Over the last decade, the concept of actionability has become a primary framework for assessing whether genetic data is useful and appropriate to return to patients. Despite the popularity of this concept, there is little consensus about what should count as “actionable” information. This is particularly true in population genomic screening, where there is considerable disagreement about what counts as good evidence and which clinical actions are appropriate for which patients. The pathway from scientific evidence to clinical action is not straightforward—it is as much social and political as it is scientific. This research explores the social dynamics shaping the integration of “actionable” genomic data into primary care settings. Based on semi-structured interviews with 35 genetics experts and primary care providers, we find that clinicians vary in how they define and operationalize “actionable” information. There are two main sources of disagreement. First, clinicians differ on the levels and types of evidence required for a result to be actionable, such as when we can be confident that genomic data provides accurate information. Second, there are disagreements about the clinical actions that must be available so that patients can benefit from that information. By highlighting the underlying values and assumptions embedded in discussions of actionability for genomic screening, we provide an empirical basis for building more nuanced policies regarding the actionability of genomic data in terms of population screening in primary care settings. As advances in genomic technology improve both the utility and cost-effectiveness of genetic testing, many have called for expanded, population-level genomic screening programs . These pIt is particularly critical to understand the qualitative complexity of actionability in genomic screening because at least 11 genomic screening programs are operating today in the United States alone, with the number of programs expected to expand . These pBecause genomic sequencing produces large amounts of data, it can be difficult to assess what data are both interpretable and valuable in clinical settings. Assessing the value of genomic data also necessitates an understanding of the relationship between that data and a set of potential actions it may set in motion. Many concepts in clinical genomics have been used to theorize this relationship, such as “clinical validity” and “clinical utility.” The National Library of Medicine defines clinical validity as how well the genetic variant being analyzed corresponds to the presence, absence, or risk of a specific disease. Clinical utility refers to whether a test can provide information about the diagnosis, treatment, management, or prevention of a disease that will be useful to a consumer . As our not being actionable is testing for a genetic predisposition to Alzheimer’s disease, because there are so few treatment options for Alzheimer’s disease  actionable would have to be a truly pathogenic change, for which there is a known treatment or surveillance recommendation that impacts a lifetime of medical management and saves lives.”—Clinical geneticistThis participant considered actionability to refer to those interventions that require serious changes in medical management and a demonstrated ability to improve patient outcomes. Others had a more expansive view:“I think of [actionability] really broadly: anything that has the potential, either now or in the future, to modify either life choices or medical treatment … We [could] say, ‘Oh, is that actionable? There’s no treatment for it.’ But then [patients] can enroll in clinical trials and things like that … And I think it’s important to consider the social and emotional aspects of what we consider actionable, too, because it’s very personal. So, some things that someone else wouldn’t consider actionable would be really important psychologically and … wouldn’t necessarily be, ‘Oh, we’re gonna change this medication,’ or do a surgery, or things like that.”—Clinical geneticistThus, despite published guidelines and metrics for actionability, we find little evidence of consensus among clinicians in practice, especially in the context of population screening. At the same time, while documenting these differences in how clinicians define actionability is important, the more informative parts of our interviews focused on the context and actual practice of declaring a gene/disease pair as being actionable or disclosing a result to patients. The following sections outline our main findings on how “actionable” results are differentially considered and practiced in context.One of the key components of actionability is an assessment regarding what types of evidence, and how much of that evidence, are required before a result should be returned to patients. This is not a straightforward question, and our discussions with participants brought up a range of factors related to the context of genomic screening that were important to different people. First, participants had different ways of balancing the tension between providing potentially life-altering data and care to patients as quickly as possible, versus waiting for more and better evidence of validity or utility. For example, some clinicians, driven by the principle of “do no harm”, were primarily concerned about the risks of overtreatment based on uncertain science:“This is how close we were to really making a big mistake. We looked at all this information, you know, we used the best databases available. We used ClinVar. We used ClinGen. We [had another laboratory perform] a separate assessment of the variants before they returned the results to us. We would look at everything and we would say, ‘Okay, these patients look like they might have a pathogenic or likely pathogenic change.’  had actually revised their classification from pathogenic to VUS [variant of uncertain significance]. And so we [did not] return this variant, but it was pretty close to us actually coming to that point. And it actually happened also with a couple of the [heart condition] genes as well, early on, that variants that we thought were likely pathogenic turned out not to be. And so that’s a really big concern, that you’re overtreating not because … you know, it was even the best standard at that time, I guess, but the standard had moved.”—Clinical geneticistThis clinician was worried that genetic screening was moving too quickly from research into clinical practice, without a solid evidence base at the population level. He cautioned that the balance of risks and benefits of genetic testing is different for someone with a family history or phenotype of disease than for an unselected, otherwise healthy patient. Many clinicians echoed this approach, preferring to wait to deliver information to patients until they could have greater certainty in its value.Still, other clinicians spent more of our discussion focused on the benefits that they could be offering patients immediately, even while recognizing that the science of genomic screening will continue to evolve. These clinicians either did not see the value in waiting for additional evidence or thought that waiting was unrealistic:“We have this weird middle line of, like: this [more evidence] is the ideal, [but] this is reality. We don’t want to leave patients hanging, because, like, the reality happens before the ideal does.”—Genetic counselorWhile this clinician would ideally prefer to have better evidence for the utility of genomic screening before implementing these programs, she sees more value in offering this service to patients now. Often, clinicians would discuss the dangers of waiting for better evidence in quite personal terms:“Sometimes the ultimate question is, ‘Think about yourself and your family and would you want to know? Would you pursue this testing if it were offered to you?’ And I think many people would say, ‘Yes.’ And then, the argument is … Isn’t this something that we should be offering for our patients now because it can have a major effect for them?”—Primary care providerIt is not surprising that participants made different calculations of how to weigh potential risks and benefits, as we see this across health and social domains. Perhaps most dramatically, the COVID-19 pandemic brought to light how people vary in their propensity for action in the face of uncertain evidence, fueling highly politicized debates about prescribing drugs such as hydroxychloroquine and remdesivir to treat COVID-19 symptoms.Where participants’ views fell along this spectrum tended to match the attitudes of others at their institution. While the methods of the current study do not allow us to explain why participants in the same institution often espoused the same balance between evidence and action, theories from other social scientists would suggest that commitment to a position is a consequence rather than a cause of affiliation . In the To summarize, the question of whether to offer genomic screening now or later was at the forefront of their minds for our participants when discussing actionability. We observed differences in how participants thought through the value of being able to help some people immediately  versus waiting until the science of genomic screening was more settled, to avoid any potential harm from incorrect interpretations. Importantly, weighing these potential benefits and harms will always be a matter of values, and the matter cannot be solved by simply “following the science”. Genomic screening programs will need to take into account these values and choices in their design and implementation.In our sample, the participants’ professional identities seemed to impact which types of evidence they deemed relevant to discussions of actionability. Participants with genetics expertise who expressed concerns about evidence were generally most concerned about the evidence of accuracy regarding a particular result, such as whether a variant was correctly classified. Many participants with genetics expertise reported investing significant effort to check and potentially even reclassify gene variants, based on their own expertise and a particular patient phenotype, matching recent research showing that this is a common practice among genetic counselors . Partici“I struggle a little bit with the fact that all labs are using the ACMG criteria but yet we can still get different answers from labs even when they’re using the same criteria.”—Genetic counselor“This is probably the biggest struggle that we have currently in the field. If I really were to go through and rank, like, what are all of my frustrations, [laboratory variability] is probably top of the list right now because there’s not a central place to collect [info about laboratories] and be able to compare back to who’s giving better reports, who’s using actual ACMG classification criteria, who’s doing whatever with whichever lab results come back.”—Genetic counselorIn contrast, primary care physicians expressed much less concern about variant classification or the accuracy of a specific result:“I don’t disagree with the genetic lab results. I wouldn’t know how to disagree with genetic lab results.”—Primary care providerInstead, primary care providers paid greater attention to population-level data showing the efficacy of genetic screening programs. They wanted to see randomized controlled trials on the efficacy of population genomic screening, along with professional society guidelines.“There’s a high bar of evidence that’s required before some big new [screening] initiative is going to be put in place. You’ll hear a lot about barriers, like, ‘Oh, our EHR isn’t ready, doctors don’t know how to do this, they don’t know how to talk to patients about it.’ All of that’s true, but all of that would fall into place pretty quickly if [a] clinical guideline from USPSTF [the U.S. Preventive Services Task Force] says, like, ‘This should be done.’ And why haven’t they said that? Maybe they haven’t asked, but I think they also know that the evidence just isn’t there, and, you know … USPSTF would have a pretty high bar of evidence that would typically rely on RCT-level evidence. So, I mean, that’s the big one … If that happens, everything else falls into place.”—Primary care provider with genetics expertiseFor some of these primary care providers, the lack of clinical trial evidence for more expansive screening programs made them reluctant to add genomic screening to their practice. Alternatively, they preferred to screen for those conditions with the most population-level evidence, such as conditions classified as CDC Tier One.Differences in perspective between genetics experts and primary care providers may also be due to the perceived tension between the tenets and realities of precision medicine versus evidence-based medicine. Below, a family physician with training in genetics explained how she understands the evidentiary standards of the two fields:“So, precision medicine is the n-of-1, right? Family medicine has been trained, evidence-based medicine has been stuffed down their throat, right? Since day one … To reach the level of what’s considered evidence-based medicine, you have to have a significant N, and that’s not what precision medicine is. And that scares them because this is where they’ve trained, this is where their comfort level is. They can go to their references, they can go to their data, they can go to the national guidelines. But precision medicine isn’t that … Precision medicine is pragmatic, right? You can’t do a huge retrospective or prospective randomized control trial, it just, it doesn’t work. And so, it’s a new comfort zone. So, while we’re educating these young docs to have the competencies, they also need to have the confidence to practice n-of-1 medicine.”