many strategies for doing outreach, with attention to Veterans not seeking VA care. This included peer mentoring, word‐of‐mouth, social media and tapping into community organizations that sponsor Veteran activities.The third priority topic focused on what happens after people get screened. Concerns were rooted in the psychological and emotional impact stemming from waiting for results, the uncertainty of the next steps and potential financial distress. PAC pointed to how results that are conveyed to patients could affect how they respond . Major concern was expressed over whether Veterans followed up after a positive screen, how to mitigate denial and the impact of military training on self‐care .3.2.2(1)What Outreach Provides the Best Outcome?Rather than generating questions in this area, PAC members focused on developing strategies  that might positively impact screening behaviour. Most members felt that raising awareness of and promoting screening was important for improving Veteran health outcomes. They suggested that researchers could help identify and test outreach approaches that would work for different Veterans. This was important for health topics that do not come up in clinical appointments and could be missed. They offered guidance such as noting that Veterans have varying backgrounds and live in different locations, and so likely require different ways of engaging. They warned that partial information could scare Veterans and lead to permanent avoidance. Awareness of lung cancer risk by younger Veterans was a particular concern.(2)Do screening results affect psychological health?This was a prioritized topic early on in PAC meetings and remained so through multiple iterations of the research agenda. Most PAC members expressed concern over waiting: waiting to see a specialist, waiting for results and waiting for further care. The emotional burden and stress of uncertainty were considered potential triggers for Veterans who are ‘already psychologically fragile’. The stress of waiting for results or having to make a decision based on complex health information can compound mental health issues. Some PAC members felt that this added anxiety might be a cause for screening avoidance.(3)Do rates of follow‐up differ if the initial scan is performed within versus outside the VA?In the three prioritized topic areas, two cycles of feedback generated 20 research questions  such as methods proposed by the James Lind Alliance (JLA) for priority settingWe anticipate that the next steps are twofold. First, policy makers, practitioners and decision‐makers within local VA and healthcare systems need to be informed about our findings. The research team has already started the dissemination process. For example, our findings were disseminated to the statewide Veterans service organizations. Second, final priorities will be disseminated with researchers so that they can utilize this information to guide the design of future research projects. In fact, our PAC members already provided additional consultation to several VA researchers outside the study. Together, we plan to investigate many of the priorities outlined here with large comparative effectiveness research studies.Patient engagement has the potential to enrich our understanding of patient priorities for research. Given the current focus on developing patient‐centred research questions, we suggest future studies to evaluate various patient engagement approaches and determine what approach works best, under which circumstances. Successful approaches will build trust in the patient–researcher partnership, ensure that patients are meaningfully engaged throughout the process and include diverse patient experiences and perspectives. We present our patient engagement approach as one option for groups interested in facilitating collaboration among multiple stakeholders to identify shared research priorities. The study highlights a hypothetical counterfactual—how different would research priorities be if selected solely by scientists versus this diverse patient advisory council. We noted that the research priorities were not the same as those initially identified by the scientists/health professionals, which points to the outcome of this hypothetical counterfactual. This study calls for future studies to assess whether lung cancer screening actually becomes better optimized by targeting research priorities identified by scientists or priorities identified by patients through a patient‐centred engagement process. While we likely will not truly know the answer to that experiment, the current study does provide valuable guidance around how to conduct a high‐quality patient advisory council.The authors declare that there are no conflict of interests.Onur Asan, Alice Yan and Jeff Whittle conceived the study and obtained the funding. Alice Yan, Katinka Hooyer and Jeff Whittle wrote the manuscript with input from all authors. Alice Yan, Katinka Hooyer, Mark Flower and Jeff Whittle contributed to the implementations of the project. Alice Yan and Jeff Whittle contributed to the revision of the manuscript. Jeff Whittle helped supervise the project. All authors were involved in critical revision of the article. All authors discussed the results and contributed to the final manuscript.


Inadequate nutrient intakes are prevalent among many populations in sub‐Saharan Africa and increasing fish consumption among pregnant/lactating women and children is one strategy to improve diets and address nutrient deficiencies. We report the nutrient content of two fish‐based recipes—fish powder and fish chutney—that contain dried small fish available in local markets in Zambia. The contribution of a serving of each recipe to the recommended daily intakes of iron, zinc, calcium and docosahexaenoic acid (DHA) for pregnant/lactating women and children 6–24 months was calculated because these nutrients are commonly deficient in African diets. We found that one 10‐g serving of fish powder provides 20% or more of the daily calcium recommendation and 37% or more of the daily DHA recommendation for both pregnant/lactating women and children. A 30‐g serving of fish chutney provides over 40% of the daily calcium recommendation for pregnant women and over 50% for lactating women. Additionally, we investigated the nutrient density  of the fish powder and compared it with the nutrient density of a small‐quantity lipid‐based nutrient supplement plus (SQ‐LNS‐plus). SQ‐LNS‐plus is designed to enhance children's diets by providing micronutrients and DHA. Fish powder is similar to SQ‐LNS‐plus in iron and zinc density and even higher in calcium and DHA density. Consuming dried small fish as part of a daily meal can be a viable strategy for combatting nutrient deficiencies in the first 1000 days. Fish‐based recipes that use locally available dried small fish have the potential to address nutrient deficiencies that often occur in the first 1000 days of life.Fish powder is a concentrated source of iron, zinc, calcium and omega‐3 fatty acids, which can greatly improve the quality of a meal when added in small amounts.Leveraging locally available and culturally acceptable foods, such as small fish, is an important consideration when designing programmes to address malnutrition in low‐ and middle‐income countries.1Consuming animal‐source foods (ASF) during the critical window of pregnancy, lactation and up to 2 years of life  is a key strategy for breaking the cycle of malnutrition , a small pelagic fish found in Lake Malawi, is high in the omega‐3 fatty acid docosahexaenoic acid (DHA)  of the foods consumed by infants 6–11 months old is especially important given their high nutrient needs relative to their low caloric needs Dewey, . This isOur study compares the nutrient density of a locally produced fish powder to SQ‐LNS‐plus, a specialized complementary food product, to add to the information on the range of tools low‐ and middle‐income countries can deploy to address malnutrition. Although there is a clear role for specialized complementary food products, foods that utilize small fish, such as fish powders, have not been adequately investigated as a potential alternative.The aim of this study was to assess the nutrient content of two locally developed fish‐based recipes—fish powder and fish chutney—and to calculate the contribution of calcium, zinc, iron and DHA to the diets of pregnant and lactating women and children 6–24 months. We also compared the nutrient density of iron, zinc, calcium and DHA of fish powder with SQ‐LNS‐plus. Our findings will contribute evidence to guide the formulation of policies and practices intended to improve the diets of resource‐poor women and children.2We chose iron, zinc and calcium as micronutrients of interest given they are often found to be inadequate in the diets of women and children in low‐ and middle‐income countries  was selected for the fish powder, and inkundu (Pseudocronilabrus philander) was selected for the fish chutney, given their availability in the districts where the recipes were developed. It was assumed that if the inkundu and dagaa fish‐based foods were acceptable as ingredients, then other small fish would also be acceptable, as there are many small indigenous species found in this region. Known as dagaa in Zambia and Tanzania, omena in Kenya and mukene in Uganda, this freshwater small pelagic fish species is endemic to Lake Victoria  to ensure the recipes could be produced at the household level. The fish were purchased dried from local markets. One 100‐g sample of each recipe was prepared. The nutrient composition of the two fish‐based recipes was analysed at the Microchem Specialized Lab Services in Cape Town, South Africa, in 2017. Recipes were homogenized prior to sampling. Only one sample was tested per fish recipe due to resource constraints. The amount of energy was determined using chemical analysis  for iron and zinc and the recommended intakes for calcium and DHA from the WHO/FAO guidelines for vitamins and minerals  of 27 mg/day for pregnant women based on the Institute of Medicine guidelines  produced by DSM Nutritional Products Ltd, a company headquartered in The Netherlands  listed in the recipe booklet, contained no DHA.The fish chutney, designed to be consumed as a condiment, provided 7% of the iron recommendation for pregnant women and 13% for lactating women Figure . It prov3.3We also found