As of May 2016, pictorial health warnings (PHWs) showing the harms of smoking were implemented in the European Union. After one year they had to be fully implemented. We studied changes in awareness of the health risks of smoking after implementation of PHWs among smokers from the Netherlands, whether the trend before the implementation changed after the implementation, and whether there were differences between subgroups.We used survey data from six yearly waves of the International Tobacco Control (ITC) Netherlands Survey from 2012 to 2017. The number of participating smokers ranged between 1236 and 1604 per wave. Data were analyzed using Generalized Estimating Equations (GEE) analyses.p = 0.059) and perceived severity  regarding lung problems. Perceived susceptibility, however, was more pronounced between 2015 and 2017 than between 2012 and 2015. Noticing information about the dangers of smoking  and knowledge about the health risks of smoking  increased between 2015 and 2017. These increases were both more pronounced when compared to 2012–2015 . Compared to high educated smokers, low educated smokers  and moderate educated smokers  were less knowledgeable about the health risks of smoking in 2016 and 2017.Indicators of awareness of the health risks of smoking that did not change between 2015 and 2017 were perceived susceptibility (β = 0.043, Introducing PHWs coincided with an increase in smokers’ knowledge about the health risks of smoking. Dutch tobacco control policy and campaigns should focus on improving Dutch smokers’ awareness of the health risks of smoking even more, especially among low educated smokers. Pictorial health warnings (PHWs) on the packet of tobacco products  were intTobacco health warnings communicate information about the health risks of smoking. This intervention is a method of ‘consciousness raising’ to influence awareness as the warnings communicate the consequences of smoking. PHWs specifically are a method of ‘imagery’ to influence knowledge about the health risks of smoking as PHWs make it easy to learn about these risks . SmokersThe THWs comprise a message with ‘Smoking causes 9 out of 10 lung cancers’ and ‘Smoking damages your lungs’. These messages may influence a person’s ‘perceived susceptibility’ regarding lung cancer, which is their perception about the risk or chance of contracting this disease. Such perceptions may play a role in smoking cessation as smokers who are feeling more susceptible to these health risks more often intend to quit smoking –25. AlsoFurthermore, three PHWs use the method of ‘fear arousal’  as they According to the I-Change model, knowledge forms a person’s awareness of the health risks of smoking, together with risk perception, and noticing advertising or information about the dangers of smoking (perceived cues) . To dateBefore the introduction of PHWs, Dutch campaigns only focused on positioning non-smoking as the social norm, quitting smoking, and prevention of smoking. Therefore, the current study aims to examine the trend in harm awareness between 2012 and 2015 and whether this trend differs from the trend between 2015 and 2017. This study may provide insights in what happens with smoker’s awareness about the health risks of smoking when there are no policies that aim to improve awareness.This paper further aims to identify subgroup differences in awareness of the health risks of smoking. In order to target campaigns at the most relevant subgroups of smokers, it is important to explore differences according to age, education, and gender. Previous research showed that lower education is associated with less knowledge about the health risks of smoking , 27–30. In sum, the current study was designed to examine three research questions: (1) Did smokers show changes in awareness of the health risks of smoking after introducing PHWs in 2016? (2) Did the trends in awareness of the health risks of smoking after introducing the EU’s PHWs differ from the trends before their introduction; and (3) Did awareness of the health risks of smoking in 2016 and 2017 differ by age, educational level, and gender? Results from our study may result in recommendations for future policy regarding health warnings and campaigns on the national and European level.This study is part of a larger PhD Project on the evaluation of tobacco control policies in the Netherlands; previously published papers from this PhD Project have used the same dataset but explore different research questions , 32. DatN = 2022), Wave 7 , Wave 8 , Wave 9 , Wave 10 , and Wave 11  were used. Those who quit smoking were excluded from all analyses because they did not receive all questions and awareness of the health risks of smoking tends to change after people quit smoking. Between survey waves, between 12.6 and 29.3% of the respondents per wave were excluded from the analyses because they had quit smoking. Attrition among smokers ranged from 17.8 to 25.4% between survey waves. Figure For our analyses, data from survey Wave 6 , knowledge about the health risks of smoking, moderators , and control variables  can be found elsewhere . Concernp-value of < 0.01 were considered as statistically significant [Data were analyzed using SPSS 23.0. To ensure representativeness of the sample, this study’s tests and statistical estimates were weighted by age and gender . For thenificant .Table p = 0.002). Separate GEE analyses showed that between 2012 and 2015 there was no change in perceived cues, while a significant increase was found between 2015 and 2017 (Table Table 17 Table .Table 3p = 0.002), aged 40–54 smokers compared to aged 15–24 smokers , and female smokers compared to male smokers .Smokers who were more likely to perceive cues to quit smoking were 55+ smokers compared to aged 15–24 smokers  = − 0.547 to − 0.122; p = 0.764). This implies that for perceived susceptibility, the increase between 2016 and 2017 was significantly stronger when compared to the increase between 2012 and 2015.The mean score for perceived susceptibility to developing lung cancer fluctuated between 3.1 and 3.2 over the years, while the mean score for perceived severity for developing lung problems fluctuated between 3.4 and 3.6 (Table p < 0.001), and aged 40–54 smokers compared to aged 25–39 smokers . Subgroups more likely to perceive developing lung problems as severe were aged 25–39 smokers compared to aged 55+ smokers , and aged 40–54 smokers compared to aged 55+ smokers .The subgroup more likely to feel susceptible to developing lung cancer was aged 40–54 smokers compared to aged 55+ smokers . The increases in knowledge about smoking causing blindness and impotence in male smokers, mouth and throat cancer, and secondhand smoke causing heart disease were more pronounced between 2015 and 2017 than between 2012 and 2015. However, only the CI’s of the trends of knowledge about smoking causing blindness were not overlapping. No changes were found in knowledge about smoking causing heart disease, lung cancer, and secondhand smoke causing lung cancer.Dutch smokers’ scores on knowledge increased from 4.3 in 2012 to 5.1 in 2017 (Table p < 0.001), and aged 40–54 smokers compared to aged 55+ smokers . Also, high educated smokers were more likely to be knowledgeable than low educated smokers  and moderate educated smokers .Subgroups more likely to be knowledgeable about the health risks of smoking were aged 25–39 smokers compared to aged 55+ smokers  Framework Convention on Tobacco Control (FCTC), which requires ratifying countries to promote public awareness of and access to information regarding the health risks of smoking . AnotherConcerning the third research question, to identify subgroup differences in awareness of the health risks of smoking, we found that, in line with previous research , youngerDifferences in sample characteristics were found over the years Table . In respDutch smokers’ knowledge levels remain low as our study for instance showed that in 2017 only 67.4% of Dutch smokers knew that smoking can cause stroke and only 85.5% knew that smoking can cause lung cancer. This implies that Dutch tobacco advocacy organisations and the government should invest more in campaigns designed to improve knowledge about the health risks of smoking’. Previous research from Australia showed that media campaigns and PHWs may operate in a complementary manner to increase awareness of the health risks of smoking and quit intentions . Our stuAlthough we cannot prove causality, the introduction of the EU’s PHWs seemed to coincide with an increase in the belief that smoking causes blindness. This finding would imply that to increase overall knowledge about the health risks of smoking, pictures may be added that cover risks from smoking that have not yet been fully communicated to the public similar to blindness. Potential risks are pregnancy complications, low bone density and hip fracture, peptic ulcer (open sores inside stomach), and diabetes mellitus type 2 . In the Even after implementing EU’s PHWs, still few Dutch smokers are fully aware of the health risks of smoking. This illustrates what can happen when countries are reticent to inform smokers about these health risks. The change from THWs to the PHWs coincided with an increase in awareness of the health risks of smoking, but reconsidering the current strategy on informing smokers about these health risks with special attention to older and low educated smokers is strongly recommended.


The development of adrenocortical adenoma and pheochromocytoma within the same adrenal gland is very rare. Furthermore, no reports have described coincident black adrenal adenoma and pheochromocytoma. We herein report a rare case of coincident black adrenal adenoma and pheochromocytoma in the same adrenal gland.A 71‐year‐old Japanese woman was hospitalized because a right adrenal tumor had been incidentally found by computed tomography. She was diagnosed with subclinical Cushing’s syndrome and underwent laparoscopic right adrenalectomy. The tumor contained two adrenal nodules. The cut surface of the larger nodule was brownish‐black on macroscopic examination. Pathological studies revealed coincident black adrenal adenoma and pheochromocytoma.To the best of our knowledge, this is the first report of coincident black adrenal adenoma causing subclinical Cushing’s syndrome and pheochromocytoma in the same adrenal gland. The mechanism of this rare scenario is unclear, and further study is necessary. ACTHadrenocorticotropic hormoneBAAblack adrenal adenomaCSCushing’s syndromeCTcomputed tomographyDMdiabetes mellitusDSTdexamethasone suppression testMCMTmixed corticomedullary tumorPCSpreclinical Cushing's syndromePHEOpheochromocytomaSCSsubclinical Cushing’s syndromeWe experienced a rare case of coincident BAA causing SCS and a PHEO within the same adrenal gland. The mechanism of this rare phenomenon is unclear. Further study is necessary to clarify the mechanism.et al.BAA is a rare benign adrenal lesion characterized by a brown or black nodule containing lipofuscin.A 71‐year‐old Japanese woman was hospitalized because a right adrenal tumor had been incidentally found by CT. She had a medical history of hypertension and hyperlipidemia. She had no physical characteristics such as central obesity, moon face, and stretch marks. CT revealed a 2‐cm mass in the right adrenal gland Fig. . Microscet al.Adrenal incidentalomas are defined as adrenal tumors that are unexpectedly discovered in imaging examinations such as CT.The mechanism of this rare scenario is unclear. However, some evidence suggests that the cortical and medullary adrenal glands influence each other by a paracrine mechanism despite the fact that the origins of the glands are different.et al.Notably, there is a category of MCMT that presents as a single tumor mass composed of an intimately admixed population of both adrenocortical cells and pheochromocytes. MCMTs have been reported in 15 cases in the literature to date.Even more surprisingly, our patient had a rare type of adrenal adenoma, namely BAA. BAA is an adrenocortical tumor with a black or brown appearance on cut sections. It may be more common in post‐mortem adrenal glands; it is rare in surgical adrenal samples. The first case of BAA was reported in 1938.We experienced a rare case of a BAA causing SCS complicated with PHEO. To the best our knowledge, this is the first report of a patient with a coincident BAA causing SCS and a PHEO. The association between these two conditions in the same adrenal gland has not yet been clearly investigated and needs further reporting.The authors declare no conflict of interest.