—Primary care provider with genetics expertiseOther participants highlighted the importance of other, “softer”, types of evidence in their practice:“I don’t think everything needs a randomized control trial. It always reminds me of a commentary that was published in a journal … talking about how you don’t need a randomized control trial to prove that parachutes work when you skydive. And so I think pharmacogenomics is kind of like that. And I think there’s a lot of soft outcomes too. I see in my patients personally, sometimes just them getting tested and knowing that we’re aware of their results makes them more open to trying a new medication or to give something a longer shot … And sometimes that will help them have a better outcome, not necessarily because the test led us to that but the act of testing helped them out.”—Primary care provider with genetics expertiseBecause evidence generation in clinical genetics tends to appear different from evidence generation in primary care, we expect to see continued disagreements about both the quality and quantity of evidence required for genomic data to be actionable in the context of population screening. Depending on professional identity, participants highlighted either the importance of generating evidence to support the proper classification of individual gene variants or the importance of collecting clinical trial data regarding population-level outcomes.Debates about evidence and actionability also depended heavily on the type of genomic screening under question. There are three main types of screening programs: screening for single-gene disorders, screening for complex disorders via polygenic risk scores, and pharmacogenomic screening. Genetic testing for single-gene disorders is the most common, and clinicians often have a good understanding of the underlying biological mechanisms that cause disease. As a genetic counselor explains:“With Mendelian [single-gene] testing, we are very grounded in an understanding of biological mechanisms … So the way that we demonstrate utility is we first establish this gene has this role and pathway in disease. We can know what [specific variants] people have; we have to, to some extent, do a discovery type of approach for each individual, and then decide if [that variant] is pathogenic in this gene that we know. So you prove utility on an individual basis almost but it’s in the context of, like, we know this disease mechanism. In a lot of medicine, those mechanisms are not as rigorously known in advance, right? So that’s worked for us, and we’ve had a lot of loopholes, I think, in how we were able to approach [evidence generation] because there’s a lot of rare diseases … or, like, just genetics, in general, has been very different from the rest of medicine.”—Genetic CounselorCollecting population-level outcome data is also, admittedly, difficult and time-consuming. A clinician describes this process for pharmacogenomic testing:“One of the criticisms of pharmacogenomics is that we’re still working on growing the evidence of outcomes data. Whenever you’re on … medication, collecting the outcome data whenever you’ve used pharmacogenomics to guide the therapies is probably not that easy. Because you have to follow people so far … to see if the outcome actually occurred or not. That’s, I think, what payers and health plans are looking for. And there is some evidence that exists for that. But it’s not totally, you know, great evidence. And so I think that’s the hardest thing.”—Primary care provider with genetics expertiseOther types of genetic testing, such as polygenic scores for complex disorders, do not have as much evidence of the underlying biological mechanisms. Instead, polygenic scores are created by comparing the genomes of individuals with and without a particular disease. Thus, the genetic counselor above explains how she sees the differences in the required types of evidence between these two forms of tests:“[For] polygenic scores … I can completely empathize with the norm that there should be randomized controlled trials. People that do randomized controlled trials are not geneticists, [geneticists] don’t know how to do that. They don’t know how to do, like, a big epi [epidemiological] study … So it’s this tension of geneticists taking the same approach of, ‘Oh, we’ve proven a relationship and that’s enough to now test people.’ And it’s like, no, it’s an association. It’s not grounded in biology. It’s a very different bit of information.”—Genetic counselorThis perspective suggests that different types of genetic tests could require different approaches regarding evidence generation and that it would be a mistake not to differentiate between them. This, in turn, could affect how clinicians and researchers understand the actionability of these tests.In addition to questions about scientific and clinical evidence, actionability also requires thought about the types of actions available to patients, clinicians, or health systems. What can patients and providers actually do with the information presented to them? If they cannot do anything, is that data worth returning? Before completing our interviews, we expected to have discussions with participants about the specific clinical decisions they make and the actions that they take based on genomic screening results. Instead, when we asked participants about how they understand the clinical actions that should or should not be taken based on genomic data, their answers were more general and focused primarily on the capacity of health systems.Specifically, the lack of institutional capacity to fully integrate population genomic screening was a primary concern for many participants. Participant perspectives varied as to why capacity was limited. Some thought that there were so many other, more immediate, population health problems to tackle that genomics just did not make the cut:“Right now, I can understand why for many [primary care providers], you know, they’re dealing with people who have COVID, they’re dealing with the flu, they’re dealing with people who … have diabetes, and so forth, and fundamentally, they wanna know if I ordered these [genetic tests], how are they gonna impact what I’m prescribing, or managing? And right now, in many cases, it doesn’t impact them.”—Genetic counselorOthers focused on the lack of genetics training for providers, making it difficult for those providers to know how to interpret and address the findings of a genetic test:BRCA1 and 2?”“I was at a clinic talking to primary care providers … And a doctor was sitting next to me and he said, ‘I won’t talk to my patients about genetics. I’m not comfortable…’ And so you’re constantly playing catch up … because the clinicians are overworked, they’re undertrained, frankly, and they don’t have the infrastructure to deal with [the question of] what is the clinical decision support tool necessary for 18- to 35-year-old women with —Clinical geneticistConcerns about capacity were sometimes the primary drivers of decision-making around actionability, rather than evidence. For example, a clinician describes how his health system chose to screen for a limited number of conditions:“And I think the smart decision we made at the time was that because we’re a safety net hospital, [with] almost no research infrastructure … no genetics department … we would return results on the CDC Tier 1 conditions. And I think that decision was probably the right one. And, subsequently, many other health systems are recognizing that, you know, the ACMG’s guidelines on incidental findings … [are] not meant for all-comers, you know, for moderate risk population screening. And that, still, the CDC Tier 1 conditions are probably appropriate to return to all-comers.”—Clinical geneticistThis clinician first mentions a lack of infrastructure, before also commenting on the higher levels of evidence for CDC Tier 1 conditions in comparison to other, more expansive lists of genes. He continued to describe his discomfort with expanding their testing because having more patients with pathogenic results would not necessarily correspond to those patients getting the care they needed:“To get to imagining returning 4% pathogenic, likely pathogenic, results in [an expanded list of genes] would be impossible. And so you’d have all these indications in the medical record and you’d have folks who were not getting the standard of care, given those indications. And so the question is, do you then interpret them and just know that?”—Clinical geneticistConcerns about capacity may vary depending on the level of resources at the health system where a participant worked, at least in our sample. For example, participants from health systems with more resources did not discuss problems related to maintaining standards of care, and instead discussed developing more expansive genomic screening that could eventually benefit a far wider number of people:“[Our hospital has] extra money sometimes to reinvest in developing new services that might not have a clear guideline behind them or clearly established utility … things we think will be important one day, and we’re gonna back it. So they decided they wanted to build more preventive genetics, population screening infrastructure in a more robust way, and really latched on to both polygenic risk scores and pharmacogenomics since the target audience for those are bigger. It’s more of a general population approach.”—Genetic counselorThis participant did not report any concerns about health system capacity—instead, her health system was working to expand testing to the largest number of patients. As genomic screening programs become more popular, we may see differences in whether health systems have the available infrastructure, both to offer testing to more patients and to support the patients that do undergo testing and need follow-up care.Generally, our responses from participants suggest that they are less interested in debating what types of specific clinical actions make a genetic result worth returning to patients and are more concerned about ensuring follow-up care for even the most straightforward screening efforts. For example, we spent less time discussing whether a genetic predisposition to Alzheimer’s disease was actionable, given the lack of clinical actions available. Instead, participants highlighted their efforts, and often frustrations, regarding providing patients with resources, even for CDC Tier One conditions with the clearest practice guidelines.In this qualitative study of clinician perspectives on the actionability of genomic data for population screening, we find that clinicians vary in how they define actionability and how they utilize the concept in practice. First, clinicians are often unable to articulate what counts as actionable information, but suggest that they “know it when they see it.” Even when participants were able to define actionability, their responses varied from restrictive to expansive. In our analysis of how clinicians implement the concept of actionability in practice, we make two main contributions to the literature on precision public health: (1) debates about the quantity and quality of evidence required for actionability are contextual, and are driven by value judgments; (2) clinicians perceive capacity and infrastructure to be major barriers to implementing genomic screening programs, and worry that this could lead to real differences in follow-up care between health systems with different levels of resources. Importantly, however, some participants argued that infrastructure to support genomic screening will “fall into place” if better evidence of clinical utility is generated. Thus, we find that the dominant concern for clinicians regarding actionability was related to standards of evidence, rather than clinical actions.As a small-scale qualitative study, our methodology and data face several limitations. First, we cannot provide evidence of how our participants’ views align with their peers across the country, because our sample is not designed to be representative of the population. Second, by focusing our interviews on genetics experts and primary care providers, we excluded the perspectives of other relevant stakeholders, such as patients, funders, and regulators. Still, our findings suggest clear disagreement and variation among clinicians who serve as key gatekeepers to population screening. Additionally, due to the depth of our interviews, this study provides greater clarity on the underlying values and assumptions that drive differences in opinion on actionability.As more population genomic screening programs develop across the United States, we encourage the designers and implementers of these programs to address these value judgments head-on, in order to develop programs that provide the ideal balance of risks and benefits, as defined by their local context. Different communities, for example, may place a different weight on the value of intervening in the moment versus waiting for better clinical evidence. This may be particularly true for rare diseases, where large-scale clinical evidence is hard to generate. While professional societies such as the ACMG provide guidance on the types of evidence and actions required to move forward with population screening , we susp