that a small amount of fish powder can improve the diets of infants and young children by providing additional iron, zinc, calcium and DHA to a given meal. A single 10‐g serving of fish powder provided 12% of the iron recommendation for infants 6–11 months and 19% for children 12–24 months Figure . Put ano4Our study found that fish‐based recipes have a high potential to address inadequate nutrient intakes in Zambia. Processing the dried small fish into powder achieved the optimum nutrient density of iron, zinc and calcium for the diets of children between 6 and 11 months old, a stage when adequate nutrient intake is highly critical and difficult to achieve. We also found that dried small fish in the form of fish powder fulfils a large percentage of women and children's daily calcium and DHA nutrient recommendation. Fish chutney can provide additional calcium, and some iron and zinc, to the diets of pregnant and lactating women, even when consumed in small amounts as a condiment. In places where dried small fish are readily available, national nutrition programmes can promote the consumption of this natural resource as a means of reducing nutrient deficiencies among nutritionally vulnerable populations.Nutrient deficiencies in infants and young children can lead to lifelong growth impairments, thus obtaining enough nutrients from the diet is paramount during the first 1000 days of life  have comparable nutrient compositions (Hicks et al., Our study is not without limitations. Due to budget constraints, we were not able to analyse a full panel of nutrients or collect information on microbial contamination in the recipes. Future studies might analyse the concentration of vitamin B12 in fish powder and chutney, because indigenous small fish have been shown to be an important source (Thilsted et al., 5Diets in many parts of Zambia provide low amounts of nutrients that are critical for early growth and development. Incorporating nutrient‐dense ASF, such as dried small fish, into recipes made with locally available ingredients is a promising strategy to alleviate nutrient deficiencies. Processing dried small fish into powder form is a viable way to increase the densities of nutrients in a given meal. Fish powder can provide an equal, or greater, density of iron, zinc, calcium and DHA than some specialized complementary food products and can be produced at home. Given the promising nutrient density of fish‐based recipes, it would be advantageous for both policy makers and civil society to promote recipes such as those presented in this study as a way to complement other approaches used to improve malnutrition within the first 1000 days of life. Additionally, programmes that work with households to promote the nutritional benefits of consuming small fish and preparing fish‐based recipes, such as those tested in this study, can help resource‐poor consumers take advantage of food resources in new ways.The authors declare no conflict of interest.KAB, LP, SMC and MMP conceptualized the study. SMC and FM developed the methodology. KAB and LP performed the formal analysis. KAB, LP, SMC and MMP wrote and prepared the original draft. KAB, LP, SMC, FM and MMP wrote, reviewed and edited the manuscript. KAB performed the visualization. KAB supervised the study. SMC did the funding acquisition. All authors have read and agreed to the published version of the manuscript.Table S1: Calcium, iron, and zinc requirements per 100 kilocalories used for calculations in Figures 2a‐dClick here for additional data file.


Social distancing measures due to the COVID‐19 pandemic will make anatomy dissecting room practicals difficult, if not impossible to run at some institutions in the upcoming academic year. The learning community that exists within physical anatomy practicals needs to be moved online. Virtual replacement of visuo‐spatial and social elements of learning anatomy pose particular challenges to educators. Our department has trialed Blackboard Collaborate, an online communication platform in conjunction with Visible Body, a 3D anatomical modeling program. We have delivered 266 hr of synchronous small group teaching to medical and physician associate students. We describe this approach and discuss the relevance of distance learning pedagogy to the design of new online anatomy teaching and development of online learning communities. Its use as a virtual classroom in anatomy education has been demonstrated previously in the literature  framework . Students were distributed non‐randomly into existing groups of between eight and 16 students who were already known to each other from previous F2F teaching. Webinars were scheduled to last 90 min, reflecting the time allocated for equivalent F2F practical classes. Many international students are enrolled on our course and scheduling took into account time zone limitations, with students allowed to swap groups for more appropriate timings. Groups were allocated the same teacher for each session to assist continuity. Each webinar centered on a slideshow presentation developed and uploaded by faculty members to the BBC platform in advance. The presentations were constructed using PowerPoint 2013  and addressed a standardized set of learning outcomes, while allowing for tailoring of content to specific group needs. Webinar slides included some more traditional text and diagrams, but emphasis was placed on incorporating clinical, radiological, and cadaveric images wherever possible.3.3Blackboard Collaborate can facilitate a bidirectional flow of written, oral and visual communication. In our webinars, tutors communicated with the students through audio‐visual means. Students were allowed to interact in whichever way they found most comfortable, which included writing or drawing on the slides, typing into an instant messaging chat bar, or by using their microphones. Where background noise became disruptive, students were asked to turn off microphones and turn on again as needed.3.4Faculty members guided students through slideshow content, which incorporated demonstrations of other programs through screensharing, and various learning activities. We broadcast Visible Body 3D models Figure  and mani3.5Most slides were designed to exploit the interactive and collaborative functions of BBC. PowerPoint files uploaded and internalized into the platform can be typed or drawn upon using a pencil tool, allowing custom slides to provide a canvas for the facilitation of a variety of learning activities. Annotations and drawings are instantaneously visible to all participants, and any number of students can perform these functions at once. We used these features to encourage students to work collaboratively in group activities Figures  and 3. T44.1Over 2 months during lockdown in the UK, our department ran a total of 266 hr of synchronous online anatomy teaching. It was feasible between five educators to provide small group webinars to cover the learning objectives that would have been addressed in larger group F2F practicals. The faculty consensus was that most students engaged well in sessions, and sometimes better than generally occurs in F2F teaching. From our perspective, the use of 3D models and group learning activities during webinars were essential to their success. Incorporating these into the BBC platform helped us to maintain student engagement, synchronously test retention and application of knowledge, and use feedback to provide real‐time tailoring of the teaching agenda.4.2Learning anatomy involves developing an understanding of complex structures and their interactions with each other in space. Three dimensional modeling packages like Visible Body have emerged as a promising and effective way of learning anatomy outside the dissecting room  including body language, facial expression and eye contact. These provide a crucial source of instantaneous feedback on the contentedness and attention of students for teachers. Non‐verbal communication can be partially replaced through videoconferencing for very small groups (Nichol & Watson, 4.4It is important to consider the impact of transitioning to online anatomy teaching on the wider medical curriculum and student experience. This academic year a significantly greater proportion of the early medical curriculum will be delivered through remote means. There may be other important benefits to developing online learner communities outside of learning anatomy. It has been acknowledged that traditional F2F anatomy practicals serve as an important forum in facilitating early development of professional skills Swick, . These i5At a time when social distancing measures are isolating students from family and friends, it may be more important than ever to provide a sense of community in our teaching. Although evidence and experience describing how best to do this in anatomy education is limited, distance‐learning pedagogy can inform the design of teaching to help develop new online communities of anatomical inquiry. We have found running synchronous teaching incorporating small group learning activities to be a viable approach, even for large student cohorts. Distance‐learning pedagogy would suggest that harnessing the collaborative and interactive functions now available in online communication platforms could develop social and cognitive presence in teaching. In our faculty's opinion these functions and the group learning activities they afforded were vital to maintain student engagement and enhance learning in webinars. The main limitations to our experience include that students and teachers were already known to each other from past F2F teaching, and that the evaluation occurred over 2 months only. Students who have not met peers or faculty members in a F2F setting, or who have more prolonged periods of online education may engage differently. Detailed investigation will be required to assess the impact of virtual curricula on anatomy education, and the student experience. Applying the Community of Inquiry framework may allow for an holistic evaluation of online anatomy education, and a survey instrument to facilitate this has been previously validated in healthcare education settings (Carlon et al., The authors declare no potential conflict of interest.William P. Flynn wrote the first draft of the manuscript. All authors reviewed and edited the manuscript and approved the final version of the manuscript.


