Extremely preterm babies are at risk of significant mortality and morbidity due to their physiological immaturity. At periviable gestations decisions may be made to either provide resuscitation and intensive care or palliation based on assessment of the outlook for the baby and the parental preferences. Health care professionals (HCP) who counsel parents will influence decision making depending on their individual perceptions of the outcome for the baby. This paper aims to explore the knowledge and attitudes towards extremely preterm babies of HCP who care for women in pregnancy in a tertiary, regional and remote setting in North Queensland.A cross sectional electronic survey of HCP was performed. Perceptions of survival, severe disability and intact survival data were collected for each gestational age from 22 to 27 completed weeks gestation. Free text comment enabled qualitative content analysis.p = 0.03 at 23 weeks, p = 0.02 at 25,26 and 27 weeks), severe disability (p = 0.01 at 24 weeks) and healthy outcomes (p = 0.01 at 24 weeks), whilst those working in regional and remote centres were more negative than those in tertiary unit for survival . Perception became less negative as gestational age increased.Almost all 113 HCP participants were more pessimistic than the actual outcome data suggests. HCP caring for women antenatally were the most pessimistic for survival (Pessimism of HCP may be negatively influencing decision making and will negatively affect the way in which parents perceive the chances of a healthy outcome for their offspring. Delivery of an extremely premature infant below 28 weeks completed gestation is uncommon, affecting less than 1 % of babies born in Australia . The decWith technological advances and enhanced quality of care, the outlook for these vulnerable babies is improving over time  and thusEarly Australian studies on the knowledge and attitudes of HCP focussed primarily on the tertiary obstetrician and neonatologists –14. HoweParents of extremely premature babies who are beyond the ‘grey area’ of decision making will also need accurate information and consistency from HCP about the potential outcome for their child, as extreme prematurity will have a considerable impact on the parents’ future lives , particuThis study aims to investigate the knowledge of HCP and ascertain their attitudes towards the provision of care for extremely premature babies, including which factors staff feel should be considered when offering, or not offering, intensive care in North Queensland.A cross-sectional electronic survey of HCPs was administered on the SurveyMonkey platform (SurveyMonkey Inc. Ca. U.S.A.). HCP at three centres in North Queensland were invited to participate.The study centres include the largest provider of tertiary neonatal care in Northern Australia, one of two regional referral centres and a remote hospital. The tertiary hospital provides care for babies of all gestations and offers care for babies with surgical and medical conditions. It cares for all babies who receive neonatal intensive care below 28 weeks gestation in North Queensland. More than half the parents delivering extremely preterm babies reside within other health districts, and nearly a quarter are retrieved following delivery at smaller health care facilities . The regFollowing identification of a pregnancy at risk of extreme prematurity, parents are counselled by senior obstetric and neonatal staff, including potential outcomes and the expected neonatal course. Those pregnancies in the ‘grey zone’ are identified, and options to provide full intensive care or palliation are discussed. Parents are also given the option to initiate full resuscitation, with the option of redirecting care either during resuscitation or on the neonatal unit, where the baby is in poor condition or appears to be significantly compromised. Where there is potential for resuscitation, obstetric actions to optimise the condition of the baby are initiated. Decisions to resuscitate often involve several discussions, during which time the woman will be cared for by midwifery staff, and the neonatal unit is toured together with a neonatal nurse. Where the family is from a peripheral centre, often only brief counselling is given prior to transfer and tertiary obstetrician review.The survey was designed with questions about the demographics of the respondent including primary location of work, work stream, experience, social contact with people with disability and whether their religious beliefs influenced their decision making. Respondents were asked: i) whether they cared for pregnant women under 28 weeks gestation who were at risk of premature delivery: ii) if they had ever been asked by a parent for their personal opinion about whether a baby should receive intensive care or palliative care: and iii) their confidence in discussing extreme prematurity with patients. Further questions explored their knowledge of rates of: i) survival: ii) severe disability: and iii) intact survival at different gestations from 22 to 27 weeks completed weeks gestation. Replies to survival and outcomes were given as one of five quintiles in 20% divisions as it was considered less intimidating to participants than asking for exact estimates, whilst still being accurate enough for analysis. Participants were asked to rank their opinion about other factors which may influence the decision to offer intensive care to extremely preterm babies, and give an opinion about the most appropriate gestation from which intensive care should be offered to premature babies, at which gestation parents could be sole decision makers, whether staff could override parents’ wishes, and the gestation at which the participant would want a potential extremely premature baby of their own to be resuscitated. Free text was allowed for participants to expand on their replies. Although similar studies are found in the literature, the questionnaire was not based specifically on any of these as none captured all the data of interest. All gestations of babies from 22 to 27 completed weeks were