A key driver of urbanization is the pursuit of economic opportunities in cities. One such opportunity is the promise of higher wages in larger cities, a phenomenon known as the urban wage premium. While an urban wage premium has been well-documented among U.S. metropolitan areas, little is known about its existence in micropolitan areas, which represent an important link between rural and dense urban areas. Here we measure the power law scaling coefficient of annual wages versus employment for both U.S. metropolitan and micropolitan areas over a 37-year period. We take this coefficient to be a quantification of the urban wage premium for each type of urban area and find the relationship is superlinear in all years for both area types. Though both area types once had wage premiums of similar magnitude, the wage premium in micropolitan areas has steadily declined since the late 1980s while in metropolitan areas it has generally increased. This growing gap between micropolitan and metropolitan wage premiums is ongoing in parallel to other diverging characteristics, such as inequality and voting behavior, suggesting that our result is part of a broader social, cultural, and political divergence between small and large cities. Finally, we speculate that if urban residents respond to the COVID-19 crisis by migrating, the trends we describe may change significantly. Globally, more than 3 million people migrate to cities each week–primarily in search of economic opportunities . Those oPast studies of the U.S. urban wage premium focus almost exclusively on metropolitan statistical areas, which are defined as urbanized areas of at least 50,000 people. Little attention has been paid to the nearly 550 micropolitan statistical areas ,9, whichWhile many studies seek to identify the causes of an urban wage premium –20, our β as the magnitude of the wage premium. A power-law relationship is superlinear if β > 1 and sublinear if β < 1. If β does not differ significantly from 1, the relationship to size is linear, meaning that the variable of interest is essentially independent of city size.To quantify annual wage premiums for both metropolitan and micropolitan areas, we adopt a power-law function relating an area’s total annual wages to the area’s average annual employment, taking the power-law’s exponent Power-law functions have been shown to describe relationships between city size and several social and economic attributes of cities ,22, inclβ = 7/6. Thus, we expected our study to find that an urban wage premium exists in both metropolitan and micropolitan areas and that it manifests as a power-law relationship between total wages and employment.In an attempt to explain these various urban scaling phenomena, Bettencourt  proposedOur spatial units of analysis are 926 core-based statistical areas (CBSAs) of the United States, covering all 50 states and the District of Columbia. This excludes 12 CBSAs of Puerto Rico that have lower quality data in the earlier years of our study. CBSAs are aggregations of one or more counties forming a coherent labor market and exhibiting a high degree of social and economic integration, particularly in terms of commuting patterns . CBSAs aWe analyzed two possible measures of city size–population and employment. However, we found data on employment to be better matched to data on wages and thus use employment throughout this study as our measure of city size .Several federal agencies publish regional data on U.S. wages and employment, including the Bureau of Labor Statistics, the Bureau of Economic Analysis, and the Census Bureau. We evaluated each of these data sources and use data from the Bureau of Labor Statistics throughout this paper .Annual wage and employment data are taken from the U.S. Bureau of Labor Statistic’s Census of Employment and Wages (CEW) for the years 1984–2020 . While CWe aggregate county-level wage and employment data to core-based statistical areas using the September 2018 county-to-CBSA delineations published by the U.S. Office of Management and Budget . BecauseW in a given year and the city’s average employment L during that yeari represents a given urban area and β is the power-law exponent which we take as the magnitude of a wage premium for a given year and category of urban area.Finally, for both metropolitan and micropolitan areas, we adopt a power-law function to represent the relationship between a city’s total wages p < 10−10 and R2 > 0.94. Example plots of individual years for both metropolitan and micropolitan areas are shown in For each year 1984–2020 we analyzed two datasets, one for micropolitan statistical areas and one for metropolitan statistical areas, for a total of 74 annual datasets. For each set we calculated a power law scaling coefficient for total wages versus total employment. We take this scaling coefficient to be a measure of the strength of the urban wage premium across a given type of urban area. In all cases β = 7/6. Furthermore, we find that while the micropolitan wage premium increased throughout the 1980s and peaked in 1989, it has decreased since that time, steadily diverging both from the theoretically predicted value and from the value in metropolitan areas. The wage premium among micropolitan areas experienced a particularly rapid decline between 2000 and 2008 and has since continued to fall at a slower rate.Results in β = 7/6 predicted theoretically in [During the same time period the wage premium among metropolitan areas increased steadily through the 1980s and 1990s before peaking in 2000 and has since been relatively stable . Yet itsOverall, results suggests that opportunities for upward economic mobility have decreased in micropolitan areas over the past 37 years and the likelihood that an urban area will increase average wages by increasing size is now limited primarily to large cities. Several mechanisms may be underlying this divergence. In the following we discuss possibilities.To explain the diverging wage premiums between micropolitan and metropolitan areas, we focus on the notion that as cities grow in size they also grow in economic complexity –30 and iTo better understand how evolving differences between metropolitan and micropolitan areas relate to high-skill industries, we explore in more detail a sector that has experienced significant change during the study period, the information sector (NAICS code 51). The information sector is characterized by firms that (a) produce and distribute information and cultural products, (b) provide the means to transmit or distribute these products as well as data or communications, and (c) process data. Here we compare information sector employment and wages of 1990 (the first year NAICS-coded data is available) to those of 2018 for both metropolitan and micropolitan areas. Wages were adjusted for inflation and are stated in 2001 dollars .Average annual inflation-adjusted wages in this industry experienced the largest percentage growth of any sector of the U.S. economy from 1990 to 2018, increasing from $42,757 in 1990 to $78,508 in 2018. However, this phenomenal growth in wages was not shared equally by metropolitan and micropolitan areas. While average information sector wages increased 85% in metropolitan areas between 1990 and 2018, they increased only 15% in micropolitan areas during the same period, a growth of less than one fifth.Perhaps even more indicative of the growing divide between metropolitan and micropolitan areas, total information sector employment has increased in metropolitan areas by 2% since 1990 while decreasing by more than 30% in micropolitan areas. Thus, the last three decades has seen demand for high wages jobs in this lucrative industry sector shift away from micropolitan to metropolitan areas.Other industries that experienced significant wage growth since 1990, including finance/ insurance and management of companies, also grew substantially more in metropolitan areas compared to micropolitan areas. On the other hand, industries which had the lowest wage increases since 1990, including retail trade and transportation/warehouse, had higher wage growth rates in micropolitan areas than metropolitan areas. A full analysis comparing all industry sectors 1990 to 2018 is presented in the Supplemental Information.While looking more deeply into individual industries may help illuminate the drivers of our result, it is also important to examine the broader socio-economic context in which the divergence in wage premiums takes place. Indeed, the divergence in wage premiums is but one of several attributes which has seen a divergence between smaller and larger cities. Following a sharp drop globally in income inequality following World War II, inequality again began to rise in the 1980s and 1990s ,38. ThisExplanations for this great divergence include the emergence of computer technology, which disrupted the skillsets demanded by workers and favored larger cities with their diverse pool of both novel skills and educational opportunities ,40. ThusAnother frequently cited driver of the great divergence is globalization, which has generally been shown to benefit large global cities to the exclusion of smaller ones –47. Anecrd wave of globalization. This so-called 3rd wave followed the fall of communism in Eastern Europe in 1989 and was marked by a global decrease in tariffs that reduced barriers to trade and altered international economic dynamics [We note that the onset of a decline in the micropolitan scaling coefficient coincides with a particular aspect of globalization which has been termed the 3dynamics –50. ThisThe metropolitan wage premium, on the other hand, continued to increase through the 3rd wave of globalization, through the launch of the WTO, and through the late 1990s. Growth in the metropolitan wage premium only stopped after 2000 as the U.S. economy experienced the detrimental effects of both the Asian financial crisis of the late 1990s and the September 2001 terrorist attacks.Finally, we note that both the halt in growth of the metropolitan wage premium as well as the onset of a period of rapid decline in the micropolitan wage premium see  coincideRecall that Bettencourt  created 2 = 0.22, p < 0.001, data for 2019). This suggests that the scaling coefficient we take as a measure of the urban wage premium may be due, in part, to higher prices in larger areas. Therefore, further investigation of this effect is warranted [The urban wage premium is often interpreted as an opportunity for workers to earn more in larger cities. Yet, previous studies are typically based on nominal wages and salaries. It remains unclear how urban wage premiums are affected when wages are adjusted for regional price differences. Using the Regional Price Parity Index (RPPI) for all items, published by the U.S. Bureau of Economic Analysis , we findarranted .Ideally, we would rerun our original analysis using the RPPI to adjust all wages for local price levels. Unfortunately, the RPPI includes local price levels only for metropolitan statistical areas and only for the years 2008–2019. Without similar price indices for micropolitan areas it is not possible to completely perform our analysis with adjusted wages. However, using what RPPI data is available we may at least probe the effects that price differences might have on our result.To do so, we use the RPPI to adjust the subset of our data for which local price levels are available  and findβ would be reduced to 1 meaning that a wage premium among micropolitan areas would no longer exist when prices are taken into account, though this could not be confirmed without actual price indices for micropolitan areas.What might this mean for micropolitan statistical areas? Faced with a similar lack of price data in Germany, Blien at al  created Taken together these studies and our limited exploration of the effect of price differences suggest that, while the premiums we measure with nominal wages are likely higher in magnitude than with real wages, it does not alter the fact that the premiums between metropolitan and micropolitan areas have been diverging for several years.The goal of this study was to determine whether a wage premium exists among micropolitan areas and, if so, how it differs from that in metropolitan areas. One of our objectives in addressing this question is the hope that it may enrich the study of other diverging social, political, and cultural attributes between metropolitan and micropolitan areas. As an example of such potential contributions of our research we link our results to another growing difference between metropolitan and micropolitan areas, voter behavior. Using county level election returns from all presidential elections 1984–2020 –57, we aResults are presented in It is important to note that we make no conclusion as to the causality of this trend but wish only to illustrate the contribution our analysis can make to better understanding a variety of diverging social, political, cultural, and economic attributes between U.S. metropolitan and micropolitan areas.Given the ongoing COVID-19 crisis at the time of this article’s writing, we feel it prudent to comment on the how the pandemic might impact the study’s main result. The pandemic has already significantly disrupted migration dynamics within the U.S., with 2021 having the lowest mover rate in over 70 years . Though If these predictions prove generally accurate it is possible that the trends shown in Finally, we re-emphasize the importance of analyzing the wage premium across micropolitan areas using price-adjusted wages. While our results show that a nominal wage premium does exist among micropolitan areas, the question of whether it continues to exist when wages are adjusted for regional price differences remains to be answered. This will require a systematic calculation of cost of living factors for U.S. micropolitan areas and thus, is a tantalizing direction for future research.While not specifically addressing the wage premium, Korpi and Malmberg studied the difference between larger and smaller urban areas in Sweden by adjusting disposable income for differences in regional prices. They found that when disposable income was adjusted for general prices, income was positively related to population size , but wheS1 File(PDF)Click here for additional data file.