OBJECTIVES/GOALS: This project aims to ensure that the age ranges of participants in OHSU studies for specific diseases match the demographics of the populations the diseases occur in, as mandated by the new NIH Inclusion of Individuals Across the Lifespan as Participants in Research Involving Human Subjects policy. METHODS/STUDY POPULATION: This study involves retrospective and prospective data. The retrospective phase (“Look Back”), reviewed all investigator-initiated OHSU studies between 2017 and 2018 with prospective consent that were disease related (N = 63). Age range per IRB protocol and per subject enrollment were graphically compared to disease demographics to determine if study age ranges were a “match” or “mismatch” to disease demographics . This data will inform the upcoming prospective phase of the study, when the study team will reach out to primary investigators of enrolling studies with education and resources, and track whether this reduces demographic “mismatch.” RESULTS/ANTICIPATED RESULTS: Of the studies, 51 were evaluated in the “Look Back” analysis. 40 studies were full matches for age inclusion matching disease demographics (78%), 40 for disease prevalence range (78%), and 38 for enrolling subjects within the disease demographic range (74%). Studies received the lowest scores in enrolling subjects that match disease prevalence, with 19 earning full points (37%) and 17 earning 0 points (33%). Limitations include difficulty in finding and applying disease demographic and prevalence ranges. In addition, in this data, 12 of the original 63 total studies could not be scored because no subjects had been enrolled or prevalence ranges were not in line with clinical expertise. DISCUSSION/SIGNIFICANCE OF IMPACT: This study highlights that many trials exclude older subjects at the upper age ranges. Future analysis of the prospective phase of the study will allow us to assist research teams in closing these gaps and will determine the Policy’s impact on the recruitment of older adults into research.


Streblospio benedicti, females make either many small eggs that develop into complex larvae that feed in the plankton or few large eggs that develop into benthic juveniles without having to feed as larvae. By isolating the contributions of maternal, paternal, and zygotic genotype to larval traits, we determined that larval anatomical structures are governed by the offspring genotype at a small number of large‐effect loci. Larval size is not shaped by the larva's own genotype but instead depends on loci that act in the mother, and at two genomic locations, by loci that act in the father. The overall phenotype of each larva thus depends on three separate genomes, and a population's response to selection on larval traits will reflect the interactions among them.The evolutionary potential of a population is shaped by the genetic architecture of its life‐history traits. Early‐life phenotypes are influenced by both maternal and offspring genotype, and efforts to understand life‐history evolution therefore require consideration of the interactions between these separate but correlated genomes. We used a four‐generation experimental pedigree to estimate the genetic architecture of early‐life phenotypes in a species with dramatic variation in larval size and morphology. In the polychaete annelid  Trade‐offs between the number and quality of offspring sit at the heart of life‐history theory, dictated by finite maternal resources that can be allocated in different ways. Mothers can produce small numbers of large, well‐provisioned offspring, or large numbers of small, poorly provisioned offspring. Ecological factors govern the optimal position of a population along this trade‐off front Vance .Heliocidaris sea urchins  eggs that develop into characteristic planktonic larvae, which feed on unicellular algae for a period of days or weeks before undergoing a metamorphosis and settling into the benthic mud. Other females produce a much smaller number of much larger eggs . These eggs develop into large larvae that do not need to feed and can immediately metamorphose and settle. In both modes of development, embryogenesis and larval morphogenesis occur in brood pouches on the female's back, and offspring are released into the water as fully formed planktonic larvae that have to feed (planktotrophs) or metamorphosis‐competent larvae that do not require planktonic food, provisioned by yolk (lecithotrophs).To understand how genetic architecture shapes the evolution of larval life‐history traits, in the context of qualitative transitions in larval form, we have developed an experimental model in the estuarine polychaete ng Levin . Some S.S. benedicti larvae differ substantially in form , made from crosses among 115 G2 females and 53 G2 males. Individual G2 males sired an average of 2 G3 families , 2 (n = 12), 3 (n = 13), 4 (n = 8)). The G2s used as parents are a subset of the 183 females and 58 males described in our earlier study , using the sim.geno function in rqtl , number of defensive chaetae, length of the longest chaeta, and presence or absence of anal cirri.To phenotype G3s to genotypes from the G2s, we employed mixed‐effect models that account for family structure and accommodate parental genetic effects. We performed a series of genome‐wide linkage scans to detect QTL. We subsequently tested the significant QTL for their transmission mode , dominance, and epistasis.To map quantitative‐trait loci (QTL) underlying trait variation in the G3 phenotype vector y asTo search the genome for QTL, we step through each genetic marker position, modeling the GQM vector holds the count of lecithotroph alleles at the marker in the G2 mother (0 to 2), the QP vector holds the count of lecithotroph alleles at the marker in the G2 father, and betas are the additive effects of maternal and paternal alleles at the marker. We model the family structure with random effects for the mother and father. Incidence matrices M and P connect each larva with the appropriate entry in the γs, vectors of random effects associated with each parent. Here the random effect of mother accounts for diverse sources of similarity among full sibs; these include background maternal and zygotic genetic effects, maternal environmental effects, and effects due to early development in a common environment. The paternal effect accounts for similarity among half sibs, which should only be present if there are zygotic or paternal genetic effects. ε is the residual error.Here, μ is the global mean vector, the We compare the fit of this model to a no‐QTL model,This model is nested within the previous model and has two fewer parameters. We use twice the difference between the models in log likelihood as a test statistic. This is the canonical likelihood ratio test statistic, asymptotically distributed as a chi‐square under the null hypothesis. For genome scans, we use permutations, described below, to generate genome‐wide null statistics.3 larva,T subscript indicates total allele count (0 to 4). We again test this model against (2), the no‐QTL null model.For purely zygotic‐effect inheritance, we can achieve higher power by using a single beta, as the effects of maternal and parental alleles should be identical. Additive zygotic effects can therefore be modeled as the effect of the sum of alleles across the two parents of each GTo directly test for parental effects at QTL detected by the tests above, we compare models 1 and 3 to one another, testing whether the model fit is significantly improved by allowing allelic effects to differ according to the parental sex. Rejection of model 3 is evidence of maternal effects, paternal effects, both kinds of effects, or a combination of parental and zygotic effects. To test a purely maternal‐effect model at such loci, we compare model 1 to a reduced model with no effect of paternal genotype at the locus:Rejection of model 4 indicates that the paternal genotype at the marker matters, rejecting a purely maternal‐effect model. Similarly, a paternal‐genotype‐only model tests whether the maternal genotype matters:2 male and G2 female genotypes independently, preserving the underlying full‐ and half‐sib relatedness among G3s. The permutations are also stratified by G2 family , as in Zakas et al.  then test for additional Qβ terms. Note that we use permutations to define genome‐wide significance for linkage scans (step 1 in the testing framework), and for subsequent tests involving the loci detected in step 1 use uncorrected p = 0.05 threshold from likelihood ratio tests.When the initial genome scan identified a significant locus , we then incorporated the genotype at that locus into a second genome scan, using the genetic model for the locus identified by comparisons among models 1, 3, and 4. This forward search continued until no additional loci exceeded a genome‐wide permutation‐based threshold of 2 male hemizygosity, unknown sexes among the G3 larvae, and unbalanced G2 genotypes due to the direction of the pedigree‐founding cross. As the sole F1 male in the pedigree carried the X chromosome from its planktotroph mother, all G2s females carry at least one planktotroph X chromosome. Denoting the lecithotroph allele L and planktotroph allele P, G2 females can never be LL homozygotes at X‐linked loci. We can nevertheless test for linkage to X‐chromosome markers by comparing the full model to the no‐QTL model (1 vs. 2), and we can test for parental effects by comparing the full model to the one‐parent‐only model (1 vs. 4).The analyses described above were applied to autosomal markers. The models are not precisely suited to the transmission genetics of the X chromosome, because of G2 population, there are four types of crosses  with X‐linked allelic affects weighted according to the sex and cross. For X‐linked markers in our Gses Fig. . Under t2 phenotypes found no evidence for sex‐linkage or for differences between sexes in any larval phenotypes , number of larval chaetae (on one side of the larva), length of the longest larval chaeta, and presence or absence of anal cirri. Number of chaetae is a mixture of a point mass at zero (24.8% of larvae) and distribution with a mode at 4 chaetae and a long right tail . We therefore modeled number of chaetae as two separate traits: presence/absence of chaetae as a binary trait, fit with a generalized linear mixed model approach , and Box‐Cox‐transformed chaetae number conditional on their presence, using the standard approach. These two approaches to the data yielded extremely similar results, and so we proceeded by combining the two models . The result is a mixed‐model analog of the