The ageing of the global population is associated with an increasing prevalence of chronic diseases and functional impairments, resulting in a greater proportion of homebound individuals.To examine the health‐care experiences of older homebound adults who have not previously received home‐based primary care (HBPC). To explore their impressions of this method of care.Cross‐sectional qualitative study using semi‐structured interviews.18 older homebound individuals in Central Virginia.Our findings revealed that homebound individuals faced significant health challenges, including pain resulting from various comorbidities. They felt that their mobility was restricted by their physical conditions and transportation challenges. These were major barriers to social outings and health‐care access. Participants left their homes infrequently and typically with assistance. Regarding office‐based care, participants were concerned about long wait times and making timely appointments. Some thought that HBPC would be convenient and could result in better quality care; however, others believed that the structure of the health‐care system and its focus on efficiency would not permit routine HBPC.Older homebound adults in this study faced high burdens of disease, a lack of mobility and difficulty accessing quality health care. Our observations may help researchers and clinicians better understand the health‐care experiences and personal opinions of older homebound individuals, informing the development of effective and empathetic home‐based care. Participant responses illuminated a need for education about HBPC. We must improve health‐care delivery and develop comprehensive, patient‐centered HBPC to meet the needs of homebound individuals. The US Census projects that by 2050, more than 20% of the total US population will consist of older adults, compared with 13% in 2010.The health‐care needs faced by homebound older adults are associated with high costs to the US health‐care system.There is a lack of qualitative research designed to investigate the health‐care experiences and needs of homebound people and their perceptions of HBPC, specifically in the United States.22.1Participants were recruited from the city of Charlottesville, Virginia, and the surrounding Albemarle, Louisa, Buckingham, Fluvanna and Orange counties. Eligibility criteria for the study included homebound status, age 50 or older, fluency in English and no apparent cognitive impairment. Participants were recruited through convenience sampling from clinical programmes, community programmes that serve homebound patients, and through a snowball sampling method. Clinical programmes included University Physicians at the Jefferson Area Board for Aging (JABA), University Medical Associates and University of Virginia (UVA) Continuum Home Health, which serves both homebound and semi‐homebound individuals. University Physicians at JABA provides primary care to patients aged 65 and older in an office setting; University Medical Associates is also a primary care office. UVA Continuum Home Health provides home health services to patients of all ages who are disabled, chronically or terminally ill, or recovering from an acute illness. Community programmes included JABA Charlottesville, Mom's Meals and Meals on Wheels of Charlottesville/Albemarle. JABA is a 501(c)(3) non‐profit that serves older adults, individuals with disabilities and caregivers in Central Virginia. Mom's Meals and Meals on Wheels are programmes that deliver meals to individuals who may have decreased mobility; Mom's Meals is a for‐profit company under PurFoods LLC, whereas Meals on Wheels is a 501(c)(3) non‐profit. Snowball sampling is a recruitment technique by which participants suggest other potential participants for the study. Homebound individuals provided a name and phone number for potential participants; the researchers then contacted these individuals about the study. Homebound status for an individual was defined as being unable to leave one's home without assistance or being confined to the home for a majority of the week, encompassing both homebound and semi‐homebound common definitions. A loose definition was used due to a lack of consensus on the definition of homebound status and because definitions based on eligibility for Medicare services may not encompass the entire homebound population.2.2During summer 2018, the primary author contacted health‐care providers and organizations in the Charlottesville community involved in homebound patient care, which advertised the study and distributed information about the study to potential participants. Interested older adults who received this information then contacted the researchers, who described the study and asked whether the individuals were willing to participate. Homebound status was verified over the phone, seeing as the eligibility criteria for the programmes from which participants were recruited were diverse and not limited to only homebound individuals. Willing participants reviewed and signed an IRB‐approved consent form and Health Insurance Portability and Accountability Act (HIPAA) authorization form.A total of 18 interviews lasting an average of 50 minutes (range 26 to 96 minutes) were conducted at the homes of participants. A qualitative descriptive design was adopted, which used semi‐structured, guided interviews to create a detailed, in‐depth summary of the intricacies of homebound life.Interviews were audio‐recorded and transcribed verbatim. To ensure participants’ privacy, pseudonyms were assigned to individual participants, identifying information was removed, and files were stored on secured devices. Descriptive statistics were used to describe characteristics of the participants. The interview data were analysed using a thematic analysis approach via NVivo 12 software  for qualitative coding. An a priori topical coding scheme was developed based on the interview guide and grounded in patient work system theory.33.1A majority of the 18 participants were female and the participants’ ages ranged from 52 to 91 years, with the average age being 68.7 years  health conditions, 2) mobility limitations and 3) health‐care experiences. While other interesting themes did emerge, such as social isolation and spirituality, these three categories were articulated by the majority of the respondents and given the greatest emphasis in this analysis, due to our research focus on participant interactions with the health‐care system. In the description of the thematic results below, refer to Tables  mobility health‐c3.2.1The homebound individuals interviewed experienced various health problems. Common physiological illnesses encompassed arthritis, diabetes, stroke, bone fractures, cardiovascular disease, heart failure, high cholesterol and urinary tract infection. Pain was mentioned by 12 participants and 16 participants considered themselves physically disabled in some manner (quotes 1‐5). Psychological conditions included depression, anxiety, schizophrenia, attention deficit disorder and post‐traumatic stress disorder. Depression was a prevalent influence on homebound individuals’ mental health; 10 participants mentioned feeling depressed, although not all of them have been clinically diagnosed (quotes 6‐8). For a majority of the older adults interviewed, the development of homebound status was due to a physical impairment resulting from medical conditions such as arthritis, bone fractures and stroke. Almost all participants experienced at least two major medical conditions.3.2.2A majority of participants were only able to leave their home 1‐3 times per week, typically only for grocery shopping or to attend a doctor's appointment (quotes 9‐12). Mobility restrictions were due to physical disabilities, lack of transportation and financial constraints. Most participants were either driven by family or friends or used public transportation services, such as a regional bus service for ADA‐certified riders providing curb‐to‐curb or door‐to‐door transport, depending on location, by appointment (quotes 20‐21). Public transportation posed challenges for study participants who were wheelchair or walker‐bound, although some individuals said transportation employees helped with access onto the vehicle (quote 20). However, getting to a public bus stop and getting from drop‐off locations to office and hospital entrances remained additional obstacles (quote 19). Participants expressed that depending on other people to get around made them feel like a burden and prevented them from engaging in community social activities (quote 22). Transportation limitations posed an even greater difficulty to rural participants, who needed to travel farther to access office‐based health care.Participants both enjoyed their outings and found them challenging. Many participants wished they could still go out socially or engage in activities they used to enjoy, and variations of the phrase ‘I wish I could do the things I used to do’ came up in a majority of the interviews . Even grocery shopping allowed participants a sense of autonomy because they could browse the store and make independent decisions about what to purchase (quote 15). However, some participants did respond that although they could not leave their homes often, they would rather stay at home regardless, as their outings caused them physical discomfort, pain, anxiety or fear (quotes 16‐19).3.2.3Participants were asked a series of questions pertaining to their personal health‐care experiences and their opinions on the concept of home‐based primary care. A common complaint among participants concerned the efficiency of the health‐care system, given that homebound older adults often faced long wait times in medical settings (quotes 26‐28) and encountered delays when trying to schedule new office‐based appointments (quote 23). Participants often felt rushed or undertreated during urgent care or emergency room visits (quotes 28‐31). From a patient perspective, rushed appointments gave the impression that the health‐care system prioritizes efficiency and profit over the needs of patients . Multiple participants referenced themselves as being experts of their own bodies and felt like some doctors formed preconceptions of their patients’ disease, failing to understand their pain (quotes 36‐38). One study participant raised a unique point regarding health‐care services for the older homebound population, saying that following a hip replacement operation, although doctors ensured she was mending physically, there was a lack of follow‐up on her mental health (quote 25). Three participants cited a lack of accessible, affordable dental care for homebound older adults, which compromised their oral health, as dental disease is a largely preventable burden (quote 24). The rural participants were especially affected by a lack of dental services.When asked which characteristics and behaviours they believed an effective care provider should display, participants referenced bedside manner and interpersonal communication skills (quotes 32‐35). Most participants interviewed had encountered some form of Medicare home health care, such as physical therapy or personal caregivers, but none routinely received home‐based primary care. When questioned about their opinion on HBPC, participants reported mixed opinions. Some participants believed that the structure of the health‐care system and its focus on efficiency would not permit routine HBPC (quote 46). A few participants said they were used to going out to see their health‐care providers, and it got them out of the house . Many participants agreed that HBPC would be convenient and could result in better health care as practitioners