The spleen is typically located in the left upper quadrant and is held in position by the suspensory ligaments, which include the gastrosplenic ligament, the splenorenal ligament, and the phrenicocolic ligament. Abnormalities within these ligaments result in the mobility of the spleen, so it may be located in the pelvis or iliac region, which is termed a wandering spleen. We present a case of a middle-aged man who presented to the emergency department with generalized abdominal pain and diffuse guarding and tenderness. The patient had a previous history of peptic ulcer disease and multiple emergency department visits for gastritis. Given the assumed diagnosis of perforated viscus, the patient underwent a computed tomography scan that demonstrated the absence of the spleen in its usual location and showed an ectopic pelvic spleen. The patient underwent successful surgical treatment with splenopexy. The wandering spleen is a rare medical condition that presents a clinical diagnostic challenge and requires a high index of suspicion. Despite its rarity, the wandering spleen should be considered in patients with recurrent abdominal pain. The spleen is normally located in the left hypochondriac region of the abdomen. It is primarily held in position by the suspensory ligaments, including the gastrosplenic ligament, the splenorenal ligament, and the phrenicocolic ligament. The absence or laxity of these ligaments leads to the migration of the spleen from its normal location to the pelvic region . This coWe present a case of a 42-year-old man who presented to the emergency department complaining of abdominal pain for one day. He reported that the pain was generalized and had a sudden onset. The pain was sharp in nature with no radiations. It was associated with nausea and vomiting. However, the pain was not related to food intake. The patient could not identify any aggravating factors. The pain was partially alleviated by the use of nonsteroidal anti-inflammatory drugs. He rated the pain as 7 out of 10 on a 10-point severity scale. The patient reported similar, but milder, episodes of such pain over the past six months that had spontaneous resolution. The past medical history of the patient was remarkable for hypertension, diabetes mellitus, and peptic ulcer disease. The patient did not undergo any previous surgery. The patient had multiple visits to the emergency department over the last five years for intermittent abdominal pain that was clinically diagnosed as gastritis. The medication history included metformin 1000 mg, amlodipine 5 mg, and omeprazole 40 mg daily. The family history was remarkable for colon cancer in his father who was diagnosed at 50 years of age.On physical examination, the patient appeared in severe pain. There were no signs of respiratory distress. The vital signs included a respiratory rate of 15 breaths/minute, pulse rate of 110 beats/minute, blood pressure of 136/90 mmHg, and a temperature of 37.1°C . Abdominal examination revealed a distended abdomen with generalized tenderness. The patient had diffuse guarding and rigidity, suggesting peritonitis. The bowel sounds had normal intensity and quality. Examination of the cardiac and respiratory systems was normal. The laboratory findings showed no significant abnormalities. Considering the patient's history of peptic ulcer disease, the provisional diagnosis was a perforated peptic ulcer disease. Hence, the patient was referred to have a computed tomography scan of the abdomen.The computed tomography scan was performed after oral and intravenous contrast administration. The scan revealed the absence of the spleen in the expected normal location and showed a homogeneous pelvic mass of similar attenuation to that of the normal spleen. There were no hypodense lesions within the mass to suggest infarction. The supplying artery was patent. Such findings represented the diagnosis of a wandering spleen Figures , 2.Subsequently, the patient underwent laparotomy with a midline incision under general anesthesia. During exploration, the spleen was seen on the right side of the abdomen and measured 12 x 14 x 9 cm in maximum dimensions. The spleen appeared viable with no evidence of torsion or gangrene. The spleen was placed at its anatomical position and fixed with absorbable mesh. The operation was uncomplicated. The patient tolerated the procedure and had an uneventful recovery. The patient was discharged on the seventh postoperative day in good condition. He required this length of stay as the patient could not fully tolerate oral intake before the fifth postoperative day due to nausea.We presented a rare case of wandering spleen in a middle-aged man who presented with a history of intermittent abdominal pain. Wandering spleen, also known as the hypermobile spleen, refers to the abnormal increase in mobility of the spleen so that it migrates from its normal position in the left hypochondriac fossa because of excessively long splenic pedicles and improper fixation of the splenic tissue to the underlying posterior abdominal wall ,5. The fThe wandering spleen may occur due to congenital or acquired etiologies. The congenital form involves the absent formation of the suspensory ligaments of the spleen. The spleen forms from the dorsal mesogastrium, which then rotates to the left side. The absence of suspensory ligaments results in long splenic pedicles, which are responsible for the increased mobility of the spleen. Furthermore, hypermobile colon and prune-belly syndrome are classic causes of the congenital wandering spleen. The acquired form of the wandering spleen has the same pathology of abnormal long splenic pedicles. The acquired form typically occurs in women of reproductive age, due to maternal hormones. It was found that both estrogen and progesterone increased the relaxin receptors on cells, promoting their action of increased ligamentous laxity . TherefoThe clinical manifestations of the wandering spleen are variable. The wandering spleen can be asymptomatic and found incidentally on physical examination or radiological investigations. It may also present with acute abdominal pain due to a splenic infarct. Furthermore, as in our case, the wandering spleen may present with intermittent abdominal pain that may be related to intermittent torsion and spontaneous detorsion of the splenic pedicle. Interestingly, our patient was clinically misdiagnosed as having gastritis and was on proton pump therapy for years. This highlights the importance of doing proper endoscopic and radiological investigations in patients with vague abdominal pain and not using an empiric treatment for common conditions without appropriate investigations. Acute presentations of the wandering spleen are likely due to torsion or mass effects on adjacent structures, causing intestinal obstruction, gastric volvulus, or pancreatitis . The difThe clinical diagnosis of a wandering spleen can be challenging, but the diagnosis can readily be made by cross-sectional imaging investigations. A computed tomography scan of the abdomen can demonstrate the absence of a spleen in its normal position in the left upper quadrant of the abdomen along with the presence of a mass in the lower abdominopelvic cavity that has similar attenuation to that of the normal splenic tissue. It can confirm the diagnosis and also identify the potential complications (e.g. splenic infarct). Ultrasound is another modality to diagnose the wandering spleen, as it is an affordable investigation and has no association with radiation risks. However, it is operator-dependent and its diagnostic accuracy is degraded by bowel gases, which may obscure the findings. In the present case, the patient underwent a computed tomography scan for a presumed diagnosis of perforated viscus given the findings of the clinical examination.Surgical treatment is the only recommended management for wandering spleen . SplenecA wandering spleen is a rare medical condition that poses a clinical diagnostic challenge and requires a high index of suspicion. Cross-sectional imaging studies can readily confirm the diagnosis and identify the possible complications. Surgical treatment, preferably with splenopexy, should be performed even in mildly symptomatic patients to prevent the complication of torsion and infarction. The diagnosis of the wandering spleen should be considered in patients with recurrent abdominal pain.