two‐part model of Broman . The larTo test for directional polygenic effects, under the assumption that loci that differ between lecithotrophs and plankotrophs will tend to affect larval traits in a predictable direction, we used expected autosomal ancestry  as a predictor in a mixed effect model that also includes the genome‐wide significant loci. We consider both biparental ancestry  and uniparental ancestry . We estimate ancestry as the fraction of lecithotroph alleles at autosomal markers, excluding the chromosomes that carry genome‐wide significant loci for the trait . This exclusion step is necessary because genotypes across the chromosomes with major‐effect loci are correlated by linkage with the major‐effect locus genotype, and so the contribution of that chromosome to polygenic effects is confounded with its major‐locus effect.3 larvae. Instead, we tested for dominance by asking whether the fit of the additive zygotic model was significantly improved by addition of a coefficient for the expected heterozygosity of the larva .For loci inferred to act via parental genetic effect, we tested for dominance effects by incorporating a term for parental heterozygosity. For loci inferred to act via zygotic effects, we cannot use this direct approach because we lack individual‐level genotypes for the G3 phenotypes, we integrated G2 and G3 phenotype data into a single genome scan for zygotic effects by summing the G2 and G3 log likelihoods at each marker under an additive zygotic model and testing against the summed log likelihoods of the relevant no‐QTL models. We also performed this genome scan after incorporating each of the established QTLs into the model, with dominance where appropriate, as covariates. Likelihoods for the G2s were estimated under simple fixed effect models. Note that this approach allows for the allelic effects of a marker to differ between the generations. Significance thresholds were estimated by structured permutation of the male and female G2 genotypes, preserving relatedness between each G2 animal and its progeny.To improve mapping resolution beyond that achieved with Gh2) using mixed‐effect parent‐offspring regressions for traits with continuous distributions . For additive zygotic‐effect inheritance, the slope of the regression of offspring phenotypes on the average phenotype of the parents, accounting for family structure with random effects, is an estimator of h2. We tested whether the heritabilities were different from zero by comparing the regressions to a null model with no effect of parental phenotype, and we tested for differences between maternal and paternal estimates by including the two parental phenotypes as separate fixed effects. Note that our heritability estimates apply only to transmission of traits from G2 to G3 in this specific cross. To estimate whether the detected QTLs account for the estimated heritability, we incorporated the QTLs as fixed effects in the regressions and compared these to no‐QTL regressions. We tested whether heritabilities were different from zero using likelihood ratio tests.We estimated narrow‐sense heritability  are genetically heterogeneous and the phenotypes of their G3 offspring therefore permit us to detect loci acting both parentally in the G2s and zygotically in the G3s.We studied the great‐grandchildren from a cross of a planktotrophic female and a lecithotrophic male. The grandchildren . These results imply that we have detected the loci responsible for the bulk of the additive genetic basis for these traits.We used mixed‐model parent‐offspring regression to estimate the heritability of chaetae length and number Table . We esti2 and G3 generations, we can achieve higher power to detect and localize zygotic‐effect quantitative trait loci. We found that the genetic signals added and background noise largely cancelled, leaving the few significant QTL defined more clearly , consistent with paternal effects. Both loci also nominally reject the uniparental paternal‐effect model , suggesting maternal effects. The direction of the inferred effects depends on the parent; lecithotroph alleles carried by mothers are associated with larger larvae, and the same alleles carried by fathers are associated with smaller progeny . However, this complication should not result in a parent‐dependent change in the sign of an allelic effect, such as we observe on the X‐linked locus affecting larval size.3 data do not allow us to simultaneously estimate maternal, paternal, and zygotic effects, but we can test for zygotic effects in a setting that controls for maternal and paternal effects. Specifically, the G2 generation varies zygotically but all individuals share heterozygous F1 parents and so parental‐effect variation is absent. We found no evidence for direct effects of the loci on X (p = 0.88) or chromosome 9 (p = 0.21) in an analysis of 240 G2 larvae , the fraction of remaining maternal genome derived from the lecithotrophic founder explains additional variation in larval size . Wild S. benedicti: its multilocus basis guarantees that parental types will be exceedingly rare among segregating cross progeny, and most advanced‐generation hybrids will be phenotypically intermediate. The genetic architecture therefore implies that the absence of intermediates in nature reflects strong selection against advanced‐generation hybrids.This result may in part be down to numbers. Although our study includes thousands of individuals, they come from only 115 full‐sib families. Particular homozygous genotype combinations are rare in this collection. For example, only three of the families involve crosses of a male and a female that are both homozygous LL at the chromosome 5 locus affecting cirri. For traits affected by multiple loci, particular parental genotype combinations may be altogether absent. This reinforces an important feature of the genetic architecture of larval life‐history in 1s, whose autosomal genomes should be equivalent: F1s with lecithotroph mothers often lacked chaetae entirely, while those with planktotroph mothers almost always had chaetae. The findings from reciprocal F1s suggest that we may be missing maternal effect loci.In addition, our findings are partly inconsistent with previous work that studied inheritance of larval traits using classical line‐cross methods, comparing phenotype distributions in reciprocal crosses and backcrosses rather than tracking individual genotypes . To test the hypothesis of a polygenic epistatic effect that manifests only at the extremes, we tested the G3 data for quadratic and cubic effects of maternal genetic background and found no significant effects. Thus, if present, maternal‐effect epistasis is likely to involve higher order interactions that are absent or rare in the G3 population. In nature, however, most hybrids will be F1s, with purely lecithotroph or planktotroph maternal backgrounds, and these animals will most often backcross to the locally common form rather than intercross with other hybrids. We therefore anticipate that higher‐order epistasis, manifest only in “pure” genetic backgrounds, could play an important role in inter‐population gene flow in wild S. benedicti.The second class of explanation for missing maternal effects invokes epistasis, specifically genes that only show their effects when a large fraction of the maternal genome comes from a single background. In reciprocal FStreblospio benedicti permits several possible mechanisms of paternal effect. Because our larval size trait is measured after embryogenesis, it may reflect both prezygotic variation, such as egg size, and postzygotic variation, such as differential allocation of yolk resources to growth and metabolism.One surprising result of our analysis is the discovery of paternal‐effect loci acting on larval size. This finding is consistent with our earlier line‐cross study of larval size (Zakas and Rockman S. benedicti males and females do not have to interact to mate; males release spermatophores onto the benthic mud and females retrieve them and store sperm for subsequent fertilizations. Plausible mechanisms for paternal effects in this case include effects of spermatophore components on oogenesis. During laboratory matings, males and females are paired in small arenas for days or weeks, during which time water‐borne chemical signaling between male and female is also possible. A suggestion of signaling between adults comes from an account of experimental crosses between genetically divergent Streblospio populations and species: incubated with distantly related females, S. benedicti males stopped producing spermatophores, even though they readily produce spermatophores when housed by themselves (Schulze et al. Although egg‐size is superficially a purely maternal trait, females may alter oogenesis in response to genetically variable signals from males. Streblospio is genetic variation in imprinting, or parent‐of‐origin‐dependent gene expression. Imprinting is fundamentally different from ordinary parental effects; the former involves gene expression in the offspring and the latter gene expression in the parents. Nevertheless, genotype‐dependent imprinting generates a genotype‐by‐parent‐of‐origin interaction whose phenotypic effects can mimic antagonistic maternal and paternal effects. Alleles at the two paternal‐effect loci may themselves be differentially imprinted, or they may act in trans to differentially regulate imprinting of other loci. To test imprinting models with transmission genetics, we would require additional data on the genotypes of individual G3s and their parental origins (Hager et al. Alternatively, paternal genetic variation may influence larval size through postzygotic effects on growth and metabolism. Although such paternal effects could be mediated directly by sperm‐borne cytoplasmic components (Seidel et al. The larvae of marine invertebrates provide a rich testing ground for models of life‐history evolution, with major transitions between planktotrophy and lecithotrophy a frequent motif Thorson  and poweBoth authors designed and conducted the experiments, performed the analyses, and wrote the manuscript.All data discussed in the manuscript are provided to readers in Supplementary File 1, along with all scripts required to reproduce the results and figures.We have no conflicts of interest to disclose.Associate Editor: T. FlattHandling Editor: T. ChapmanSupplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Table S1. G3 linkage scan results.Table S2. Heritability analyses from parent‐offspring regressions.Table S3. Joint G2‐G3 additive zygotic linkage scan results.Click here for additional data file.