may better understand one's living environment (quotes 42‐44); however, other participants were uncomfortable with the prospect of having clinicians in their home. In addition, some individuals wanted to continue seeing their trusted long‐time personal physician, in office .4Participants shared a collection of compelling narratives regarding their experiences as homebound older adults in relation to the health‐care system. Although responses to more sensitive personal topics were likely influenced by participants’ unique experiences and personality, common opinions that fell within the thematic clusters of health conditions, mobility limitations and health‐care experiences still arose often. By speaking about their health conditions, participants helped characterize the heavy medical burden faced by older homebound individuals. Medical conditions that resulted in functional limitations and homebound status seemed to limit access to office‐based care. This challenge was exacerbated by public transportation inaccessibility. In describing their interactions with the health‐care system, participants indicated issues with office‐based care, desirable health‐care provider characteristics and varied opinions of home‐based primary care. These themes were supported by existing qualitative research and are important considerations for health‐care providers and policymakers involved in the care of homebound older adults.In accordance with published research on the homebound population, the participants interviewed faced significant physical and mental health challenges.Homebound individuals wanted to be more mobile and independent, and felt restricted by their physical conditions and transportation challenges.Among the most prevalent concerns regarding office‐based care were difficulty with making timely appointments and long wait times. Participants highly valued health‐care providers who listened closely to patients and displayed genuine care and concern. Coordinated HBPC where patients’ goals and preferences are clear, patients are provided community resources, and collaboration exists between health‐care providers, social workers and medical equipment agencies has the potential to circumvent office‐based care issues.As homebound older adults possess widely varying opinions towards HBPC, one's individual needs and preferences should be considered when deciding whether to implement HBPC for the particular individual. Opposition and confusion regarding HBPC may be influenced by a lack of exposure, experience and education about this system of medical care, which has only recently experienced a resurgence.4.1While this research illuminates important perspectives of the homebound older population, it has various limitations. There are limitations to the recruitment method; participants learned about the study from common programmes they utilize, so they may have shared characteristics. For example, participants recruited through UVA Continuum Home Health may tend to have more positive conceptions of home‐based care, having received some form of in‐home care, whether it be physical therapy or home‐based social work services. Disadvantages of snowball sampling include lack of representativeness and sampling bias, as participants may refer potential participants with similar characteristics due to homophily. The recruitment method did not appear to exhibit any association with severity of participant health problems based on interview data; however, no formal analysis based on recruitment method was performed. Increased diversity of the participant population might have revealed additional relevant perspectives that are culturally significant or unique. Although the proportion of Asians and Latinx individuals in Central Virginia is low (around 3%‐6% based on estimates for various counties), additional data should be collected from these groups to obtain opinions that are more representative of these populations. The relatively large number of rural and African American participants in this study is a strength, as it illuminates important, often underrepresented viewpoints.Due to the qualitative nature of our research, no causation nor statistically significant correlations can be drawn from the study's observations. Furthermore, variations in regional culture and preferences prohibit this qualitative investigation from being generalizable nationwide. Study participants mentioned financial difficulties in relation to transportation, hospital visits and office‐based care, but no empirical data on socio‐economic status were collected. In future studies, it would be valuable to collect this information and investigate correlations between particular participant characteristics and their opinions on health care. Additionally, future research involving a larger sample could explicitly compare the experiences of urban versus rural populations. Future research on this overarching topic should focus on investigating a specific thematic category highlighted by this study, using quantitative statistical methods and a larger, more diverse sample that is more representative of and generalizable to the population.5The key thematic categories of health conditions, mobility limitations and health‐care experiences characterized by this study provide a framework for further investigation into factors that affect the lives of homebound older adults. Homebound individuals in this study and in the literature faced a higher disease burden than the rest of the older population. They drew specific attention to the physical pain they commonly experienced and the manner in which it was addressed by their health‐care providers. Mobility restrictions resulting from physical impairments and lack of transportation limited individuals’ access to health care and social activities, and rural individuals were especially disadvantaged. Homebound older adults utilize health care at high rates, so it is imperative to consider their opinions on the quality and efficacy of health‐care services. These results highlight a lack of knowledge regarding home‐based primary care. This research may help researchers, clinicians and policymakers better understand the health‐care experiences of older homebound adults in the United States. This study should inform the development of comprehensive patient‐centered home‐based care, as well as new approaches for overcoming identified health‐care challenges.The authors declare that there is no conflict of interest.Yao and Cheng conceived and designed the study. Cheng acquired the data and drafted the manuscript. All authors analysed and interpreted the data and critically revised the manuscript.


Self‐incompatibility (SI) is a self‐recognition genetic system enforcing outcrossing in hermaphroditic flowering plants and results in one of the arguably best understood forms of natural  selection maintaining genetic variation over long evolutionary times. A rich theoretical and empirical population genetics literature has considerably clarified how the distribution of SI phenotypes translates into fitness differences among individuals by a combination of inbreeding avoidance and rare‐allele advantage. At the same time, the molecular mechanisms by which self‐pollen is specifically recognized and rejected have been described in exquisite details in several model organisms, such that the genotype‐to‐phenotype map is also pretty well understood, notably in the Brassicaceae. Here, we review recent advances in these two fronts and illustrate how the joint availability of detailed characterization of genotype‐to‐phenotype and phenotype‐to‐fitness maps on a single genetic system (plant self‐incompatibility) provides the opportunity to understand the evolutionary process in a unique perspective, bringing novel insight on general questions about the emergence, maintenance, and diversification of a complex genetic system. At the phenotypic level, SI is typically determined by the segregation of a finite (often large) number of mating types, corresponding to as many distinct pollen–stigma recognition specificities. Beyond the sole avoidance of self‐fertilization, SI thus also prevents fertilization between unrelated individuals expressing identical recognition specificities  and mount a specific and appropriate cellular response (pollen tube rejection vs. acceptance). Identification of the SI genes in a handful of plant families revealed entirely different molecular and physiological processes, making it clear that SI arose independently several times  by which plants favor outcrossing and avoid selfing or crossing between related individuals , respectively , where SI is controlled by a non‐a et al.  recentlyS‐loci can arise through different evolutionary trajectories, possibly using functionally related gene families and a repertoire of “pre‐adapted” S‐receptor‐like genes in Brassicaceae. Similarly, independent origins of two distinct S‐RNase‐based gametophytic SI systems have been suggested in the Rosaceae family, generating the coexistence of distinct self‐recognition (genus Prunus) and non‐self‐recognition systems  within the family  and identification of new S‐allele sequences by de novo assembly . The very large proportion of shared alleles among closely related species can be explained by the strong negative frequency‐dependent selection expected to maintain the ancestral polymorphism as well as by the adaptive introgression of S‐alleles after speciation  is initially unique in its population and therefore benefits from maximal mating success due to the advantage of the rare .An important limitation of these models is that they consider single isolated populations only, while population subdivision is expected to affect strongly the overall segregation frequency and ultimate fate of SC mutants  or ancestral versions of SRK alleles  has retained the same recognition specificity as the ancestor, while the other one (28S) has functionally diverged and now represents a true evolutionary novelty. This observation shows that SI specificities can indeed be maintained over long evolutionary timescales and allow to reject models predicting rapid allelic turnover , by computing the likelihood of alternative models taking into account drift only or drift with negative frequency‐dependent selection acting on the S‐locus, which confirmed the occurrence of selection. Focusing on the maternal success in experimental conditions of limited pollen availability, Leducq et al.  than homozygotes for the recessive S‐haplotype 1S (where no load was detectable), consistent with the theoretical prediction. A genetic load associated with S‐haplotypes was also detected in A. lyrata, although the link with dominance was not confirmed  suggests that the deleterious mutations are more likely to be found in the immediate flanking genes rather than within the S‐locus itself.While the theoretical prediction is clear , empirical evidences of the sheltered load are scarce, notably because distinguishing the effect of inbreeding depression due to the mutational load spread throughout the genome from the effect of the genetic load specifically linked to the 55.1S‐locus. It is tempting to speculate that the evolution of the network of pairwise dominance interactions between S‐alleles should converge toward a linear hierarchy for two main reasons. First, theoretical studies showed that a mutation changing the dominance level without changing the allelic specificity  should be favored as long as it does not lead to co‐dominance , produced by a hairpin RNA within the S‐locus, and acting in trans‐ via de novo methylation of the promoter of the recessive SCR allele  and a diversity of targets located in different SCR regions  and