Sense of presence and extraneous cognitive load (ECL) are the two psychological effects widely employed to explain the cognitive outcomes caused by high-immersive media . This study identified the concepts of both technological affordance  and the psychological effects of VR learning. It investigated the mechanism by which immersion leads to better or worse communication in the context of art education. We operationalized the concept of immersion into two levels: a high-immersive VR system (HTC VIVE Cosmos) and a low-immersive tablet system (iPad). Through a between-subject experiment, we found that higher immersion not only led to a greater sense of presence but also lowered extraneous cognitive load. Enjoyment and attention increased as a sense of presence rose but were not necessarily predicted by extraneous cognitive load. This study found that sense of presence was a more robust explanatory variable than ECL and that cognitive load could be lower in a high-immersive environment with content specifically designed for VR. Immersive media plays an increasingly vital role in the field of multimedia learning. Some researchers claimed that immersive virtual reality (IVR) has unique advantages in promoting learning outcomes in terms of learning motivation, enjoyment, interest, knowledge retention, and skill transfer  is processed as peripheral cues that can trigger some psychological experience  and has positive effects  explore the effectiveness of IVR in the context of art learning and 2) examine the underlying mechanism and compare the competing explanatory paths . A between-subject experiment was carried out to examine the effects of immersion on enjoyment, attention allocation, and retention, as well as the mediating effects of sense of presence and extraneous cognitive load (ECL).The concept of presence was introduced to the field of VR in the 1990s , technological affordance can trigger heuristics through psychological cues to impact users' ways of processing content Sundar, , and preTo summarize, most scholars in HCI tend to classify immersion as an attribute of a technical system that is an important factor that triggers presence. However, in a broader context, presence is not exclusively associated with high immersion. It is a psychological status that could be achieved by attributing more mental effort to a less immersive environment. Meanwhile, people can perceive different levels of presence even in the same media environment.Enjoyment refers to the degree of positive emotion thatusers feel when interacting with a digital system. Sherry  explainsTo a certain extent, VR offers users a virtual space and a fully occupied view that reduces the user's perception and attention to physical space. Compared with framed screens, users are less likely to be distracted by information other than media content. The sense of presence has been widely used as the psychological reason why immersion can make people focus. Kim et al.  found thControversy remains regarding the memory effect  of presence  in terms of retention  and 2D media  are different in the first place. The difference between VR and graphic learning materials is not only their immersive nature. It is also possible that the recording of VR content may lose some information in the side or back view. Some content of low quality  may cause high cognitive load, low presence, or other poor cognitive outcomes due to inappropriate content organization, which should not be blamed on the technological affordance of immersion per se . They were randomly assigned to two conditions, with 29 in the low-immersion group and 30 in the high-immersion group.https://store.steampowered.com/app/1172310/Mona_Lisa_Beyond_The_Glass/). This VR content can be played on three types of media, including HMD VR, mobile VR, and iPad versions, enabling researchers to present the same content through different media techniques. The device used by the high-immersion group was VIVE Cosmos, which supports a wide field of view (FOV) and six degrees of freedom (6DoF); the low-immersion group used iPad Pro 2018, with which participants could view content by swiping the screen or turning the device.An art VR program called the Mona Lisa: Beyond the Glass, jointly developed by Le Louvre and HTC VIVE, was adopted as the content material  as a reward for participation.Outcome variables: The participants were asked to report whether they had enjoyed the viewing experience, the viewing method, and the viewing content by answering three questions adopted from previous research  questionnaire developed according to the stimuli  together with likeness .t-tests showed significant main effects of immersion on presence  and extraneous load . Viewing the content in a high-immersion environment  elicited a higher level of presence than viewing on an iPad , together with a lower level of extraneous load  than viewing the content in the low-immersion group . Therefore, the answer to RQ1 in this study is that immersion increases presence but simultaneously decreases ECL. Enjoyment  and attention allocation  also showed significant differences between the two groups, with the high-immersion group reporting greater enjoyment  and higher attention  than the low-immersion group . However, no difference in retention  was observed between the two groups .The results of independent sample via presence and cognitive load  while demonstrating a significant negative impact on extraneous load ; thus, the first step was supported. Then, we found that presence elicited enjoyment  and attention allocation . Immersion's indirect effect on enjoyment  and attention allocation  via presence was significant. However, no impact of ECL on enjoyment  or attention  was found. In addition, no impact of presence  or extraneous load  on retention was demonstrated in the presented experiment. These results responded to RQ2.The results showed that immersion had a significant positive impact on presence . Hence, similar to Birbara et al. . As we measured immediate retention as a reflection of memory level, our results suggested that, when the message in the viewing material was the same, the information conveyed would not be impacted by the media platforms. This is consistent with the finding of Makransky et al. , which sa et al.  suggestiThis research also adds to the body of literature on the mechanism of how presence and ECL explain the relationship between immersion and final cognitive effects. We tested multiple mediator models and found a mediation effect of presence between immersion and enjoyment. Presence is also the mediator between immersion and attention. However, no significant mediation effect was found for ECL, and neither of these two potential mediators could predict the memory of factual knowledge about the Mona Lisa. Our findings draw more parallels to the immersion–presence–benefit route and fewer parallels to the immersion–extraneous load–downside route. This suggests that the sense of presence can explain more in our case, while ECL does not have to be a downside of high immersion.Our study has practical implications for IVR content production. With the development of interactive VR content .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Synthetic analogues of the DNA-alkylating cytotoxinsof the duocarmycinclass have been extensively investigated in the past 40 years, drivenby their high potency, their unusual mechanism of bioactivity, andthe beautiful modularity of their structure–activity relationship(SAR). This Perspective analyzes how the molecular designs of syntheticduocarmycins have evolved: from (1) early SAR studies, through tomodern applications for directed cancer therapy as (2) prodrugs and(3) antibody–drug conjugates in late-stage clinical development.Analyzing 583 primary research articles and patents from 1978 to 2022,we distill out a searchable A0-format “Minard map” posterof ca. 200 key structure/function-tuning steps tracing chemical developmentsacross these three key areas. This structure-based overview showcasesthe ingenious approaches to tune and target bioactivity, that continueto drive development of the elegant and powerful duocarmycin platform. Several total syntheses have beenreported,5 and biochemical research has shown how their site-selectivityof DNA alkylation depends on structural features and stereochemistry.7 Clinical drug8 and prodrug9 candidates for cancer treatment quickly advancedto phase I and II clinical trials.13 Even after initial trials werediscontinued due to narrow therapeutic index or strong side effects,an entire “duocarmycin family” of synthetic analogueswith a broad range of aims and applications have been pursued. Thisminireview aims to distill this diversity of duocarmycin developmentinto a rapidly grasped, yet comprehensive, format.The natural products CC-1065 and duocarmycinSA are irreversible DNA alkylators that react after docking in theminor groove. Since their isolation from (1) SAR studies have explored the relationship of pharmacophorestructure to DNA alkylation, and simplified synthetic analogues suchas the cyclopropabenz[e]indoles(CBIs)14 have been developed, to retainthe parent functionality but with greater chemical tractability.15(2) Prodrugs aiming to direct activitybetter toward target cancer cells have explored activatable alkylationmotifs and bifunctional conjugates.16(3) Antibody-drug conjugates (ADCs) have also been developedfor improved targeting, and in this incarnation the first duocarmycinderivative was recently FDA-approved.17Medicinalchemistry around duocarmycins has focused on three keyareas 1. (1) SA18Poster S1) which groupsand analyzes structural design features, with particular focus onduocarmycin family (1) SAR, (2) prodrugs and bifunctional conjugates,and (3) ADCs.In this Perspective we present a focused digest of the chemistryin these areas, collating most of the prolific research on duocarmycinsusing a systematic literature review workflow SLR;18,then gr218 was conducted to collate and groupthe vast majority of experimental literature concerning duocarmycins.Two groups with low structural diversity were split off: (a) reportsof the isolation, characterization, and mechanism of action of naturalproducts structurally related to CC-1065; and (b) reports of preclinicaland clinical trials of early cancer drug candidates. Three groupswith high structural diversity are analyzed here: (1) SAR: synthesis and cellular evaluation of derivatives in structure–activity-relationship(SAR) studies; (2) Prodrugs: synthesis, evaluation, and/ortherapeutic use of prodrugs, mainly based on bioactivation of the seco-duocarmycin latent alkylator functional unit, or ofbifunctional small molecule conjugates bearing at least one (seco-)duocarmycin; (3) ADCs: synthesis, conjugation,and therapeutic efficacy of (multi)functional ADCs incorporating asynthetic duocarmycin or its seco-precursor.SLR24 were harvested for additional references. Thus, a comprehensiveduocarmycin structural library was assembled, from 583 reports—mainlyof primary research  with major contributions by Boger (125),Sugiyama (58), and Lee (25). Innovation increasingly focused on targeting,with activatable prodrugs and bifunctional small molecule conjugatestaking off during the 2000s and 2010s  led by Denny and Tercel (49), Tietze (35), Saito (23),and others. Finally, since the 2010s, conjugates of duocarmycins withmonoclonal antibodies  haveopened up a new future for this class of bioactives. Combining thetunable