We describe maleic‐acid derivatives as robust cysteine‐selective reagents for protein labelling with comparable kinetics and superior stability relative to maleimides. Diamide and amido‐ester derivatives proved to be efficient protein‐labelling species with a common mechanism in which a spontaneous cyclization occurs upon addition to cysteine. Introduction of chlorine atoms in their structures triggers ring hydrolysis or further conjugation with adjacent residues, which results in conjugates that are completely resistant to retro‐Michael reactions in the presence of biological thiols and human plasma. By controlling the microenvironment of the reactive site, we can control selectivity towards the hydrolytic pathway, forming homogeneous conjugates. The method is applicable to several scaffolds and enables conjugation of different payloads. The synthetic accessibility of these reagents and the mild conditions required for fast and complete conjugation together with the superior stability of the conjugates make this strategy an important alternative to maleimides in bioconjugation. Maleic acid derivatives are studied as cysteine‐selective reagents for protein labelling with comparable kinetics and superior stability to existing reagents. A novel mechanism is described to readily access stable chloro‐maleamic acid. The mild conditions, fast kinetics, and irreversible conjugation make this method an appealing alternative to existing cysteine‐targeted methods. Over the last decade protein site‐selective conjugation has become a thriving research field.However, it is well known that thio‐succinimide adducts can undergo fast and uncontrolled disruptive cleavage by thiol‐exchange in plasma, which ultimately compromises the safety and efficacy of the conjugate.There are also acyclic reagents, such as benzoyl acrylic reagents11, 25We hereby report on diamide and amido ester derivatives from maleic acid as new, efficient reagents for Cys‐selective protein conjugation. These reagents undergo fast Michael addition reactions with free Cys‐containing proteins in stoichiometric amounts under physiological conditions. Conjugation occurs through an unprecedented mechanism in which, upon Cys addition, spontaneous cyclization is promoted by attack of an amide moiety to the distal carbonyl group to form a succinimide link Scheme . By expaWith the goal of finding alternative approaches to access homogeneous conjugates with fully stable linkages in free Cys‐containing proteins, we set out to investigate maleic acid derivatives with labile functionalities, which we envisioned could undergo fast Michael addition with thiols under physiological conditions and, upon conjugation, would hydrolyze to form succinamic acid structures or equally stable analogues.1–10, Figure , Conditions A, Figures S3–S6) to assess their reactivity as Michael acceptors. Having determined the reactivity of 1–10 in small molecule experiments, we now exposed these reagents to the presence of a free Cys‐containing protein . As a model protein we chose Ub, which was engineered with a reactive Cys at position 63 (K63C). Surprisingly, the reactivity of the different species varied depending on the setup. Some species showed high reactivity in small molecule reactions but no protein conjugation reactivity, whereas others proved to be very efficient protein conjugation reagents but were unreactive in a small molecule setup. In successful conjugations, Cys‐selectivity was confirmed after digestion by MS/MS studies  and by using Ellman's reagent as a chemical control .The reactivity of β‐mercaptoethanol (BME) was evaluated with different maleic acid derivatives  to determine if the cyclization was induced as a result of the pH of the solvent or the thiol addition. Amido esters 2 and 8 cyclized spontaneously in the presence of D2O pH 8 to form a maleimide species . However, this was not the general behavior for amido esters; compound 6 together with diamides 5, 7, 9, and 10 was completely stable under these conditions. This aqueous stability supports the idea that thiol addition in the protein environment is responsible for triggering the cyclization. By using diamide 10 and Ub as a model, the reaction showed great tolerance for pH  and temperature [25–37 °C ].Mass analysis of the protein conjugates formed revealed a unique conjugate with a mass of 8754 Da (protein mass + 188 Da) for all the examples. Interestingly, the mass of the newly formed conjugate did not correspond to the addition of the full reagent. Instead, we observed the formation of a thio‐succinimide linkage. The origin of such a linkage is through intramolecular attack of the amide to the distal carbonyl group Scheme , which c9, is conjugated, a regioselectivity issue arises. The presence of two secondary amide moieties allows for two possible cyclizations, each driven by one of the amides .When a reagent with two different secondary amide groups, such as s Scheme ; each paAnother appealing aspect of this conjugation reaction is the controlled release of small molecules in a particular microenvironment of the protein sequence. Upon cyclization, an amine (diamides) or alcohol (amidoesters) molecule is released, which could open new approaches for monitoring reactions (release of fluorophores), Cys‐targeted decaging of payloads or site‐directed bifunctionalization of proteins. We are currently investigating this directed release in our laboratories and will report it in due course.The thio‐succinimide linkages obtained are known to hydrolyze over time under physiological conditions to give a linear and stable N‐substituted thio‐succinamic acid linkage, but the kinetics of hydrolysis is extremely slow and often yields mixtures of conjugates. Harsh hydrolyzing conditions, such as high pH and temperature, could ultimately lead to the complete hydrolysis to succinamic acid linkages, but such conditions are not compatible or desirable with proteins because they can result in unfolding and loss of functional properties. To address this problem, we focused on accelerating the hydrolysis of the linkages created by the conjugation of maleic acid diamides.11–14, Figure 11 (3 equiv) to a solution of Ub resulted in complete conjugation 7. In all examples a Michael addition/cyclization mechanism to yield a succinimide was observed, which supports the generality of the mechanism in different protein sequence microenvironments. Analysis of the circular dichroism (CD) profiles of the conjugates relative to native proteins confirmed that overall the tertiary structure of the proteins was maintained throughout conjugation .The generality of the method was demonstrated by conjugation of 11–14). Alyphatic 11 and aromatic 12 amide species could be accessed as well as amides with an incorporated alkyne 13 or azide 14 functionality. These new functionalities were incorporated into the protein structure to enable further functionalization through copper(I)‐catalyzed alkyne–azide cycloadditon. Compounds 12–14 showed similar reactivity to 11 upon reaction with Ub to form stable, linear chloro‐maleamic acid linkages . However, when compound 14 was tested with different proteins, upon conjugation–cyclization, different reactivity was observed for some of these proteins. In Ub, C2AmUb‐14; the conjugate remained stable in buffer, and no degradation was observed after 48 h at 37 °C in the presence of glutathione (1 mm) or in human plasma .An array of dichloro butenediamide analogues with different functionalities were synthesized ], but all others formed selectively one of the two potential linkages. Conjugation of HER2‐targeted IgG Trastuzumab V205C with 14 led to a similar result .The enhanced plasma stability displayed by the conjugates formed from the reaction of Cys with reagents 14 . By mutating lysine 207 into an alanine, the new Trastuzumab V205C+K207A was conjugated to 14 and this time complete selectivity to the linear linkage was obtained . This result supported the idea that nearby lysine residues were the driving factors for the formation of the cyclic linkage, allowing us to predict the final linkage formed and if mixtures were to arise, avoid the formation of the cyclic species by introducing cysteines in the appropriate place or mutating the problematic lysine residues. We found, however, that mutation of lysine 207 in Trastuzumab V205+K207A reduces the nucleophilicity of cysteine 205 and only approx. 50 % conjugation is achieved under the previously described conditions .To explain this result at the atomic level, we performed molecular dynamics (MD) simulations on the Fab fragment of Trastuzumab conjugated to compound  Figures  and S87  Figures . The inv14, the Fc fragments with 274C and 289C reacted completely and selectively formed the linear linkage . These simple maleic acid derivatives enable installation of modifications on antibodies at specific sites to form homogeneous and stable antibody conjugates.To find suitable positions to genetically encode a reactive Cys that allow complete conversion, yet selective formation of the stable linear linkage, we produced Fc fragments of an IgG with Cys insertions at different positions . Under identical reaction conditions with 11 and its analogous maleimide reagent were conjugated to Fc fragment 289iC. Under the same conditions, 11 and maleimide showed comparable conjugation kinetics as a 1:1 mixture of Fc‐11 and Fc‐maleimide constructs was obtained . However, when we compared the stability of the constructs in the presence of GSH (1 mm) after 66 h at 37 °C, the constructs  conjugated to 11 showed higher stability than the corresponding species conjugated with maleimide . While the conjugates formed with reagent 11 remain intact in the presence of a thiol, constructs built using maleimides form a mixture of hydrolyzed maleimide and unconjugated antibody. These data show a key advantage of our reagents to build stable conjugates and illustrate how enhanced hydrolysis kinetics of the cyclic linkages allows the formation of fully stable species.To compare the kinetics of conjugation between maleic acids and maleimides, an equimolar mixture of We describe the protein conjugation reactivity of a series of maleic acid derivatives and identify amido esters and diamides as viable conjugation reagents to produce succinimide linkages through a new stepwise mechanism. The strategy was optimized to use dichloro butenediamides, which promote a further hydrolysis step to form completely stable linkages to proteins. This latter species shows comparable conjugation kinetics relative to maleimides yet benefits from superior stability. The general mechanism described composes a Michael addition followed by spontaneous cyclization to form cyclic linkages similar to those obtained by conjugation to maleimides. Introduction of Cl atoms into the cyclic linkages promotes subsequent hydrolysis, leading to fully stable linkages. Nearby nucleophilic residues can promote an alternative mechanism where a second nucleophilic attack leads to the formation of a cyclic linkage, but this pathway can be controlled and depleted if undesired conjugate mixtures are obtained. The approach allows for the creation of conjugates with azide/alkyne moieties and opens up possibilities to create tailored conjugation for a number of relevant proteins, including nanobodies or IgGs relevant for the formation of ADCs.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