its unique target. An important difference between the models by Durand et al. , while additional sRNA regulators may be necessary to allow the regulatory system to deal with the increased complexity of highly multiallelic systems .This simple regulatory model was later shown to extend to more complex topologies of the dominance network, that is, when more than two levels of dominance exist. In d et al.  and Yasud et al.  is how nd et al. , the domd et al.  proposedS‐alleles reached the top of the hierarchy by evolution of the sRNA precursors they carry toward more generalism (enabling them to interact with more SCR targets), rather than by increasing their total numbers. On the other hand, the most recessive alleles tend to have more targets that are on average also more generalist because they tend to be targeted by multiple dominant alleles. In addition, cases of apparent redundancy have also been observed, with some dominant haplotypes targeting twice the SCR allele of the same recessive haplotype  with defects in at least the SCR gene. While loss‐of‐function mutations are typically expected to be recessive  is apparently still producing sRNAs predicted to be able to transcriptionally silence the more recessive SCR allele (SCR2) brought by its A. arenosa parent. Hence, the shift to selfing in this recently formed hybrid species may have been an indirect consequence of the identity (and dominance) of the SI alleles carried by the two progenitor parents. How much the evolution of the mating system of this hybrid species was contingent upon the dominance of the two particular SI alleles it inherited is an open question.In a similar twist, the tetraploid a et al.  recently5.4S‐locus pollen dominance encode miRNA‐like hairpin precursors, the sRNAs from the dominant S‐locus appear to function by targeting RdDM methylation to the promoter of the recessive SCR allele . This raises the intriguing possibility that they may be using different mechanisms from those proposed in Brassica (and from each other). Finally, in contrast to the situation in pollen, S‐locus dominance in pistils does not appear to be due to differences in transcriptional regulation (of SRK)  could be achieved in spite of extensive divergence of the underlying molecular components.In spite of intensive efforts to decipher the SI response, the molecular and physiological processes by which plants discriminate self‐ from nonself pollen remain incompletely described , and because of some of their most salient evolutionary properties , but it is clearly an objective worth pursuing for several reasons. Beyond the clear implications for applied research to facilitate the creation of stable varieties with agronomic characters of interest Box , the comBOX 2S‐allee effect (Wagenius et al., Although our own work is mostly geared toward fundamental research, understanding how SI is controlled and evolves has clear relevance for applied research. First, SI has been used as a way to control the production of F1 cultivars in Brassica crops (Nasrallah, S‐linked genetic load relative to the overall genomic load, and how demographic variations and mating system shifts affect these factors in return typically remain poorly documented. Further theoretical and experimental studies will provide exciting novel insight to understand the factors favoring and maintaining SI or leading to SC and eventually selfing, and understand the evolutionary dynamics of S‐alleles.Inbreeding depression is a key factor in to predict the evolution of mating systems in natural populations. At present, however, important aspects of its genetic architecture are crucially lacking. In particular, the distribution of fitness effects and dominance of segregating deleterious mutations as well as the contribution of the


Aphis glycines) as a case study. Foliar insecticide and host‐plant resistance are effective management tactics for preventing yield loss from soybean aphid outbreaks; however, pyrethroid‐resistant aphid populations pose a management challenge for farmers. We evaluated eight scenarios relevant to soybean aphid management in Iowa with varying probabilities of aphid outbreaks and insecticide‐resistant aphids occurring.The profitability of farming varies based on factors such as a crop's market value, input costs and occurrence of resistant pests, all capable of altering the value of pest management tactics in an integrated pest management program. We provide a framework for calculating expected yield and expected net revenue of pest management scenarios, using the soybean aphid (Our equation suggests that insecticide use is profitable when the probability of an aphid outbreak is ≥29%, and soybean production will become more costly with increasing probability of pyrethroid‐resistant aphids. If farmers continue to use pyrethroids, they will not experience financial consequences from pyrethroid‐resistant aphids until the chance of insecticide resistance is 48%. Aphid‐resistant varieties provided consistent yield and offered the highest net revenue under all conditions.This framework can be used for other crop–pest systems to evaluate the profitability of management tactics and investigate how resistance impacts revenue for farmers. Including the cost of resistance in crop budgets can help farmers and agronomic consultants comprehend these impacts and enhance decision‐making to increase revenue and curb resistance development. We developed a framework to evaluate the profitability of management scenarios across varying probabilities of a pest outbreak occurring and probabilities that the pest is resistant to the management tactic. Agricultural decision‐making is particularly complex, because behavioral differences based on the individual farmer's perception of risk leads to variation in the decision‐making process and differences in farm enterprises often present unique choice options.For insect pests, insecticide use is expected to decrease when resistance is sufficiently prevalent that an alternative management tactic becomes more cost‐effective.Integrated pest management (IPM) seeks to prevent economic damage from pests by coordinating multiple tactics that promote positive biological interactions without negative impacts on productivity.Greater emphasis on financial evaluations of pest management strategies is needed in the future, especially with increasing occurrence of resistance to management tactics. In systems where crops are economically injured by few pests, such as the Midwest United States, pest management budgets could help farmers comprehend the impact of decision‐making on revenue and guide adoption of IPM principles. Financial models and budgeting techniques for pest management options are most useful if designed to be easily adjusted for different environments or crop–pest systems.Aphis glycines Matsumura (Hemiptera: Aphididae)] was used as a case study to test the framework because multiple effective management tactics are available for this sporadic pest. We used Iowa as the basis of a snapshot analysis, both in terms of the pest's status and the inputs required to produce soybeans [Glycine max (L.) Merr.]. The pest status of the soybean aphid varies within North America and in other soybean growing regions where the aphid is native.The objective of this research was to develop a decision‐making framework to maximize expected yield and revenue in the presence of a pest outbreak. Soybean aphid  also are available but are not considered separately in our framework. Insecticides are either not applied (X), applied prophylactically  or applied only when an ET is reached (T). Prophylactic applications would not require the farmer to monitor the population in the field (N), but ET‐based applications require monitoring (M) to make a timely insecticide application. Monitoring implies scouting the field during a growing season, but it also can be performed in a more limited manner to determine if the insecticide was effective. We refer to this as efficacy scouting (E), which occurs only following the application of an insecticide to ensure the pest was controlled. The pest could be either susceptible, deemed wild‐type (W), or resistant to insecticides (I), although resistance to another management option could be substituted. To demonstrate how to use these components to form a pest management scenario, SHXN‐W represents a scenario where a susceptible, herbicide‐tolerant variety is planted, no insecticide is applied, no scouting occurs and the pest is susceptible to insecticides Table .2.32.3.1Soybean aphid is an invasive pest of soybean in the United States, first documented in Wisconsin in 2000.Insecticides and host‐plant resistance are equally effective management tactics for managing soybean aphid in Iowa, and aphid‐resistant varieties eliminate the need for insecticides when management is required.Profitability of host‐plant resistance and foliar‐applied insecticides for soybean aphid management can be compared using the equations in Section E(Y), we multiplied EYyield, the outcome of Eqn (1), by the ten‐year average yield in Iowa (3537 kg ha−1).Eight scenarios are discussed in this manuscript, although more scenarios are possible . Aphid‐resistant varieties were not expected to experience aphid populations that result in yield loss (q = 1) based on previous research.Yield loss was calculated in each site‐year with the equation 2.3.3Costs associated with soybean aphid management tactics differ because more inputs are required to manage populations with an insecticide application (e.g. insecticide and application costs), and aphid‐resistant varieties are not yet available in highly desired herbicide‐tolerant backgrounds.–1 was assumed for each variety .The total cost of producing soybean in Iowa, from planting through harvest, was accounted for in all scenarios using a snapshot analysis from 2018 . Typical management practices for soybean production were outlined, and costs were appraised for each practice Table . The 201–1) was used in our calculation of expected net revenue.Finally, because 53% of Iowa farmland is rented, primarily using cash rental agreements, the 2018 state average land rent was included in the total cost of soybean production.2.3.4excel file in the online supporting information.Analyses were performed to evaluate how sensitive expected net revenue is to changes in yield, soybean price, aphid‐resistant seed cost and overall input costs because these factors often influence management decisions. If the results are sensitive to these variables, optimal decision‐making could depend on these conditions each year, which are often unpredictable. All factors except the one of interest were held constant in each analysis. These data are not shown but can be recreated using the −1) and high (4035 kg ha−1) yields in Iowa were used: the original assessment accounted for the ten‐year average yield (3537 kg ha−1).–1) and ten‐year average (US$0.40 kg–1) soybean prices: the original assessment accounted for the ten‐year low soybean price (US$0.33 kg–1).In order to understand how yield might affect the results of the case study, the ten‐year (2009–2018) state averages for the low (3026 kg ha high 403 kg ha−1 33.13.1.1E(Y) was calculated over a range of aphid outbreak probabilities (po) for scenarios that assumed aphids were susceptible to insecticides  decreased as the probability of an outbreak increased without management to protect yield ) parameters and estimates for all soybean aphid management scenarios analyzed.Click here for additional data file.Supporting Table S2. Expected net revenue (E[R]) parameters and estimates for all soybean aphid management scenarios analyzed.Click here for additional data file.