potency and molecular flexibility of the duocarmycins, withthe potential for enriched delivery to cancers, has led to a new waveof duocarmycin antibody–drug conjugates in clinical trials,driven by Byondis B.V. (22), Medarex Inc. (7), and others. With theirincreasing therapeutic relevance, the share of patents in the lasttwo areas of research is also significantly higher and the DNA-docking motif (“segment B”) led to our currentunderstanding of the structural features that need to be arrangedfor DNA association and sequence-selective alkylation (succinctlydescribed by Hurley7).The lead naturalproduct CC-1065 was isolated in 1978,e]pyrroloindole(CPI) of duocarmycin SA25 can serve assegment A. Much research has focused on the chemically tractableCBI, with optional substitutions;14 cyclopropaindole26 (CI) and others27 also alkylate DNA with the reactivity trend (CBI ∼ CPI >CI) (Many heterocyclicsystems beyond the native cyclopropa[PI >CI) 4a.S)-configuration for DNA alkylation,29 but high potency can be maintained with “proagent” seco-variants, that use in situ intramolecularWinstein spirocyclization to unfurl their activated cyclopropane,relying on the para-phenol.30 Good leaving groups 31 and severalalternatives to the dihydroindole 32 are all tolerated. Alternatively, masking thisphenol suppresses spirocyclization:33 adisruptive step that opened the door for rational tuning of prodrugcandidates in later years (see below). The group of Lee also introducedachiral seco-variants that are similarly reactive,but structurally simpler and more accessible.35The activated cyclopropane electrophile must bein its native ,29 and monoalkoxylated (DEI),37 or even simple mono/oligoindoles; and other heterocycles38 can also be used. These do impact DNA binding,alkylation site-selectivity, and potency; but overall, the tolerancefor segment B variance is high (Segment B heterocycles have mainly clustered around indole-basedrings that strengthen DNA binding. Stepwise simplification of thenative dimeric segment B (in CC-1065) gave variously the deoxygenatedCDPI dimer, is high 4a.39 Using syntheticoligo-pyrroles/imidazoles from the minor-groove binder distamycinA as segment B binding domains gave potent duocarmycin analogues allowingsequence-selective alkylation in specific areas of DNA.41 “Standard” duocarmycins consist of segments A andB linked by an amide bond: but the natural product Yatakemycin43 has revealed that multiple B segments may be used, and randomlyshuffled around without losing bioactivity.44 Dimeric bisalkylators with two A segments, allowing interstrandDNA cross-linking, also give extremely high potency.46Assembling the A and B segments has alsoreceived attention. Aremarkable class of hairpin duocarmycin conjugates was driven by Lownand Sugiyama in the 2000s.3.2seco-duocarmycin bioactivity was to be triggered in situ by unmasking a para-phenol, toavoid parasitic loss of a preformed cyclopropane en route to target tissues. These carbamate hydrolysis designs  were discontinued in clinicaltrials due to side effects and low therapeutic index.50 Follow-up work mined esters, solubilized carbamates, phosphates,and others as other hydrolytic activation methods -reactiveperoxides,66 photoactivated designs,68 and oxidizable naphthalenes.69 Recently,cyclic dichalcogenides have joined this panoply of prodrugs.71 Finally, bifunctionalconjugates of duocarmycins with other pharmaceuticals  showthe wide applicability and adaptability of this unique class of bioactives.Key steps toward cancer-selective prodrugs were initiated by thelab of Denny in the late 1990s. They introduced nitro-kylators 4b.57−59 iazepine 4b,75,76 3.3seco-duocarmycinphenol with a linker conjugated to the antibody: allowing spirocyclization-basedactivation after linker cleavage. Intracellular cleavage of theselinkers  can directly liberatethe key Winstein cyclization phenol, but additional self-immolativespacers, that undergo cyclization or elimination cascades to liberatethis phenol, are common.83 Type B designs attach a phenolic prodrug of the duocarmycin, viaa peripheral site, to the antibody: permitting an extra layer of prodrug-basedselectivity if prerelease activation can be avoided chemical mechanisms for tumor-selective prodrug activation, and (iv)antibody-based mechanisms for tumor-selective prodrug delivery. Solubilizationand self-immolative spacers have also proven critical. It is perhapsno accident that the first duocarmycin to be clinically successfulhad built in nearly all these strategies  SAR simplification for synthetic access, (ii) protectingthe cyclopropane warhead by forming it rategies 5. We see4Duocarmycins have undergonegreat efforts toward developing targetedcancer therapeutics. A careful understanding of their unusual mechanismof bioactivity, leveraging spirocyclization and docking for high-potencysite-selective DNA alkylation, has enabled many creative approachesusing the duocarmycins as a modular bioactive platform. Here we haveprovided a structured literature review tracking the chemical developmentsof the last 40 years, that have led from isolation to basic understanding,early trials and setbacks, re-engineering, and ultimately a firstclinical anticancer agent.100 with the A0-size We hope this concise overview willpromote a structure/function-basedunderstanding, allowing rational design and use of duocarmycin-basedbioactives. It also follows the didactic tradition of Njardarson’sPostersà la carte. A structure-based overview to guide the choice and understandingof these features, with easy direction to the corresponding references,may be very helpful for gaining a coup d’oeil when entering new scientific territory: particularly where the frontiersof research are increasingly interdisciplinary. We can still expectmuch from the duocarmycins; and we hope this Perspective and its Posterbring a graphic understanding of how to design, incorporate, and exploitthis powerful molecular class.The modularity of duocarmycinbioactivity should encourage researchersto design in structural features


Dirofilaria immitis is a zoonotic parasite that infects canids and other vertebrates. We expanded the use of long-read nanopore-based sequencing technology by performing genomic de novo assembly of a D. immitis specimen retrieved from a canine cardiopulmonary dirofilariasis case by using the ONT MinION platform. We also identified loci previously characterized as being associated to macrocyclic lactone resistance selection pressure. The identification of a resistant zoonotic parasite alerts for the overuse of macrocyclic lactone in the region.Dirofilaria immitis is a zoonotic parasitic nematode that infects domestic and wild canids, among its vertebrate hosts. The genetic analysis of D. immitis nowadays transcends the need for genetic taxonomy of nematodes, such as the study of resistance to macrocyclic lactone. We expanded the use of long-read nanopore-based sequencing technology on nematodes by performing genomic de novo assembly of a D. immitis specimen retrieved from a canine cardiopulmonary dirofilariasis case using the ONT MinION platform, followed by the study of macrocyclic lactone resistance. The assembled genome of D. immitis consists of 110 contigs with an N50 of 3687191. The genome size is 87899012 and contains a total of 9741 proteins; 6 ribosomal RNAs, with three belonging to the small subunit (18S) and three to the large subunit (28S); and 73 tRNAs. Subsequent analysis of six loci previously characterized as being associated to macrocyclic lactone resistance selection pressure showed that four have a genotype associated with either some loss of efficacy or the resistance phenotype. Considering the zoonotic potential of D. immitis, the identification of a resistant parasite alerts for the overuse of macrocyclic lactone in the region, which poses a potential risk to both veterinary and human public health. Dirofilaria immitis is a parasitic nematode that infects domestic and wild canids as well as other animals, including humans. The associated disease typically occurs in temperate, tropical, and subtropical areas of the world, with the agent being transmitted by several mosquito species such as those belonging to the Culex, Aedes, and Anopheles genera [s genera . These ms genera . Femaless genera .Dirofilaria immitis worms can cause canine cardiopulmonary dirofilariasis , a widespread disease that can have a fatal outcome if animals are not treated [D. immitis in dogs represents a risk for the human population, who may suffer from pulmonary dirofilariasis and, in many cases, pulmonary nodules that can be misdiagnosed as malignant tumors [ treated . Moreovet tumors .D. immitis, which can easily be distinguished from other filarioids, taking into account the cardiac location of adults. Nonetheless, genetic analysis nowadays transcends the need for genetic taxonomy of nematodes. An example is the requirement for ascertaining resistance to macrocyclic lactones, known to have a genetic origin [D. immitis stages in dogs and cats. However, loss of efficacy has been described since 2005, and whole-genome analysis has been performed on D. immitis isolates to characterize their genetic profile and differences that could potentially be associated with evident loss of efficacy and resistance [Morphological analysis is commonly used for the differentiation of nematode species, but not without its drawbacks such as scarce distinguishable characters, a circumstance that may hamper their classification to the species or even genus level . This isc origin . Macrocysistance .Nematode characterization based on markers such as the internal transcribed spacer (ITS) regions of the ribosomal RNA locus, the 28S large subunit ribosomal RNA gene (28S LSU rRNA), the 18S small subunit ribosomal RNA gene (18S SSU rRNA), and the cytochrome oxidase I gene (coi) followed by Sanger sequencing is a widespread, low-cost approach to ascertain genetic profiles . HoweverD. immitis specimen retrieved from a canine cardiopulmonary dirofilariasis case using the ONT MinION platform.The aim of the present work was to expand the use of long-read nanopore-based sequencing technology on nematodes by performing genomic de novo assembly of a D. immitis worms (n = 32) were observed at routine parasitological investigation in the right heart and pulmonary artery of a dog that died from an unrelated cause in the municipality of Caminha, northern Portugal, November 2020. The city of Caminha is located by the mouth of the Minho River (circa 340 km in length), under a wet Atlantic climate, being the area of the country with the highest precipitation, reaching around 1800 mm on average and peaking at more than 3500 mm [D. immitis worms was kept in a municipal kennel since birth and had not received any macrocyclic lactone treatment. No ethics permission was obtained since the parasites were taken during regular post-mortem evaluation. One nematode was selected, washed three times in phosphate-buffered saline at pH 7.2, frozen at −80 °C, and subjected to mechanical disruption with a disposable pestle. Nucleic acid extraction followed the previously described procedures [Adult  3500 mm . The dogThe 1D genomic ligation (SQK-LSK109) library preparation kit  was used imputing 1.2 μg of extracted genomic DNA, and libraries were then developed as instructed by the manufacturer with a calculated final library quantity assessment at 467 ng. Then, 79.3 ng was loaded onto the MinION sequencer using an R9.4.1 flow cell managed