SWEET genes encode sugar transporter proteins and often function as susceptibility (S) genes. Consequently, the recessive alleles of these SWEET genes provide resistance. This review summarizes the available literature on the molecular basis of the role of SWEET genes (as S genes) in the host and corresponding transcription activator‐like effectors (TALEs) secreted by the pathogen. The review has four major sections, which follow a brief introduction: The first part gives some details about the occurrence and evolution of SWEET genes in approximately 30 plant species; the second part gives some details about systems where (a) SWEET genes with and without TALEs and (b) TALEs without SWEET genes cause different diseases; the third part summarizes the available information about TALEs along with interfering/truncated TALEs secreted by the pathogens; this section also summarizes the available information on effector‐binding elements (EBEs) available in the promoters of either the SWEET genes or the Executor R genes; the code that is used for binding of TALEs to EBEs is also described in this section; the fourth part gives some details about the available approaches that are being used or can be used in the future for exploiting SWEET genes for developing disease‐resistant cultivars. The review concludes with a section giving conclusions and future possibilities of using SWEET genes for developing disease‐resistant cultivars using different approaches, including conventional breeding and genome editing. SWEET genes function as susceptibility genes. They are activated by binding of TAL effectors (secreted by the pathogen) following a code and can be exploited for developing resistant cultivars. The third part deals with the molecular mechanism involved in host–pathogen interactions involving SWEET genes and/or TALEs; the code that is used by pathogens’ TALEs in binding to effector‐binding elements (EBEs) available in the promoters of SWEET genes will also be covered in this section. The fourth part of the review discusses possible approaches that are being used for developing resistant cultivars using SWEET genes. At the end of the review, conclusions are provided and manipulation of these SWEET genes to achieve disease resistance in crops is discussed.In contrast to the above gene‐for‐gene model, the S genes follow an inverse gene‐for‐gene model, where the virulence/toxin gene of the pathogen can cause infection only when the host carries a dominant allele of a sensitivity/S gene. This system operates in the case of wheat diseases including tan spot, Stagonospora nodorum blotch, and spot blotch, where the host S gene like 2Eriobotrya japonica; Wu et al., Triticum aestivum; Gautam et al., A genome‐wide survey of SWEET genes has been undertaken in >30 species Table . The num3Arabidopsis thaliana , which provides resistance against bacterial blight caused by X. oryzae, as well as sheath blight caused by Rhizoctonia solani, is most important, although the functional mechanisms differ (see next section). The role of this gene in susceptibility to sheath blight was confirmed by the reduced susceptibility of transgenic plants carrying the mutant allele Ossweet11 and the increased susceptibility due to overexpression of the dominant allele OsSWEET11 , whose expression is induced by AvrBs3 in response to attack by Xanthomonas campestris pv. vesicatoria  and a clubroot‐susceptible (CS) line for the clubroot resistance gene CRb in Chinese cabbage (B. rapa), it was shown that infection by the pathogen led to a significant increase of glucose and fructose content in the roots of the CS line relative to that in the CR line. In another study involving field mustard (B. oleracea), higher expression levels were observed for six BoSWEET genes in roots of a CS cabbage cultivar (CS‐JF1) at 7 days after inoculation. Localization of a subset of BoSWEET proteins in the plasma membrane was also confirmed in CS‐JF1 , which is involved (as a cofactor) in the activation of S genes; its recessive allele xa5 encodes a protein that carries a single amino acid substitution (V39E) in TFIIAγ5, thus providing resistance against bacterial blight. Another unknown gene encodes TFIIAγ1, which compensates for the absence of TFIIAγ5, but no corresponding R gene like xa5 is known for TFIIAγ1  is activated by the effector PathXO6 and the gene encoding TFIIAγ1  is activated by the effector PathXO7; the genes encoding both these effectors are carried by PXO99A and cause bacterial blight.CsLOB1 is the target gene for citrus bacterial canker in citrus fruit, which is caused by Xanthomonas citri pv. citri and X. citri pv. aurantifolii. The expression of this gene is induced by each of three TALEs  from X. citri pv. citri and also by PthB and PthC, which are associated with susceptibility to citrus canker , involved in bacterial streak in wheat, which is caused by Xanthomonastranslucens pv. undulosa, is another example where the corresponding TALEs bind to the promoter of the gene. TaNCED‐5BS encodes the enzyme that catalyses the rate‐limiting step in abscisic acid (ABA) biosynthesis and has been shown to be responsible for susceptibility to this disease.The S gene UPA16, which is a SWEET gene, and UPA20, which encodes a bHLH TF) are activated by a TALE (AvrBs3), which binds to the UPA box available in the promoter of each of these genes  domain and an activation domain (AD)  through the T3SS into the host cell, so that TALEs represent a special class of these T3Es  that is activated by AvrBs3  N: R; (e) NS: N; (f) NK or NH: G ‐EBE (DNA) code was resolved in 2009 by two independent groups. The first group, led by Adam Bogdanove, searched for patterns of alignment between amino acid sequences of TALE proteins and nucleotide sequences of target promoters of SWEET genes using a database of genes up‐regulated by TALEs . The code has also been used for developing designer TALEs (dTALEs) that have been used to activate or repress the expression of SWEET genes and to explore their functions  are also available, which can predict the amino acid sequence of the central repeat region of TALEs and the nucleotide sequences of EBEs that are available in the promoters of host SWEET genes. These tools and their essential features are summarized in Table 4.3GhSWEET10 gene for susceptibility to bacterial blight in cotton ; Xa13 rendered the resistant rice cultivar IRBB13 (xa13/xa13) susceptible to Xoo  for specific EBEs; these dTALEs may be delivered through the bacterial secretion system to activate the host genes for susceptibility/resistance or for the purpose of their functional analysis. For developing dTALEs, modification of only the RVD is required; non‐RVD sequences have no major effect on binding to EBEs , causing infection, or interact with R genes , providing resistance. In rice, the Xa1 gene is responsible for resistance against all strains of Xoo that otherwise cause bacterial blight; Xo1 is a similar gene for resistance against both bacterial blight caused by Xoo and bacterial leaf streak caused by X. oryzae pv. oryzicola (Xoc) and was discovered later , which have been discovered during the last 5 years , Xa14, CGS‐Xo111, and Xa45(t). It was shown that all rice plants carrying Xa1, including transgenic rice plants carrying Xa1, were resistant to all such Xoo strains that lacked genes encoding iTALEs.In a subsequent study on the gene i et al.  cloned aXo1 gene for resistance against bacterial leaf streak was also studied in parallel by Read et al.  depends on the NLS domain of its C‐terminal region and the central repetitive transcription AD described earlier in this review. A TALE binds to an EBE available in the promoter of an ER gene, inducing transcription of this gene for providing resistance. Thus, a TALE may either bind to an EBE in a SWEET gene, causing susceptibility, or bind an available ER gene, providing resistance. While inducing ER gene expression, a TALE behaves like an Avr gene, suggesting a gene‐for‐gene relationship between TALE genes in the pathogen and ER genes in the host. Five ER genes have so far been cloned: 5SWEET genes provide only one of the several systems for disease resistance available to plant breeders. One possible approach to achieve resistance is to utilize the system (in the host) that prevents or reduces sugar supply. This mechanism has been described as “sugar starvation for disease resistance” or “starvation‐mediated resistance”  and OsSWEET14 (xa41), which are direct targets of the following five major TALEs: PthXo2, PthXo3, AvrXa7, TalC, and TalF  will not be activated. The majority of such mutants have been shown not to have any negative effect on plant phenotype, thus making these mutants useful for developing resistant cultivars . Heterologous expression of OsSWEET13 in Nicotiana benthamiana leaf cells also elevated sucrose concentrations in the apoplast, thus supporting the conclusion that Xoo enhances the release of sucrose from host cells, helping the pathogen in the compatible interaction. However, the gene OsSWEET13 of two japonica rice cultivars, namely Nipponbare and Kitaake, lack EBEs for PthXo2, so that these two cultivars are resistant