Endotoxin is recognized as a major trigger of the immune response leading to pro‐ and anti‐inflammatory cytokine release, activation of the coagulation cascade, vasoplegic shock, and multiple organ dysfunction syndrome. A beneficial effect could be achieved through extracorporeal adsorption of circulating endotoxins in the blood as adjunctive treatment for unresponsive endotoxic shock. However, the precise clinical indication for its initiation is widely debated in the literature. Similar to the source control, microbiological cultures and antibiotics administration, endotoxin activity assay evaluation at regular intervals, and the targeted use of PMX‐B hemoperfusion could be lifesaving and adequate within the golden hour for the diagnosis and treatment of endotoxic shock. Sepsis is the devastating result of a complex and dysregulated host response to an infection. It leads to an imbalance between pro‐ and anti‐inflammatory mechanisms and predisposes to the development of multiple organ dysfunction syndrome (MODS).For this reason, a beneficial effect could be achieved through extracorporeal adsorption of circulating endotoxins in the blood as an adjunctive treatment for unresponsive endotoxic shock. Toraymyxin  is a cartridge containing polymyxin B (PMX‐B) immobilized on polystyrene fibers conceived for binding and neutralizing endotoxins from patients' blood through direct hemoperfusion. Beyond the pathophysiological rationale of the use of PMX‐B hemoperfusion (PMX‐B HP) for endotoxin removal, the precise clinical indication for its initiation is widely debated in the literature. According to the most updated evidence available from large randomized trials, septic shock patients with MODS score >9 and endotoxin activity assay (EAA) level ranging from 0.6 to 0.9 are those who may benefit the most from PMX‐B HP treatment in terms of improvement of survival.Apart from the general agreement to personalized indications for this complementary therapy, an appropriate timing indication for its initiation has not yet been proposed.According to our experience and clinical practice, the early phase of hemodynamic instability (vasoactive inotropic score [VIS] 10 < x < 30) during endotoxemia is usually associated with a slight decrease in renal function , biomarkers positivity and with an EAA level of 0.3 < x < 0.6. However, several concerns might be raised on the delayed approach of extracorporeal endotoxin removal in these situations. In particular, a rapid increase in endotoxin levels should be expected, particularly for patients with Gram‐negative sustained sepsis, due to endotoxin release during bactericidal treatment. Research and clinical protocols should consider an assessment of EAA at regular intervals to identify this rapid endotoxin increase.Based on our experience, we strongly suggest to start extracorporeal endotoxin removal within 4 hours after source control and starting antibiotic therapy Figure A. An oveThree stages of a “golden hour” might be thus recognized in which the patient with a high risk of endotoxic shock might probably benefit most from endotoxin extracorporeal removal.The timing becomes a crucial factor in the management of sepsis and the consequent MODS. Based on accurate monitoring and biological markers, an adjust prescription and appropriate delivery of what we defined sequential extracorporeal therapy in sepsis (SETS) might be guaranteed Figure B.9 In thIn a comprehensive approach to the endotoxic shock treatment, the evaluation of kidney function, vasopressor requirements, and EAA levels with source control and antibiotic therapy based on the time from sepsis diagnosis, may have a role to personalize the treatment for each specific patient. The dynamic monitoring and prescription could further refine the treatment personalization adequately responding to the criteria of precision medicine. Thus, not only a specific treatment could be provided for every single patient, but even a more specific treatment shall be provided for every moment that patient has a particular need during his/her ICU stay.This article does not contain any studies with human participants or animals performed by the author.The authors have no conflicts of interest to declare related to this manuscript.All authors contributed equally to the writing of this editorial. All authors read and approved the final manuscript.


P = .018), pancreas  and colorectum , but with lower odds of multiple myeloma . Suggestive evidence of association of genetic liability to long‐sleep duration with lower odds of pancreatic cancer  and kidney cancer  was observed. However, none of these associations passed the multiple comparison threshold and two‐sample MR analysis using FinnGen data did not confirm these findings. In conclusion, this MR study does not provide strong evidence to support causal associations of sleep duration with risk of overall and site‐specific cancers. Further MR studies are required.Studies of sleep duration in relation to the risk of site‐specific cancers other than breast cancer are scarce. Furthermore, the available results are inconclusive and the causality remains unclear. We aimed to investigate the potential causal associations of sleep duration with overall and site‐specific cancers using the Mendelian randomization (MR) design. Single‐nucleotide polymorphisms associated with the sleep traits identified from a genome‐wide association study were used as instrumental variables to estimate the association with overall cancer and 22 site‐specific cancers among 367 586 UK Biobank participants. A replication analysis was performed using data from the FinnGen consortium . There was suggestive evidence that genetic liability to short‐sleep duration was associated with higher odds of cancers of the stomach (odds ratio [OR], 2.22; 95% confidence interval [CI], 1.15‐4.30;  What's new?With the exception of breast cancer, the impact of sleep duration on site‐specific cancer risk remains largely unknown. Here, using Mendelian randomization analysis, the authors investigated the causal role of sleep duration for overall cancer and for 22 site‐specific cancers in more than 367 000 UK Biobank participants. Suggestive associations were detected between certain cancer sites and genetic liability to short or long sleep duration or genetically predicted sleep duration. However, no statistically significant associations were identified between sleep duration and overall or site‐specific cancer risk, indicating that sleep duration does not have a causal influence on cancer. BMIbody mass indexCIconfidence intervalFDRfalse discovery rateGWASgenome‐wide association studyIGFinsulin‐like growth factorIVWinverse variance‐weightedMRMendelian randomizationORodds ratioSNPsingle‐nucleotide polymorphism1Sleep is essential for maintaining optimal physiological processes. Sleep‐related problems are becoming increasingly prevalent in modern society, affecting 20% to 40% of the general population.Several observational studies have examined the association between short‐sleep (commonly defined as <7 hours/night) and long (commonly defined as ≥9 hours/night) sleep duration and cancer risk.Mendelian randomization (MR) is an epidemiologic technique that utilizes genetic variants that are reliably associated with potentially modifiable risk factors to determine their causal role for disease risk.22.1https://www.finngen.fi/fi).This MR study is based on data from the UK Biobank study.2.2P < 5 × 10−8) among 446 118 UK Biobank participants of European ancestry.r2 > 0.1 in European populations) between SNPs was evaluated using LDlink.rg = −0.89) and long‐sleep (rg = 0.68) duration; the corresponding correlation between short sleep and long sleep is modest (rg = −0.28).We selected all single‐nucleotide polymorphisms (SNPs) previously shown to be associated with the sleep duration traits at the level of genome‐wide significance P < 5 × 0−8 among2.3P value <.05, IVW method), sensitivity analyses using the weighted median and MR‐Egger approaches were conducted.The analyses were performed using the mrrobustFTO gene (one SNP for the long sleep duration trait and another SNP for the continuous sleep duration trait), which has pleiotropic effects with, for example, body mass index. In a separate multivariable analysis, we additionally adjusted for smoking initiation, that is, the obtained estimates represent the direct effect of sleep duration, which is not driven by smoking initiation. Summary statistics for data for smoking initiation were obtained from the published GWAS.Additionally, we conducted a sensitivity analysis omitting sleep‐related SNPs in the P values that passed a critical value corresponding to false discovery rate (FDR) of .05 were considered as strong evidence of associations. P values that did not pass a critical value but were less than .05 were considered as suggestive evidence of associations.Reported odds ratios (OR) with their 95% confidence intervals (CI) are per genetically predicted one‐unit increase in log odds of short‐ and long‐sleep duration and per genetically predicted additional hour of sleep in every 24 hours. The Benjamini‐Hochberg method was applied in the main analysis based on the UK Biobank data to correct for multiple testing of all 69 associations. 3P = .018), pancreas  and colorectum , but with lower odds of multiple myeloma . Genetic liability to long‐sleep duration was associated with lower odds of pancreatic cancer  and kidney cancer  and with higher odds of testicular cancer . Genetically predicted continuous sleep duration showed a suggestive inverse association with kidney cancer . Removing the SNP in the FTO gene did not alter the observed associations except for testicular cancer  and long sleep with pancreatic cancer  were in the same direction as in UK Biobank.Replication analyses based on the FinnGen consortium did not reveal significant associations of sleep duration traits with any cancer site from the discovery stage  who reported sleep duration of 5 to 6 hours per night compared with those who slept 7 to 8 hours.In contrast to several prospective studies,There are several potential pathways that can explain the role of sleep in cancer development. Sleep duration may be a surrogate for exposure to light at night and be closely related to melatonin production, a hormone mainly produced by the pineal gland primarily during darkness. Experimental evidence suggests that besides the important role of melatonin in regulation of circadian rhythms, it is also involved in inhibition of tumor development of a wide variety of cancers, including stomach,Another potential pathway of the relationship between short sleep and gastrointestinal cancers is that excessive body weight, insulin resistance, type 2 diabetes, altered gut microbiota, inflammation and impaired immune function may mediate this association. For instance, several experimental studies have demonstrated that acute sleep deprivation may cause an alteration in glucose and insulin metabolismA strength of this study is the MR design, which reduces confounding and reverse causality. In addition, we could assess the association between sleep duration and overall cancer in a large cohort, thereby providing high power to detect a weak association. The restriction of the population to European‐descent individuals minimized stratification bias. A limitation of this study is that the precision of the results was low or modest in analyses of site‐specific cancers and we are unable to discount the role of chance underlying these findings. Both the sleep‐related traits and cancer outcomes were assessed in a single population (UK Biobank), which might have resulted in bias toward the direction of the observational association between sleep duration and cancer. We could not replicate the results using the FinnGen data. This disparity in results could potentially be related to overestimation of the associations in UK Biobank or the smaller number of cases in FinnGen. In addition, due to a genome‐wide genetic correlation of three sleep traits used in the MR, there is an overlap in variants between these genetic instrumental variablesIn conclusion, this MR study does not provide strong evidence to support causal associations of sleep duration with risk of overall and site‐specific cancers. The suggestive associations of short‐ or long‐sleep duration with certain cancers merit further investigation in other large MR studies.The authors declare no conflict of interest.Studies included in the consortia were approved by local research ethics committees and all participants provided written informed consent. The analyses were approved by the Swedish Ethical Review Authority.AppendixS1: Supplementary MaterialClick here for additional data file.