by the MinKNOW software . The ONT MinION Mk1B platform was used with active channel selection performed at every 1.5 h, resourcing to no script modifications. Refueling of the flow cell was performed 24 h after initiation by first extracting excess liquid from the waste chamber, followed by the addition of 37.5 μL of SQB and 37.5 μL of H2O on the SpotON sample port. An additional 24-hour run was then performed.Canis lupus, Homo sapiens, or Wolbachia and therefore removed from the dataset for further analyses. Contigs potentially belonging to D. immitis were corrected by mapping the raw reads against the assembled contigs through the Pilon software (version 1.24). Confirmation of the species was double-checked by using the small subunit of the ribosomal RNA (18S) against the NR database using NCBI blast  and blast against the SILVA SSU database, where the best match was again AB973231.1 belonging to D. immitis. The corrected contigs were compared against the three available D. immitis genomes at NCBI  to verify the integrity of our assembly. The completeness of the assembly was verified through BUSCO software (version 4) using the nematode database. Final contigs were annotated in order to locate coding regions using Genemark-ES software (version 4.65) with self-training and default parameters. Non-coding regions belonging to rRNAs and tRNAs were also located with RNAmmer software (version 1.2) and tRNAscan-SE (version 2.0), respectively. Functional annotation for the coding regions was performed using Sma3s (version 2), retrieving GO terms and EC numbers for enzymes. Deposition of the Whole Genome Shotgun project was performed at DDBJ/ENA/GenBank under the accession code JAKNDB000000000, with the current version (described in the present paper) being JAKNDB010000000.After completion of the sequencing run, Guppy  was used for base-calling signal data (.fast5 files), with the generated fastq files used to generate statistics with NanoPlot (version 1.19.0). Raw reads were processed using Porechop software (version 0.2.4) with the default parameters to trim sequences from all known Oxford Nanopore adapters. After trimming, all reads were used to perform a complete genome assembly using Flye software (version 2.8.3) with the parameters “-nano-raw” to specify the input type data and an expected genome size of 100 Mb. Contigs obtained at the assembly step were processed using Kraken2 to locate potential contaminants in the sample. Some contigs were identified as belonging to D. immitis populations, previously referred to as markers for macrocyclic lactone resistance in D. immitis in the United States [D. immitis genome with the Basic Local Alignment Search Tool (BLAST) and Interactive Genome Viewer (IGV) for full characterization of the sample.Single-nucleotide polymorphism (SNP) loci genotypes that differentiate loss of efficacy/resistant populations from susceptible d States  and in Ad States , were idD. immitis consists of 110 contigs with an N50 of 3687191. The majority of the contigs had a very good match with one or more of the compared genomes without any sign of fragmentation. The genome size is 87899012 and contains a total of 9741 proteins; 6 ribosomal RNAs, with three belonging to the small subunit (18S) and three to the large subunit (28S); and 73 tRNAs. The results obtained for BUSCO show that the genome is complete to a high level, showing the following values . We therefore present evidence that a single ONT MinION flow cell can produce sufficient data to assemble a contiguous, high-level, near-full-length genome of D. immitis. Noteworthily, parasite genome assemblies used as references are still today considered to be vastly fragmented, frequently hampering more in-depth analysis [The assembled genome of D. immitis genome, with the corresponding SNP associated with resistance selection pressure or at least loss of efficacy for various macrocyclic lactones [A total of 42 loci in the lactones ,10, wereD. immitis worm collected for this study was kept in a municipal kennel since birth and had not received any macrocyclic lactone treatment, a fact which suggests that resistance-associated SNPs were likely acquired from ancestral D. immitis previously circulating in the region. It is assumed that macrocyclic lactone resistance first appeared circa 1998. At that time, macrocyclic lactone D. immitis preventives had been recommended for use as the first line of treatment to prevent heartworm for over 10 years, having been registered as 100% effective [D. immitis are suggested to have been circulating prior the use of macrocyclic lactone preventives [Interestingly, the dog hosting the ffective ,13. Hencventives ,13.D. immitis whole genomes from resistant and susceptible lines could help detect genetic markers for resistance [True drug resistance has a genetic basis; hence, continuing efforts to analyze sistance . Long-resistance . The seqsistance ,15,16. Tsistance .D. immitis genome using long-read nanopore-based sequencing technology. We show that a single ONT MinION flow cell can produce sufficient data to assemble a contiguous, high-quality genome from a complex nematode. The data from this study also provide information suggesting the circulation of macrocyclic-lactone-resistant D. immitis in northern Portugal. Considering the zoonotic potential of D. immitis, the identification of a resistant parasite alerts for the overuse of macrocyclic lactone in the region, which poses a potential risk to both veterinary and human public health.In conclusion, this work provides, for the first time, the de novo assembly of the


Lactobacillus strains using a cranberry oligosaccharide fraction as the carbon source. In addition to arabino-xyloglucan structures, cranberry oligosaccharides included pectic rhamnogalacturonan I which was methyl-esterified, acetylated and contained arabino-galacto-oligosaccharide side chains and a 4,5-unsaturated function at the non-reducing end. When grown on cranberry oligosaccharides, ten Lactobacillus strains reached a final culture density (ΔOD) ≥ 0.50 after 24 h incubation at 32 °C, which was comparable to L. plantarum ATCC BAA 793. All strains produced lactic, acetic, and propionic acids, and all but three strains produced butyric acid. This study demonstrated that the ability to metabolize cranberry oligosaccharides is Lactobacillus strain specific, with some strains having the potential to be probiotics, and for the first time showed these ten strains were capable of growth on this carbon source. The novel cranberry pectic and arabino-xyloglucan oligosaccharide structures reported here combined with the Lactobacillus strains that can metabolize cranberry oligosaccharides and produce short-chain fatty acids, have excellent potential as health-promoting synbiotics.Numerous health benefits have been reported from the consumption of cranberry-derived products, and recent studies have identified bioactive polysaccharides and oligosaccharides from cranberry pomace. This study aimed to further characterize xyloglucan and pectic oligosaccharide structures from pectinase-treated cranberry pomace and measure the growth and short-chain fatty acid production of 86  Consumption of foods containing sufficient amounts of dietary fiber has been associated with a wide variety of health benefits, including the alleviation of gastrointestinal diseases . DietaryE. coli. Cranberry arabino-xyloglucan oligosaccharides with SSGG structure (arabino-xylo- side chains) blocked the adhesion of E. coli O157:H7 to colon HT29 cells and E. coli CTF073 and UT189 to urinary tract T24 cells , wh, whLactoBifidobacterium breve 2141 and Bifidobacterium longum subsp. infantis 3300 were also investigated for their ability to grow on cranberry A6 as a sole carbon source, with B. breve 2141 shown to reach a final culture density (OD600) > 0.50 (B. longum subsp. infantis 3300 (ATCC 15697) was observed to reach a final OD600 < 0.40, which was not considered a significant level of growth in our study. In a previous study, it was also reported that B. longum subsp. infantis ATCC 15697 was unable to grow on cranberry xyloglucans [Lactobacillus strains utilized FOS or inulin better when grown at 32 °C instead of 37 °C, including strains L. casei 4646, L. paracasei B4564 and to a lesser extent L. reuteri 1428 which were identified as growing on cranberry A6 in this study [L. rhamnosus GG was reported to not utilize cranberry A6 in both the current study and the Ozcan et. al. [600 > 0.30 suggesting a higher level of background growth using our specific growth conditions (data not shown). Previously, B. longum subsp. longum (UD401) was the only Bifidobacterium strain reported to grow using cranberry xyloglucan as a sole carbon source, reaching a final OD600 = 0.15 [B. breve may also be able to utilize this potential prebiotic.) > 0.50 . B. longoglucans . The higLactobacillus strains and Bifidobacterium breve 2141, which showed optimal growth on cranberry A6 , and all but L. fermentum 1925, L. rhamonosus LRCLR1 and L. reuteri 1428 produced butyric acid (white bars). L. plantarum ATCC BAA 793 was one of the highest producers of both propionic (4.10 mM) and butyric (0.88 mM) acids, even though the other cultures appeared to grow better on cranberry A6 based on their final culture densities , L. delbreuckii subsp. lactis B735 (3.82 mM), and L. plantarum B4496 (3.42 mM) produced the highest concentrations of propionic acid; and L. buchneri (0.91 mM) and L. bulgaricus LB11 (0.74 mM) produced the most butyric acid. B. breve 2141 produced the highest concentration of butyric acid (1.05 mM) in our study, which was a similar concentration observed when grown on FOS (0.98 mM) or inulin (1.01 mM) [B. breve 2141 was shown to produce a higher concentration of propionic acid (≥3.50 mM) on FOS or inulin, when compared to growth on cranberry A6 (1.58 mM). Additionally, L. casei 4646, L. delbreuckii B735 and L. rhamnosus LCR1 were previously reported to produce less propionic acid when grown on FOS (≤0.45 mM) or inulin (≤0.71 mM) as compared to cranberry xyloglucan, while butyric acid production was not detected using FOS or inulin. However, L. reuteri 1428 was reported to produce higher concentrations of propionic (≥2.74 mM) and butyric (≥1.22 mM) when FOS or inulin was provided as a sole carbon source [From the ten lactobacilli identified to grow on cranberry A6, n source , studiesn source ,30,40,44n source ,45,46.Lactobacillus or Bifidobacterium strains in our study would not be sufficient to elicit this type of response but could potentially serve to raise the concentration produced by the indigenous microbiota if they could be for development of a synbiotic.Propionate has been reported to reduce fatty acid synthesis within hepatocytes and lower serum cholesterol levels; contribute to weight control as a regulator of satiety; and to induce apoptosis of colorectal carcinoma cell lines . The conIn humans, the intestinal microbiota produces less butyrate than acetate or propionate ; howeverLactobacillus strains identified for fermenting cranberry xyloglucans have not been investigated for potential probiotic activities, other than the production of SCFAs described within this study. L. casei 4646 is the most well-studied strain for its potential to contribute to the formation of dental caries since it was originally isolated from a carious lesion [L. casei 4646 preferentially colonized the tongue and saliva, and when it was associated with the plaque microbiota at > 1% there was