included although resuscitation is usually provided at the older gestations.The survey was piloted with a group of senior nursing and medical staff and a psychologist involved in neonatal care to assess face validity and adapted to ensure clarity.An email link was sent by the primary investigator to all neonatal, paediatric and obstetric medical staff specialist or doctors on college training programs at the tertiary centre. Senior nursing managers sent the link to registered midwives and neonatal intensive care nurses at the tertiary centre and a research co-ordinator at each of the smaller centres sent the link to obstetric, midwifery and paediatric staff. A second email was sent two weeks later to promote participation. It was not possible to identify which staff had responded to the link, beyond the demographic data related to work stream.p < 0.05. A comparison was made between HCP who care for women primarily prior to delivery - obstetrics and midwifery staff , and after delivery – neonatologists, neonatal nurses and paediatricians , referred to as postnatal HCP. Questions about factors which may influence opinions positively or negatively towards resuscitation were given as a Likart score, with scores of very likely and likely to imply a positive influence to offer intensive care, a score of neutral was considered to indicate that the factor was not contributory to the opinion, whilst an unlikely or very unlikely score was considered to indicate the factor would make the HCP less likely to agree with resuscitation. Missing data was excluded from analysis. Content analysis was performed on the qualitative data using a process of coding for thematic classification.The survey data were imported directly from the survey tool and were analysed using IBM SPSS 25 . Analysis used frequencies for numerical data. Chi square was used for categorical variables. Where categorical data with multiple ordinal responses occurred, Kruskal-Wallis H test to compare means was utilised. Significance was defined as The tertiary unit studied had outcomes for survival and all short-term morbidities within the expected range for units within the Australian and New Zealand Neonatal Network (ANZNN). The ANZNN data collection is a collaborative network established under the recommendation of the National Health and Research Councils Expert Panel on Perinatal Morbidity . For thiThe study was approved by the Townsville hospital human research ethics committee HREC/15/QTHS/194, and acknowledged by James Cook University (JCU) ref. 6485. Governance approval was given by all participating sites and JCUE-mails were sent to 174 potential participants, with 113 replies . Demographic details are shown in Table Some participants did not complete all aspects of the survey – with midwives and those from outside of the tertiary centre less likely to answer all questions. For different gestations, 81–91% of antenatal HCP, and 90–98% post-delivery HCP answered survival questions, 64–72% of antenatal HCP and 83–90% postnatal HCP answered severe disability questions, 59–67% of antenatal HCP and 77–87% postnatal HCP answered questions about intact survival. There was no clear pattern in gestational age for the missing data. Survival questions were answered by 89–97% by the tertiary group and 79–90% by the non-tertiary participants, Severe disability questions were answered by 78–84% by the tertiary group and 62–74% by the non-tertiary group, and the intact survival questions answered by 69–81% by the tertiary and 56–69% of the non-tertiary group  was an appropriate lowest gestation to offer parents, with midwifery and paediatric staff considering 25 weeks (IQR 24–26) and obstetricians and neonatal nurses choosing 26 weeks (IQR 25–26 and 24–26 weeks respectively). There were insufficient neonatologist response to analyse. For all HCP, a choice from a gestational age of 25 (IQR 24–26) compared to offer for patient 24 (IQR 24–25) was significantly different 2 [1] 4.64), 25 weeks p = 0.02 (χ2 [1] 4.49), 26 weeks p = 0.02 (χ2 [1] 5.05), 27 weeks p = 0.02 (χ2 [1] 6.76), as well as significant differences in perception for severe disability at 24 weeks p = 0.01 (χ2 [1] 4.64), and intact survival p = 0.01 (χ2 [1] 7.35), with the antenatal HCP more negative for each parameter.Comparison was made between the antenatal HCP (58 participants) and the HCP caring for the baby after delivery (53 participants). Analysis showed a significant difference in the perception for survival for most gestations from 23 to 27 completed weeks; 23 weeks p = 0.03 (χp = 0.03 (χ2 [1] 5.07), 24 weeks p = 0.03 (χ2 [1] 5.13), and 25 weeks p = 0.03 (χ2 [1] 3.95), but there were no other significant differences for estimates of severe disability or healthy outcomes.Analysis of tertiary hospital HCP (74 participants) compared to regional and remote HCP (39 participants) showed that the regional and remote HCP were significantly less optimistic about survival at 23 weeks Participants were asked at which gestation parents should be the final decision maker Table .Table 3One hundred and twenty free text comments were received. These were divided into six themes Table .Table 4Given that accurate information is essential for collaborative decision making by parents and medical staff around the treatment for periviable babies this study demonstrates that there is greater pessimism about the outcomes of the most premature babies by all HCP groups than is indicated by the actual outcome figures. Information is also important for enabling parents of babies at older gestations who will still require tertiary level intensive care for their babies to understand the risks to their offspring, and at older gestations, HCP are more accurate in their knowledge. HCP who have the most contact with parents prior to delivery, are the least accurate in terms of both mortality and the risks of a poor outcome at the lowest gestations. This discrepancy is concerning, as proactive antenatal care improves neonatal outcome, and where the antenatal team disagrees with the neonatal team in the provision of care, the