TFE) tend to focus on large plots that can be expensive to establish and maintain and may be unsuitable for large trees or indigenous forests. We set out to establish a relatively inexpensive TFE method in a natural forest with large trees.Drought‐induced tree mortality is an emergent threat to forests worldwide, particularly to large trees. Drought‐manipulation experiments involving throughfall exclusion  under long‐term simulated drought. We measured fluxes of water (sap flow) and carbon  as indicators of drought effects.We designed a novel 2 area around individual boles, causing reduced soil moisture and reduced sap flow in droughted trees.Throughfall was cut off to a 22.25‐mTFE method centered on individual, large trees in native forest and is highly customizable to fit other forest and species types. It can be used to assess physiological responses to drought of individual trees independent of stem size.Our new  Under global climate change, drought‐induced tree mortality is rising worldwide . This procedure provides detailed designs, measurements, and sampling regimes, and allows for flexibility with certain constraints. However, many forest types, especially dense mature forests, have complicated spatial arrangements of trees and understory plants, which makes installation of established protocols particularly challenging. None of these methods are practical or appropriate for understanding drought impacts in indigenous or old growth forests occurring in inaccessible terrain, which is common in areas of the world, such as New Zealand, that are underrepresented in the literature.Forest‐based TFE experiments range in size from small‐scale single‐species plantations  Loudon) is one of the largest (by volume) and longest‐lived tree species in the world , a pathogen responsible for widespread death of A. australis  TFE was initialized in mid‐2017 at Huapai Scientific Reserve  in western Auckland, New Zealand  were placed near the base of each tree, and litter was collected and sorted every three weeks  were drilled into the ground on the ascending and descending sides of each tree near the trunk  specializing in manufacturing waterproof outdoor shelters to produce the custom tarpaulins for the TFE. The material used is commercially known as Canvacon, a woven, polyethylene‐based polyfabric that is UV‐resistant for extended life outdoors. Tarpaulins were 4.5 × 4.5 m draped as a diamond in order to maximize area while avoiding adjacent plants. Each tarpaulin had a hole in the center measuring the circumference of drought tree stems—plus an additional ~4 cm to allow for growth—and ringlets along the perimeter to allow for tying down. Around the stem hole, a ~20‐cm‐tall “collar” runs from the plane of the tarpaulin up the stem in order to deflect stemflow onto the exclusion plane  drainage gutters (inner diameter of 250 mm) were laid so that each gutter had a 90‐degree bend and covered two edges each of the perimeter of the tarpaulin. Those were individually fed into collection drums  with a semi‐circular cut made in the lid at the bottom vertex. The volume can be collected after small rain events to quantify exclusion efficiency. We decided against trenching as a precaution to prevent the spread of kauri dieback. A full breakdown of costs is outlined in Table −2 s−1) with a LI‐COR quantum light sensor , rainfall by a tipping bucket rain gauge , and wind speed and direction with a cup anemometer and vane, respectively . Apart from the tipping bucket, sensors were mounted on a weather station tripod (MEA) and logged onsite . Additional cylinder rain gauges were kept at the meteorological station and in the open close to the study site to compare precipitation variance. There was minimal difference between the manually measured rainfall at the meteorological station and closer to the forest.Meteorological data were collected at a station located approximately 1 km from the experimental site. Data were recorded in 30‐min data intervals for air temperature (°C) and relative humidity (%) with a Vaisala HUMICAP probe  protected in a radiation shield, photosynthetically active radiation  values were derived from temperature and relative humidity. Soil moisture was averaged for drought and control trees (six sensors for drought and control each as there were two per tree). We only show a selection (depths for 20 cm and 40 cm) of soil moisture for clarity. For each tree, sap flow values from the base of the stem and the base of the canopy were averaged. These were then averaged to produce representative sap flows for drought and control trees. For point dendrometers, we followed a similar process as sap flow to produce representative radial change for drought and control trees.A. australis) show some clearly visible differences between drought and control trees.The tarpaulins were installed in December 2018. Here, we present three days of summer data (from January 2019) after approximately one year of imposed drought. There is compelling evidence in our preliminary data set that our experimental design induced drought effects Fig. . There iThe sap flux patterns present convincing evidence that VPD and PAR are drivers of transpiration. The prominent dips in sap flux for both drought and control trees at around midday on 13 January coincided with temporary declines in VPD and PAR, and the diminished peaks in sap flux over the subsequent two days were to be expected after the rain event in the morning of 14 January. Comparing sap flux between drought and control trees, the lower flux rates by the drought trees  over the three‐day period indicate less soil water uptake , further measures of plant responses and traits , and imaging of canopy changes .Drought responses often occur at the leaf scale; therefore, in addition to the whole tree measurements reported here, we are also carrying out seasonal canopy sampling. For A. australis (Macinnis‐Ng and Schwendenmann, A. australis avoid drought stress.The experiment will extend until at least August 2020. By this time, we expect that differences in sap flow and radial change will become more pronounced between drought and control trees. We expect a stem water deficit to develop as the drought progresses (Kaplick et al., In conclusion, we have overcome challenges of expense and logistics to produce a method that is suitable for iconic and ecologically significant large trees. Large trees dominate forest water cycles and ecosystem carbon storage (Stephenson et al.,


Disparities have been documented in maternal mortality rates between women from different ethnic, age and socio‐economic groups in the UK. It is unclear whether there are differential changes in these rates amongst women from different groups over time. The objectives of this analysis were to describe UK maternal mortality rates in different age, ethnic and socio‐economic groups between 2009 and 2017, and to identify whether there were changes in the observed inequalities, or different trends amongst population subgroups.Maternal mortality rates with 95% confidence intervals (CI) in specific age, deprivation and ethnic groups were calculated using numbers of maternal deaths as numerator and total maternities as denominator. Relative risks (RR) with 95% CI were calculated and compared using ratios of relative risk. Change over time was investigated using non‐parametric tests for trend across ordered groups.Women from black and Asian groups had a higher mortality rate than white women in most time periods, as did women aged 35 and over and women from the most deprived quintile areas of residence. There was evidence of an increasing trend in maternal mortality amongst black women and a decrease in mortality amongst women from the least deprived areas, but no trends over time in any of the other ethnic, age or IMD groups were seen. There was a widening of the disparity between black and white women .The clear differences in the patterns of maternal mortality amongst different ethnic, age and socio‐economic groups emphasise the importance of research and policies focussed specifically on women from black and minority ethnic groups, together with other disadvantaged groups, to begin to reduce maternal mortality in the UK. Are there differences in maternal mortality rates amongst women from different ethnic, age and socio‐economic groups in the UK and are these differences changing over time?The maternal mortality rate overall has been static in the UK for more than 10 years. There are known differences in mortality rates between women from different ethnic, age and socio‐economic groups in the UK, but changes in mortality rates over time amongst different population subgroups have not been examined.This study shows that maternal mortality amongst black women in the UK is increasing, with evidence of a widening inequality between black women and white women. Maternal mortality amongst women living in less deprived areas is decreasing.1Maternal mortality surveillance has been conducted in the UK since the early 1950s, but the methods and focus of reports have evolved over time. The epidemiology of maternal mortality has almost completely changed from two‐thirds of deaths being due to direct obstetric causes in 1952‐54,Maternal mortality rates in the UK have largely been static over the period 2009‐17,From the 2009‐11 triennium onwards, maternal mortality data have been published on an annual basis in the UK, presented as rolling 3‐year average rates. The report published in 2018, including data for 2014‐16, highlighted the wide disparity in mortality rates between black, Asian and white women.2This analysis is based on data extracted from published maternal mortality reports.Denominator data were obtained from published data from the Office for National Statistics, National Records Scotland, Northern Ireland Statistics and Research Authority, NHS Digital, or from national birth data. Denominator maternities  data classified by ethnic group and by area deprivation score were only available for England, and therefore analyses investigating ethnic group and socio‐economic deprivation were conducted only for England. Additionally, denominator data in the five census classification groups were only available from 2012; therefore, ethnicity data for mixed and Chinese/other groups are only presented from 2012 to 2014 onwards. Maternities for which ethnicity was not stated were included in the “white European” group because the re‐distributed proportions matched with the estimated ethnic profiles in the UK population census.2.1stata version 15 (Statacorp).Maternal mortality rates with 95% confidence intervals were calculated using numbers of maternal deaths as the numerator and total maternities in each specific group as the denominator. Relative risks with 95% confidence intervals were calculated to compare maternal death rates between groups of women. A non‐parametric test for trend across ordered groups was used to investigate the change in three‐yearly rolling maternal mortality rates over time.2.2No approval was required for this secondary analysis of published data.3During the study period, there were 662 maternal deaths amongst 7 001 210 maternities; an overall maternal mortality rate of 9.46 per 100 000 maternities . There was no difference in overall maternal mortality rate in the UK between 2009‐11 and 2015‐17  and a decrease in mortality amongst women from IMD quintiles I (P = .054) and II (P = .029), but no trends over time in any of the other ethnic, age or IMD groups. There was a widening of the disparity between black and white women .Denominator data for women of mixed or Chinese/other ethnicity were not available in the same format for 2009‐11, therefore, 2012‐14 and 2015‐17 data could not be compared with 2009‐11. However, there was some evidence of an increasing disparity in mortality rate comparing mixed ethnicity women with white women between the two later triennia . Note there was no clear evidence of an increasing trend in mortality rate amongst women of mixed ethnicity over the period 2012‐17 (3.13.1.1There has been no overall change in the UK maternal mortality rate over the period 2009‐17. However, this analysis suggests there have been changes in