to this strain . Using this information, it was also possible to use gene editing involving CRISPR/Cas9 technology to achieve resistance through mutation in the causal SNP in the promoter of the S gene OsSWEET13 . In this context, one can use computational approaches to allow identification of genes carrying EBEs in the dominant SWEET alleles’ promoters. Once validated, the EBEs provide an opportunity for finding insensitive promoters, as in the case of i et al. . In addi5.3Two important tools for genome editing include TALE nucleases (TALENs) and CRISPR/Cas9; both have been used successfully for the modification of EBEs in the promoters of SWEET genes Table .5.3.1OsSWEET11 and OsSWEET14 , but involving binding sites for only two of the four TALEs (AvrXa7 and PthXo3) in the case of OsSWEET11. The mutations obtained in this study provided resistance against the relevant strains of the pathogen. In the other study, Blanvillain‐Baufumet et al. , but not those used by TalC, were resistant to bacterial strains. The TalC EBE mutant line was, however, resistant to a strain expressing a dTALE inducing OsSWEET14 expression, whose EBE was also edited in this line. It was speculated that TalC may have additional targets, suggesting that TALE‐mediated plant susceptibility may result from induction of several, genetically redundant, host S genes by a single effector.There are at least two studies involving TALEN‐mediated genome editing of SWEET genes, including LF Table  obtainedt et al.  edited O5.3.2OsSWEET13 (xa25), so that Xu et al. (OsSWEET11 and OsSWEET14) for editing, leading to the production of an edited genotype named MS14K, which carried broad‐spectrum bacterial blight resistance. When MS14K was tested using 131 Xoo strains, it was found to be resistant to only 121 strains and susceptible to 10 Xoo strains, suggesting that these 10 Xoo strains may carry novel TALEs that activate S genes. Therefore, MS14K was used once again for editing the third gene (OsSWEET13). Xu et al. ; this objective was successfully achieved by both studies, although it remains unclear whether the edits introduced in the two studies are different. It is likely that there are some minor differences leading to the same result in terms of failure of binding of all known TALEs to the promoters of the three genes. One may also wonder whether it is desirable to combine the edits obtained by the two groups to further enhance the level of durable broad‐spectrum resistance against bacterial blight.The objective of both studies was to achieve durable broad‐spectrum resistance against bacterial blight through editing of EBEs for all the three SWEET genes  for resistance against bacterial blight represent these Group 2 ER genes, each containing multiple transmembrane domains functioning as a promoter trap  in its promoter ‐mediated knockdown of an S gene that facilitates infection. For instance, the dominant allele 6Tsn1, which also follow inverse gene‐for‐gene relationships and are used by some necrotrophic pathogens causing wheat diseases like tan spot, spot blotch, and Stagonospora nodorum blotch. In this case also (as in case of SWEET genes), the plant–pathogen interaction depends upon the availability of a dominant S gene in the host.Plant breeders develop disease‐resistant cultivars using a variety of approaches including pyramiding of R genes to provide durable resistance. The most important of these approaches is the use of dominant R genes, which follow gene‐for‐gene relationships. However, in recent years, several classes of dominant S genes have been discovered, so that the recessive alleles of these S genes are deployed for developing resistant cultivars. An important class of these S genes are those that are used by specific virulence genes in the pathogen causing the disease. One of the classes of these host S genes is the SWEET genes (encoding sugar transporters), whose dominant alleles are presumably used by the pathogens for supply of nutrition in the form of sugars. Another class of such sensitivity or S genes includes genes like SWEET genes have been identified in approximately 30 plant species, but they are only exploited in approximately 10 species as S genes by pathogens. Hopefully, more SWEET genes and species carrying these genes that are used by the pathogens will be discovered in the near future. Therefore, there is a need to continue the search for pathosystems involving SWEET genes functioning as S genes. In cases where SWEET genes are already known to function as S genes, there is a need to study the genetic variability of individual SWEET genes to (a) identify naturally occurring alternative recessive alleles for the dominant SWEET S genes, and (b) utilize them in crop breeding programmes for developing resistant cultivars. These SWEET genes carry binding sites in their promoters for a specific type of pathogen effectors called TALEs. TALENs and the 2020 Chemistry Nobel Prize winning approach CRISPR/Cas9 have also been used for genome editing, leading to the development of resistant genotypes.The knowledge about the molecular basis of pathogenesis involving SWEET genes has also been used for designing strategies for developing disease‐resistant crops. These strategies will be the focus of attention in future plant breeding programmes and may include a number of approaches discussed in detail in this review. The most important example of the use of SWEET genes to achieve disease resistance is bacterial blight of rice. Two important papers and a commentary published recently (Eom et al., Another important area of current and future research is the study of the origin and evolution of the T3SS, which is used by pathogens to secrete T3Es. The structure of the T3SS is known to play a crucial role in prokaryote–eukaryote interactions during infection, such that diversification of T3SSs in bacterial pathogens is also an important area of research; at least seven distinct T3SS families are already known (for a review, see Merda et al., TABLE S1 Number of SWEET genes reported following genome‐wide studies in different plant speciesClick here for additional data file.TABLE S2 Alternative genes used as targets by pathogens using TALEsClick here for additional data file.TABLE S3 Bioinformatics tools for identification and classification of EBEs and TALEs and also for designing TALEsClick here for additional data file.TABLE S4 A summary of studies conducted involving the use of natural/induced variations in SWEET genes to achieve disease resistance through loss of susceptibility in plantsClick here for additional data file.TABLE S5 A summary of natural variants in EBEs in the promoters of SWEET genes in riceClick here for additional data file.


The comparative utility of physiological reserve measures in predicting important clinical outcomes following liver transplantation (LT) requires further study. The aim of this work was therefore to compare the utility of physiological reserve measures in predicting early adverse clinical outcomes post‐LT.A single‐center, retrospective cohort study of LT patients consecutively recruited between 1 January 2015, and 31 August 2020. Outcomes measured were sepsis and death within 12 months of LT, hospital length of stay (LOS), and intensive care LOS. Physiological reserve measures were handgrip strength, mid‐arm muscle circumference, and cardiopulmonary exercise testing (CPET) measures. Analysis was performed using univariate and multivariate logistic regression for sepsis and death, and univariate and multivariate Cox regression for hospital and intensive care LOS.versus lowest tertile . Hospital LOS was linearly associated with handgrip strength  in multivariate analysis. Intensive care LOS was associated with peak VO2  and VE/VCO2 slope  in multivariate analysis.Data were obtained for 109 subjects. Patients were predominantly (64%) male with a median (interquartile range [IQR]) age of 57 (49–63) and median (IQR) Model for End‐Stage Liver Disease score of 16 (11–21). In multivariate analysis, the odds of sepsis were lower in patients in the highest Handgrip strength and CPET both identify candidates at high risk of adverse outcomes after LT. The authors aimed to compare the associations between the physiological reserve measures handgrip strength, mid‐arm muscle circumference, and cardiopulmonary exercise testing (CPET) with early adverse clinical outcomes post LT. A single‐centre, retrospective cohort was performed with LT patients recruited consecutively between 2015 and 2020. Handgrip strength and CPET both identify candidates at high risk of adverse outcomes post LT. The prediction of important clinical outcomes  following liver transplantation (LT) remains complex and challenging and is likely to be determined by multiple factors. Knowledge of the importance of physiological reserve and its impacts on clinical outcomes has been generated initially from the geriatric and surgical literature.Frailty is defined as “a state of vulnerability to poor resolution of homoeostasis after a stressor event and is a consequence of cumulative decline in many physiological systems during a lifetime”.Sarcopenia, a term that describes the loss of muscle mass and strength with increasing age,2 have been associated with both pre‐LT mortality and with increased post‐LT intensive care length of stay (LOS) and mortality.Cardiopulmonary exercise testing (CPET) has gained popularity as an objective measurement of “fitness” and is considered the gold standard for assessing physiological reserve. Lower anaerobic threshold and low peak VODespite the availability of a wide number of physiological reserve tests to assist in prediction of post‐transplant outcomes, there is a lack of consensus about the most useful and cost‐effective test to use in routine care. For example, to the best of our knowledge, there are no studies comparing resource‐intensive gold standard tests (CPET) with simpler bedside test .The aim of this study was therefore to compare these tests  performed during LT assessment in their ability to predict adverse outcomes after LT.This was a retrospective cohort study of patients who underwent liver transplant assessment and subsequent liver transplant at the South Australian Liver Transplant Unit, located at Flinders Medical