a reduction in Streptococcus mutans-induced dental caries. These results suggest that L. casei strains have potential as oral probiotics, with L. casei strain Shirota reported to prevent plaque-induced gingival inflammation [Lactobacillus and Bifidobacterium strains, as well as strains of Streptococcus thermophilus, Streptococcus salivarius, Lactococcus lactis and Enterococcus faecium on oral health [L. casei group, which consists of L. casei, L. paracasei and L. rhamnosus, have been studied for their ability to prevent allergic reactions, obesity, pathogen colonization and cancer; as well as modulate the immune system and enhance cognitive functions [L. rhamnosus GG being commercially available as a probiotic. Other species described in this study included L. fermentum, from which strains have been studied for their antimicrobial activity for food preservation and their cholesterol-lowering and anticancer potential [L. plantarum and L. paraplantarum [L. delbreuckii subsp. bulgaricus [L. buchneri [L. reuteri [B. breve 2141 was also shown to grow on cranberry xyloglucan, and several strains of this species have been investigated for treatment of targeted pediatric diseases, including diarrhea, infant colic, celiac disease, obesity, allergies, and neurological disorders [Lactobacillus and Bifidobacterium strains identified as growing on cranberry xyloglucans to determine if they are feasible candidates for the development of synbiotics to improve human or animal health. Previously our laboratory demonstrated the survival and subsequent production of SCFAs by L. reuteri 1428 and B. breve 2141 within a synbiotic matrix containing FOS, inulin, or pectic-oligosaccharides [The ten s lesion . Howevers lesion  study reammation . Severall health . Additiootential . Similarlantarum ; L. delblgaricus ,68; L. bbuchneri  and L. r reuteri . B. brevisorders . Taken tcharides , but addp < 0.05), which agreed with our previous study that reported FOS and citrus pectic-oligosaccharides induced Bifidobacterium growth within a mixed fecal fermentation [Bacteriodaceae and a reduction in Enterobacteriaceae [When fecal fermentations were performed using the preparative HPLC fraction 2 from cranberry A6 (extract 2), no significant growth was observed for any of the bacterial populations monitored . For theentation . The abseriaceae . Anothereriaceae . Unfortueriaceae  reportedp < 0.05) compared to the negative control at 24 h  ≥ 0.50 after 24 h incubation at 32 °C. All of these ten Lactobacillus strains produced lactic, acetic and propionic acids, and all but three strains produced low levels of butyric acid. This was the first report that the Lactobacillus ability to metabolize cranberry oligosaccharides is strain specific, and this is the first study to demonstrate that several Lactobacillus species, other than L. plantarum, can utilize this potential prebiotic. In mixed culture fecal fermentations using FISH analysis with 16s rRNA probes, no bacterial class had significant growth when the cranberry preparative HPLC fraction enriched in arabino-xyloglucan was included yet acetate, propionate and butyrate increased in these fecal fermentations. The even higher level of SCFAs produced when crude cranberry pomace was included in fecal fermentations suggests that other cranberry compounds may contribute to its microbial modulating effect. More research is necessary to completely determine the cranberry prebiotic structure/function relationships.The carbohydrate composition and structure of cranberry pectic oligosaccharides was described as rhamnogalacturonan I structures which were methyl-esterified, acetylated with arabinogalacto-oligosaccharide side chains and a 4,5-unsaturated function at their non-reducing ends. Since arabinose-rich pectic oligosaccharides were previously reported with prebiotic activity, this suggested that the cranberry A6 fraction could also stimulate the growth and short-chain fatty acid production of health-promoting bacteria. Of the 86


Evaluation of micromotion in various activities in daily life is essential to the assessment of the initial fixation of cementless short stems in total hip arthroplasty. This study sought to evaluate three-dimensionally the micromotion of two types of cementless short stems.Two types of stems were used: the Fitmore stem with a rectangular cross-section (rectangular stem) and the octagonal-oval GTS stem with fins (finned stem). Finite element analysis was used to calculate the micromotion of two activities that place a heavy load on the stem (single-leg stance and stair climbing). Three values were measured: the magnitude of micromotion (mean and 95th percentile), the location of micromotion above the 95th percentile value, and the directions of the micromotion vector.1. There was no significant difference in the magnitude of the micromotion between the rectangular stem and finned stem groups for single-leg stance or stair climbing. 2. In both groups, the micromotion was greatest at the proximal and distal ends. 3. The direction of the micromotion was similar in both groups; internal rotation occurred from the distal to the middle of the stem during stair climbing.The rectangular stem had comparable initial fixation to that of the finned stem. In both models, the micromotion was greater at the proximal and distal ends. The direction of the micromotion was not dependent on the stem shape but on the direction of the load on the artificial femoral head. These results will be important for stem selection and future stem development.The online version contains supplementary material available at 10.1186/s13018-022-03329-5. Cementless short stems have been used for hip arthroplasty in an increasing number of cases due to their superiority in femoral bone preservation. Although the medium-term results for cementless short stems are stable, there are some cases in which the stem loosens within a short period due to insufficient initial fixation , 2. To aThe subjects consisted of patients who underwent total hip arthroplasty (THA) with a Fitmore or GTS stem  Fig.  at SappoThe Fitmore stem (rectangular stem) is characterized by its rectangular cross section and medial curve, and the GTS stem (finned stem) is characterized by its elliptical octagonal cross section with fins. The surgical approach was antero-lateral (AL), also known as the Orthopädische Chirurgie München (OCM) approach, in all cases. This study was approved by the Institutional Review Board of our hospital . CT images  were obtained at 120 kV, 70mAs, and a slice thickness of 0.5 mm. CT images were taken from the pelvis to both knee joints before THA and within 2 weeks after THA.As postoperative CT images show strong artifact of the stem, postoperative femur-stem models were created using the following method. First, the pre- and postoperative CT images of all selected patients were converted into standard triangulated language (STL) data using three-dimensional (3D) imaging software . The femurs and the implanted stems were then three-dimensionally reconstructed from the CT images; values of 200 Hounsfield units (HU) or more were defined as bone –16, and ρ (g/cm3)) using the standard calibration curve of the finite element analysis software, as in previous reports [E (MPa)) was calculated from the BMD of each mesh based on the prediction equation of Morgan et al. [Elastic analysis was used for the analysis. Micromotion was defined as the displacement difference at the element nodes of the contact surface between the femur and the stem –19. The  reports , 21, andn et al. , 22  of the micromotion of the stem calculated by finite element analysis, the area in which micromotion with a value greater than the 95th percentile was distributed, and the direction of the micromotion vector. The 95th percentile value was used to minimize the influence of outliers, as the maximum value could be affected by outliers. The vectors were displayed as vectors from the starting point of each node, and the length of the vectors was multiplied by 150 and illustrated using graphing software   with a mean age at surgery of 62.4 ± 7.9 years and a mean body mass index (BMI) of 24.7 ± 3.0 were included. Ten patients had rectangular stems and 10 had finned stems (Table p < 0.01) . There were no significant differences in the 95th percentile values of the micromotion between the two groups.In the rectangular stem group, the mean value of the micromotion was 29.9 ± 5.0 μm for single-leg stance and 37.4 ± 7.2 μm for stair climbing, with that for stair climbing being significantly larger (In both groups, micromotion above the 95th percentile value was found at the proximal and distal ends during single-leg stance and stair climbing Fig. . MicromoThe micromotion vectors were similar for the same activity in the two groups Figs.  and 8. TThis study is the first to evaluate the direction of the micromotion of the stem in three dimensions. To evaluate the initial fixation of the cementless short stems, we need to evaluate micromotion under load , 10, 12.For the initial fixation of cementless stems, it is important that they have excellent rotational stability . HoweverAl-Dirini et al.  reportedNo reports to date have evaluated the direction of micromotion three-dimensionally during multiple activities. In the present study, the direction of micromotion was assessed in three dimensions using three-dimensional vectors. In this study, it was found that micromotion with internal rotation from the middle to the distal part of the stem, regardless of the stem shape, occurred more during stair climbing than during single-leg stance. This result suggests that the direction of micromotion in a cementless short stem is not dependent on the shape of the stem but on the direction of loading on the bone head by the activity. By evaluating the micromotion using three-dimensional vectors, it became possible to evaluate the behavior of the stem during early postoperative activities.One limitation of this study is that the number of cases was small: ten rectangular stems and ten finned stems. However, the strength of this study is that it compares not only a single activity but two different activities with different degrees of rotational torque in daily life. In addition, this study only uses finite element analysis and no validation based on cadaver experiments. However, the same method was used in previous reports where validation was obtained by finite element analysis and cadaver experiments , 19. FurUsing a patient-specific postoperative femoral model, we evaluated the three-dimensional micromotion as obtained from finite element analysis of two types of cementless short stem. The rectangular stem without fins had a comparable initial fixation to the octagonal-oval finned stem. In both models, micromotion was greatest at the proximal and distal ends. Furthermore, the direction of the micromotion was similar for the same activity in both models, suggesting that the direction of the micromotion may depend on the type of motion regardless of the stem shape. By using the three-dimensional vector evaluation of micromotion, we could analyze not only the magnitude but also the three-dimensional direction of the micromotion. The three-dimensional vector evaluation of micromotion in this study is thought to be helpful for stem selection and stem development.Additional file1. Figure S1: Mesh convergence Analysis of maximum deflection and number of elements. The largest number of elements is in this study.