specific population subgroups, with evidence that the maternal mortality rate is increasing amongst black women and decreasing amongst women resident in less deprived areas. There has been a widening of the disparity in mortality rate between black and white women, with black women experiencing a fivefold higher maternal mortality rate in 2015‐17, compared to a 2.5‐fold difference in 2009‐11 when compared to white women. There may also be a widening of the disparity in maternal mortality rate between women of mixed ethnicity and white women, with a threefold higher mortality rate in 2015‐17 compared to no difference in 2012‐14.3.1.2This study used national data collected over 9 years with enhanced case ascertainment to identify maternal deaths, thus estimates of mortality rates in the whole population and within population subgroups are likely to be robust. Data on women's characteristics were collected from medical records or from published national vital statistics data and therefore have a high level of completeness and validity.3.1.3Although this analysis uses national data collected over several years, maternal death remains an uncommon event. It is possible that some clinically important differences were not identified due to limited study power. The national confidential enquiry reports estimate denominator ethnicities by inclusion of any women with missing ethnic group information into the white ethnic group, since this most closely approximates the ethnic group distribution seen amongst birth registrations. It is possible therefore that there has been some misclassification of women's ethnicities in the denominator. This misclassification will not occur, however, amongst women who have died, since the information on ethnic group has been extracted from self‐reported ethnic group in women's medical records. Given changes to the ethnic groupings in which denominator data are published, the study was only able to examine trends over a period of 2012‐17 amongst women from mixed, Chinese or other ethnic groups, which further limits the power of the study to identify important trends. Denominator maternities data were not available at an individual level, therefore, an adjusted analysis was not possible.3.1.4These findings suggest that there needs to be a particular targeting of research and action to address maternal mortality amongst black women in the UK, which appears to be increasing, and potentially also to address mortality amongst women of mixed ethnicity. In contrast, maternal mortality appears to be decreasing amongst women resident in less deprived areas. In England, a halving of maternal mortality between 2010 and 2030, subsequently revised to 2025, has been a national ambition since 2015.Previous studies in the UK have shown that pre‐existing medical co‐morbidities, anaemia during pregnancy, previous pregnancy problems, inadequate use of antenatal care, substance misuse, unemployment and maternal age are all independently associated with maternal death.A qualitative study of the maternity experiences of women with multiple disadvantages giving birth in England, most of whom were from black and minority ethnic groups, highlighted that women brought feelings of powerlessness and low self‐esteem to their maternity appointments, which could be made substantially worse by disrespectful care.Similar disparities in maternal mortality have been identified in the USA, and addressing structural racism has been emphasised as one of the actions to begin to tackle this.4The maternal mortality rate is static in the UK. This analysis has identified clear differences in the patterns of maternal mortality amongst different ethnic, age and socio‐economic groups in the UK. There appears to be no change in the patterns of disparity between most groups over time. However, there is evidence that the maternal mortality rate amongst black women in England is increasing and that the gap between black and white women in terms of their mortality rate is increasing. This emphasises the importance of research and policies focussed specifically on women from black and minority ethnic groups, together with other disadvantaged groups, to begin to reduce maternal mortality in the UK.All authors declare they have no conflicts of interest. Click here for additional data file.


In South Africa, HIV‐infected men are less likely than women to test and know their status (the first UNAIDS “90‐90‐90” target), and men have worse outcomes across the HIV care cascade. HIV self‐testing (HIVST) may address this testing disparity but questions remain over the most effective distribution strategy and linkage following a positive test result. We implemented a men‐focused HIVST distribution programme to evaluate components contributing to participation and retention.We conducted an implementation study of multi‐venue HIVST kit distribution in rural and peri‐urban KwaZulu‐Natal (KZN), South Africa. We distributed HIVST kits at community points, workplaces and social venues for on site or take‐home use. Clients could choose blood‐based or oral‐fluid‐based HIVST kits and elect to watch an in‐person or video demonstration. We provided a USD2 incentive to facilitate reporting test results by phone or SMS. Persons with reactive HIVST results were provided immediate confirmatory tests (if used HIVST on site) or were referred for confirmatory testing (if took HIVST off site) and linkage to care for ART initiation. We describe the testing and linkage cascade in this sample and describe predictors of reactive HIVST results and linkage.Between July and November 2018, we distributed 4496 HIVST kits in two regions of KZN . Most participants (58%) chose blood‐based HIVST and 42% chose oral‐swab kits. 11% of men were testing for the first time. A total of 3902 (83%) of testers reported their test result to the study team, with 314 (8%) screening positive for HIV. Among 274 men with reactive HIVST results, 68% linked to ART; no significant predictors of linkage were identified. 10% of kit users reported they would prefer a different type  of kit for repeat testing than the type they used.HIVST is acceptable to men and rapid distribution with optional testing support is feasible in rural and peri‐urban settings. HIVST kits successfully reached younger men and identified undetected infections. Both oral and blood‐based HIVST were selected. Scaling up HIVST distribution and guidance may increase the number of first‐time testers among men and help achieve the first UNAIDS “90” for men in South Africa. AcroEarly, large‐scale demonstrations of HIVST kit distribution showed success in increasing HIV testing among men in three sub‐Saharan African countries using approaches including community‐based, workplace‐based and health facility‐based distribution . Studies2We conducted and evaluated a multi‐venue distribution programme of HIVST kits in peri‐urban and rural districts of KwaZulu‐Natal, South Africa, that targeted adult men. In order to understand the impact of HIVST in resource‐limited settings with a view towards programmatic scale up, we designed the distribution to be pragmatically implemented, with lay staff providing most services and minimal participation incentives.2.1Teams of 3‐6 lay counsellors set up distribution sites (4‐8 events per week during the study period) in community centres, at male‐dominated workplaces , and at venues including sporting events, tuckshops, bottle shops and other public gatherings. Two types of HIVST kits were available at distribution events: a fingerprick blood‐based kit  and an o2.2Counsellors obtained informed consent from men interested in participating prior to distributing kits. The only eligibility criterion was being age 18 or older. Participants completed a baseline questionnaire about demographics, HIV testing history, sexual behaviours, alcohol use, test kit preferences and cell phone number. They received a test kit labelled with a study identification number.Participants were asked to report the results of their HIVST to the study team. All participants received a callback card with their HIVST kit to report the results of their self‐test to the study team, as well as a voucher for cellphone airtime  to be redeemed at the time of reporting results.. Participants who tested on site presented to study staff after testing to voluntarily report their results or could elect to leave the site and report results later using the callback card. Participants who were directly assisted by study staff in conducting the HIVST reported their results directly as they received them. Persons taking kits off site reported results by calling or sending an SMS/text message to the number on the callback card for a free callback by a study staff person. Staff then contacted the participant and ask for the HIVST result. Study staff provided post‐HIVST counselling including referral for confirmatory testing and ART if the HIVST was reactive, or HIV prevention information and referral for voluntary male medical circumcision if the HIVST was nonreactive.Participants received a non‐identifying reminder text message after 2 weeks if they had not yet reported their result. . The reminder was repeated at 1 and 2 months. If no result was reported by 2 months, staff made an outreach call to assess test use and results.After participants reported their results (either in person or by phone) and received counselling and referrals, staff distributed the airtime incentive and administered a brief questionnaire to assess experience, usability, acceptability and preferences about HIVST.Distribution events also provided confirmatory HIV testing on site  and linkage to ART for persons with positive confirmatory tests. Linkage was offered either through referral to a local clinic of the participant’s choice or referral into an ongoing ART delivery study .2.3All participants who reported their HIVST results received a phone call 2 months after results reporting to assess whether they had linked to confirmatory testing and ART or preventive services. We defined linkage to ART as attending clinic (or enrolling in the DO ART study) and initiating ART. Study staff validated a portion of self‐reported ART initiation by confirming registration with ART clinics.2.4Percentages were calculated for descriptive statistics. Crude and adjusted relative risks of positive HIVST results were calculated among men with a known HIVST result for selected predictors. Crude and adjusted relative risks of linkage to care were calculated among men with a reactive HIVST. Relative risks were, adjusted for potential sociodemographic confounders: age, study site, testing venue type, testing location, HIV testing history, circumcision status, educational status, marital status, employment status and self‐reported alcohol use. Adjusted relative risks were calculated in R using modified Poisson regression for binary outcomes with robust variance estimation.2.5The study was reviewed and approved by the institutional review boards or ethics committees of the HSRC, University of KwaZulu‐Natal, and the University of Washington. All participants provided informed consent.33.1Between July and November 2018, study teams distributed 4496 HIVST kits  to South African adults in two districts.Persons receiving HIVST kits at the distribution sites were overwhelmingly men (96%) by design Table . The medAlmost all men (86%) stated that the reason they elected to test using HIVST was that they wanted to know their HIV status. Seven percent stated their reason was HIVST was more convenient than facility‐based HIV counselling and testing, and 3% stated they participated to receive the airtime incentive.3.2Test results were reported for 3486 (81%) of HIVST kits distributed to men. Overall, a total of 8% of reported test results were reactive for HIV antibodies. Among 274 men with reactive HIVST results, linkage outcome was determined for 81% ; 72% of men with reactive HIVSTs received a confirmatory test (6 confirmatory tests were negative), and 95% of men with a positive confirmatory test linked to care and initiated ART. Overall, 68% of men with reported reactive HIVST results successfully linked to ART.Retention along the cascade differed between