Centre, Adelaide, South Australia. The South Australian Liver Transplant Unit provides LT services to a population of approximately 2 million people across South Australia and the Northern Territory. All patients who underwent LT between1 January 2015 to 31 August 2020 were eligible for inclusion in the study. All LT patients are followed up long‐term by the unit via both face‐to‐face and telehealth consults.Patient information was collected from both paper and electronic medical record systems. Information on physiological reserve assessments, biochemistry, Model for End‐Stage Liver Disease (MELD), and Child–Pugh scores were recorded at the time of transplant assessment.2), and minute ventilation/carbon dioxide production (VE/VCO2) slope.CPET was performed in a subset of 20 patients using a ramped exercise protocol aiming for 10 min of loaded exercise on a stationary bicycle with continuous monitoring of cardiac rhythm, expired gas, and pulse oximetry.et al.Handgrip strength was measured with a single spring dynamometer, as described by Klidjian et al.: MAMC = MAC − (3.14 × TSF thickness); where MAC is mid‐arm circumference and TSF is triceps skinfold.MAMC was calculated as described by Lohman To reduce measurement variability, handgrip strength and MAMC were performed by the same operator (ward dietician), and CPET testing was performed by the same operator (respiratory physiologist) during the study period.Length of ICU stay and overall LOS were calculated from the date of transplant to the date of discharge from ICU and hospital, respectively. Sepsis was defined as any severe infection requiring antibiotic treatment or hospital admission less than 12 months from liver transplant. Follow‐up time was taken from the date of transplant until death or 31 March 2021, whichever occurred earlier. There were no prehabilitation type interventions offered to patients on the waiting list beyond routine dietetic advice and management as appropriate for any patient with advanced liver disease.Descriptive statistics were performed using frequency and percentages for categorical data and mean (SD) or median (interquartile range [IQR]) for normally and non‐normally distributed continuous variables. Univariate and multivariate binary logistic regression were used to assess 12‐month sepsis and death. Length of hospital stay and length of ICU stay were assessed using univariate and multivariate Cox regression models with the Breslow method for ties. A hazard ratio below 1.00 indicated longer LOS. The multivariate regression models were adjusted for age, sex (model 1) and fully adjusted for age, sex, MELD score, Child–Pugh score, etiology, and the Charlson Comorbidity Index (model 2). For the outcome “sepsis within 12 months” an additional analysis was performed using handgrip strength as a categorical variable (using tertiles) and using predictive margins for tertiles of handgrip strength. Adjusting for gender is necessary to ensure the physiological reserve–outcome associations are not simply a result of confounding due to there possibly being differences in physiological reserve according to gender. However, there may still be differences in the strength of the physiological reserve–outcome associations according to gender. An additional stratified analysis was performed to assess for this.This study was approved by the Southern Adelaide Clinical Human Research Ethics Committee (AUD/20/SAC/211).There were 126 transplants performed on 124 patients during the study period. There were 15 patients who lacked data points for any of the physiological reserve measurement variables, so they were excluded from the final analysis of 109 transplants in 109 individual patients. Patient characteristics are shown in Table P = 0.014). Tertiles of handgrip strength were a significant predictor of sepsis within 12 months (Table P = 0.036) or highest  tertile when adjusting for age and gender. This relationship was also present when adjusting for multiple confounders for both the middle  and highest  tertiles. The observed rate of sepsis was higher in the lowest tertile (28.1%) compared with those in the middle (12.2%) or highest (10.8%) tertiles. The predicted probability of sepsis within 12 months, following full adjustment for patients in the lowest tertile for handgrip strength was 60.4%, for the middle tertile 14.6%, and the highest tertile 6.74%  on univariate analysis. This association persisted with full adjustment . Lactate threshold was associated with hospital LOS  after univariate analysis. When adjusting for age and gender, lactate threshold  and peak VO2  were both associated with hospital LOS. Although a shorter hospital LOS post‐LT with increased lactate and peak VO2 was seen when adjusted for age and gender, this was not significant on multivariate analysis with full adjustment. Hospital LOS was not associated with MAMC on any analysis.The associations between hospital LOS and physiological reserve measures are shown in Table 2  and longer with increased VE/VCO2 slope . ICU LOS was not associated with MAMC.The associations with physiological reserve measures with intensive care LOS are shown in Table In order to exclude any further association between gender and the outcomes, a further stratified analysis was performed, shown in Table This study examined the predictive value of three physiological reserve measures that were routinely used at our center during the assessment of liver transplant candidates. The data demonstrated that both handgrip strength and CPET provided useful prediction for clinically important post‐LT outcomes. Specifically, CPET measures were independently associated with intensive care LOS, and handgrip strength was independently associated with hospital LOS and sepsis within 12 months. MAMC was not a useful instrument in this study and was not associated with any of the clinical outcomes assessed.This study adds to the growing literature supporting the concept that physiological reserve measures have independent utility in predicting surgical outcomes,et al.,The data confirm the clinical usefulness of handgrip strength, a simple, single bedside test of reduced muscle strength. Handgrip strength was associated with multiple key outcome measures and this finding is consistent with the emerging concept that measures of functional muscle strength may be more clinically useful than those of muscle mass. Measures of muscle mass cannot provide information about quality of muscle, and it has been shown that the correlation between muscle strength and mass is only modest.CPET measures were also useful in predicting intensive care LOS. These are measures of cardiorespiratory fitness and ventilatory efficiency, and the results demonstrate the significance of these factors for patients undergoing LT. CPET however has several limitations relevant to LT assessment. Firstly, it requires complex equipment and time resource, which may not be widely available for all transplant units. Secondly, a percentage of LT candidates are too unwell to perform this testing. In comparison, handgrip strength is simple, inexpensive, and can be performed at the bedside without the need for complex training or significant time allocation during a busy transplant workup schedule. It was also found to be associated with multiple significant post‐LT early adverse outcomes , whereas CPET was associated with only one (ICU LOS). Due to these factors, handgrip strength would appear to provide greater clinical utility and cost‐effectiveness as a tool for the assessment of physiological reserve in LT candidates. To the best of our knowledge, this is the first study to compare the clinical utility of both handgrip strength and CPET measures and their ability to predict post‐LT adverse outcomes.The underlying biological mechanisms by which frailty and sarcopenia contribute to poor clinical outcomes in this setting are unknown. However, several plausible mechanisms have been proposed, including the involvement of “myokines,” which are cytokines or peptides produced during skeletal muscle contraction. Irisin, a myokine that is released during exercise and improves mitochondrial function, is suppressed in sepsis.Our study had several limitations. Firstly, CPET measures were only available in 20 patients, as CPET was introduced for LT candidates from 2018. This limited the statistical power of the results for these variables. In addition, there was a low number of deaths during the first post‐transplant year, and consequently limited statistical power to detect associations of our physiological reserve measures with 1‐year mortality. Despite these limitations, we were able to find several statistically significant associations for the majority of outcome variables.Interpretation of study findings is also limited by the single‐center, retrospective observational design of the study. For example, the accuracy of data collection for sepsis events was limited to events recorded within the medical record databases. We therefore cannot exclude the possibility of missed cases of sepsis that occurred outside our hospital networks that may have led to underreporting of this outcome. As with all non‐randomized studies, there was potential for important unmeasured residual confounding. However, this risk of bias was reduced by the statistical analyses adjusting for known potential confounders.Despite these limitations, we believe the study provides useful information for LT teams and is likely to be generalizable to typical LT candidates given the limited exclusions from the study . This pilot study provides justification for larger, prospective, multicentred studies comparing the relative utility and cost‐effectiveness of CPET and handgrip strength during LT assessment.In conclusion, Handgrip strength and CPET in liver transplant candidates both provided clinically useful prediction of important adverse events following LT. Low handgrip strength was associated with early post‐LT sepsis and prolonged hospital stay on fully adjusted analysis. Poor performance at CPET was associated with prolonged intensive care stay on fully adjusted analysis. Particularly in low resource settings, handgrip strength is a useful tool to identify LT candidates with higher risk of early adverse events following transplantation.