men who tested on site and men who took kits off site to test Figure . ResultsWe evaluated predictors of a reactive HIVST among men whose test results were known Table .Men who took an HIVST kit off site to test had a lower risk of a reactive HIVST compared to men who tested on site . Factors associated with increased risks of a reactive HIVST included older age, uncircumcised status and unmarried status. Self‐reported alcohol use of seven drinks or more per week was associated with 72% higher risk of a reactive HIVST compared to less alcohol use . Men who reported previously testing for HIV had a decreased risk of a reactive HIVST , compared to men who reported being first‐time testers.Men testing off site were slightly more likely to link to care than men testing on site, , but this was not statistically significant. The increased likelihood disappeared after adjusting for demographic features ; no statistically significant predictors of linkage to ART were found among men with reactive HIVST results with linkage data available (Table 3.3Men reporting HIVST results (N = 3488) completed a questionnaire about their experience using the kits and learning their HIV status. Most  reported that using the test kit was “easy” or “very easy.” Most men  reported that they learned to use the kit from the in‐person demonstration, and 2937 (84%) reported that they did not require additional help in using the kit. 697 (20%) reported receiving help from a study counsellor using the kit or interpreting results and 26 (1%) requested help from a friend or family member.Men were asked what type of HIVST kit  they would prefer to use in the future, based on their experience with the type they used. Over 90% of men reported a preference to use the same type they had used initially: 1428 (90%) of Oraquick users reported preferring to do a repeat test with Oraquick, and 1928 (93%) of the blood‐based iTest users stated they would prefer to use the iTest again.At the two‐month outreach call to ascertain HIVST results, 92/3576 (2.6%) men reported they did not use the kit they received. The main reasons cited for not using the HIVST were: being unsure how to use it, not having time to use it, and losing the kit. Five men stated that they attempted to use the blood‐based kit and could not obtain blood with the lancet, so could not complete testing. Two men disclosed they were already taking ART (which they did not disclose at the time of receiving the test kit), so did not use the kit.4Offering HIVST kits in community‐based settings successfully reached men in South Africa for HIV testing, including young men, and resulted in 68% linkage to care among men reporting a reactive HIVST. Small cadres of lay health counsellors distributed over 1000 HIVST kits per month to men, of which nearly 12% were used by first‐time testers. Team members were able to provide support for HIVST as needed by clients. These findings support that HIVST strategies found to be efficacious in randomized controlled trials are effective in increasing HIV testing in men in a quasi‐programmatic implementation setting , 19. TakIn addition to the variety of locations targeted for HIVST distribution, providing men with options for test type  and support during testing  was valued. We observed differences in the proportion of first‐time HIV testers, prevalence of circumcision and kit type preferences between the two study districts, highlighting the need for options to address preferences in blood versus oral‐based HIVST, locations for testing and support needs among the diverse group of men we tried to reach , 22. StuFirst‐time testers, men who elected to test on site, and men who used alcohol heavily were more likely to have a reactive HIVST than repeat testers, men who tested off site and men who had lower alcohol use. These findings are encouraging in showing that HIVST can reach men with these risk factors, and also confirms these risk factors for undetected HIV infection. Alcohol use is a known risk factor for HIV transmission, but many men in South Africa who use alcohol heavily report barriers to accessing HIV services, in some cases related to their alcohol use , 26. HIVIn all distribution settings, HIVST distribution was acceptable. A small incentive was adequate to promote reporting of HIVST results for over 80% of participants. Without additional incentives to further engage in care, we observed that 68% of men with reactive tests linked to care and initiated ART. This is encouraging, and consistent with community and household‐based HIV testing efforts in the region . We antiIn this study, men who elected to test on site (with or without direct assistance from a study team member) were more likely to be HIV‐positive, but less likely to link to care compared to men who took HIVST off site to test at home or by themselves. Much of the discrepancy in linkage was explained by important demographic differences in these two groups. However, the discrepancy suggests ways to target interventions to men at higher risk of not linking to care. For example providing on site rapid ART initiation for men with a reactive HIVST and confirmatory test could overcome the initial linkage to care barrier in this population, a service not required for all men needing to link to care, but may benefit this particular at‐risk population. The CASCADE study in Lesotho demonstrated that immediate ART initiation after community‐based HIV testing increased linkage to care and viral suppression compared to referral to clinic facilities to initiate treatment, and an early study of linkage after self‐testing found that home‐based confirmatory testing and ART initiation was associated with greater ART initiation than clinic referral , 29. Add4.1Our program had several notable strengths. We conducted HIVST distribution in a pragmatic way designed to be adapted and scaled up to a programmatic setting, where resources for individualized services and follow‐up may be limited. Although our project used additional resources to follow up participants beyond what may be feasible in a programmatic setting, these were limited to ascertainment of outcomes: incentives were provided for participants to report results to assess outcomes, and follow‐up phone calls to assess HIVST use and linkage to care. Because incentives to report results were not contingent on the result itself, we do not anticipate significant bias was introduced as a result of providing the incentives. In summary, we demonstrated the feasibility of rapid, large‐scale distribution of HIVST to reach men, including men who have not yet accessed HIV testing, and high rates of results ascertainment, and linkage‐to‐care.Our study had some limitations. HIV testing history, HIVST results and linkage to care data were obtained by self‐report, which is subject to responder bias and social desirability bias. For a variety of reasons, people who know their HIV status, including persons on ART, do not always disclose this knowledge in testing settings . There i5Men continue to contribute disproportionately to undiagnosed PLHIV in South Africa. HIVST distribution to men in community settings, including workplaces and social venues, is a feasible strategy to increase the number of men learning their HIV status in South Africa. This strategy can reach thousands of men per month with limited staff and resources, though cost‐effectiveness data are lacking and should be urgently pursued as a priority. Essential components of the acceptability of HIVST were access to multiple testing modalities  and availability of staff support. Although HIVST distribution increased the number of men who learned their positive HIV status (“first 90”) and resulted in moderate linkage to ART initiation (approximately 68%), HIVST distribution alone was not sufficient to ensure complete linkage to care. Additional strategies and support are needed to increase engagement of HIV‐infected men in linkage to ART after a reactive HIVST result to optimize the HIV cascade; these should be pursued aggressively in combination with continued access to HIVST.The authors declare that they have no competing interests.AES, CLC, HvR and RVB designed the study. MK, AvH, KS, OK and NS conducted the project and collected the data. TTS and AES analysed the data. AES and RVB drafted the manuscript. All authors contributed to revisions and content of the final manuscript.


Visualizing the functional interactions of biomolecules such as proteins and nucleic acids is key to understanding cellular life on the molecular scale. Spatial proximity is often used as a proxy for the direct interaction of biomolecules. However, current techniques to visualize spatial proximity are either limited by spatial resolution, dynamic range, or lack of single‐molecule sensitivity. Here, we introduce Proximity‐PAINT (pPAINT), a variation of the super‐resolution microscopy technique DNA‐PAINT. pPAINT uses a split‐docking‐site configuration to detect spatial proximity with high sensitivity, low false‐positive rates, and tunable detection distances. We benchmark and optimize pPAINT using designer DNA nanostructures and demonstrate its cellular applicability by visualizing the spatial proximity of alpha‐ and beta‐tubulin in microtubules using super‐resolution detection. A modified implementation of DNA‐PAINT microscopy is used to detect spatial proximity of biomolecules with super‐resolution capabilities. The new technique, called Proximity‐PAINT, features a precisely tunable detection range, high sensitivity and low false‐positive rates. The implementation can be applied to visualize cellular protein‐protein interactions and other biomolecules of interest, such as nucleic acids. The coordination of the myriad of processes occurring within a cell relies on direct interactions among their molecular components, such as nucleic acids and proteins. In order to understand life on the molecular level, it is thus paramount to develop techniques that are able to visualize and quantify proximity of biomolecules. For example, mechanisms that regulate protein activity and their structural arrangement require components to be in close spatial proximity.Imaging‐based methods, on the other hand, offer the advantage of spatially resolved characterization of PPIs in the native context of a cell. Chief among such techniques is Förster Resonance Energy Transfer (FRET), which allows sensitive distance measurements between two molecules of interest.Here, we report the development of a versatile and programmable method for in situ proximity detection between two molecular targets with super‐resolution readout capability and call it Proximity‐PAINT (or pPAINT). The pPAINT approach is based on DNA‐PAINTTo faithfully detect the interaction between two targets of interest using pPAINT, each target is labeled with one of the two DNA strands that comprise the pPAINT system Figure . If the To quantify the detection sensitivity under different experimental conditions, we placed the pPAINT sensor (consisting of the two strands described above) at the center of the DNA origami . The edge of the origami was decorated with 52 DNA‐PAINT docking strands  resulting in a frame around the pPAINT site at the center. Using Exchange‐PAINT,In a first proof‐of‐principle experiment, we designed the pPAINT sensor without a stem or leash region in order to quantify pPAINT′s capability of detecting immediate spatial proximity with no spacing between the two split docking sites Figure . To achiIn order to probe larger molecular distances, a semi‐stable stem region was thus a necessity for pPAINT. However, the inclusion of a complementary stem region between the two halves of the pPAINT system could lead to false positives, if the stability of the stem would be increased to a point where it could force the two entities of the pPAINT sensor together, even in absence of spatial proximity of an underlying biomolecular system under investigation. In order to assay the stem stability with regards to possible false positives, we designed one DNA origami structure in which the two pPAINT entities were spaced 0 and 20 nm apart  should be addressed to the authors.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.