outcomes for the baby are seen to be worse . Where aIt is possible that the information as understood by HCP’s is merely out of date, however, whilst survival data has improved with time, there have been only modest improvements in the rates of severe disability seen in some studies . Even inExtremely preterm babies will remain in the neonatal intensive care for months before going home. Parents who have experienced neonatal intensive care have been shown to have high rates of anxiety, depression, stress and trauma , 30 whicStaff based at smaller centres were found to be more negative about survival below 26 weeks than the tertiary HCP, but there was no difference in their perceptions of rates of disability. The origins for this are unclear. This has been noted in the Australian context in previous studies . Most HCClinicians who are involved in the care of women prior to delivery are significantly more negative than those who care for the baby in the short and long term. This confirms previous work done and has previously been shown to adversely affect the antenatal care of the extremely preterm fetus , 18. CliAll clinicians would offer care for patients at significantly lower gestations than they would wish for themselves, which is not unexpected given their negative perceptions of outcome. This has been described previously in trainee doctors  and may The difficulty in predicting an outcome for an individual pregnancy from large epidemiological studies was reflected in several free text comments. Whilst statistics may be important to clinicians, these reflections of uncertainty may be important factors for parents to understand. In a pilot study of 15 clinicians giving antenatal counselling, Prentice et al.  showed tMost HCP recognise a need to support autonomy in parental decision making. However, this attitude was not consistently reflected in the answers to the range of questions asked. Where parents wanted intensive care provision for their baby but the clinician did not feel that it was in the best interests of the baby, 69.1% of respondents said that this care should be provided whilst 21% said that it should not. However, where parents did not want intensive care for the baby, but the clinician did, 37.5% would follow the parental request, but 40.6% would provide resuscitation despite this preference. Below 25 weeks, over half respondent felt that parents could be the sole decision makers, but 64.6% also said that clinicians could disregard parental choice at this gestation. It seems recognised that risks of death and disability decrease with increasing gestational age, but specific gestational cut offs are relatively artificial. Ethical dilemmas in the relative roles of parents and clinicians are reflected in these findings, with a range of opinions from complete parental autonomy to decline intensive care, even at gestations over 25 weeks, and those which deem that parents should not always be the final decision makers, even if intensive care then occurs for babies whose parents did not want this for their child. The data suggests that the trend is towards clinicians as the final arbiters of decisions. Further research could clarify the underpinnings of HCP beliefs.Parental involvement in decision making can only be based on accurate information. Most guidelines currently in use in Australia, include parental discretion around the resuscitation of babies below 24 or 25 weeks gestation , 4, 39. There are some limitations to this study. It is a relatively small study based on a self-designed cross sectional survey from one area of Australia only which may limit the generalisability. However, local examination is important and the findings are consistent with those found in previous studies both historically and more recent. Another potential limitation is the use of the long term follow up data from the ANZNN. Follow up rates at the tertiary unit are relatively poor, and highest rates of follow up occurs for local babies within the immediate tertiary unit area and only one other regional centre where standardised tests are available.The strength of this study is that it there was a good response rate, and that participation was invited from regional and remote centres where many patients initially presented with complications in their pregnancy. There are few studies which examine referring HCP knowledge. The survey also included staff whose contribution towards parental knowledge might previously have been ignored such as midwifery and neonatal nursing staff, as well as more junior obstetric staff. Midwifery and neonatal nursing staff will contribute to the parents’ perception of the long term with much closer daily contact whilst providing care both antenatally and postnatally and can influence the hope that parents need to cope with their neonatal experience. The inclusion of paediatricians who see these babies long term is also uncommon, but important as they will often have a long-lasting relationship with the children. A further strength of the study is that it has been done in an area with a high Indigenous population where Indigenous babies are over-represented on the neonatal unit. A strength of the study not reflected in most studies is the content analysis of the qualitative data. Qualitative data adds to the richness of the quantitative data in studies of knowledge and attitudes.Clinicians who work with pregnant patients need to give accurate information about the chances of survival and long term disability of babies who deliver at extreme prematurity if they wish to have collaborative decision making. This is most important for the senior clinician providing counselling but also important for other staff who may find themselves in a situation where their opinions will be revealed to the parents. Message framing will influence the parents decision making, but also their positivity during the neonatal unit stay. Enhanced positivity, without giving false reassurance, will improve parental experience of neonatal care and reduce the risk of poor mental health outcomes for the parent. Clinician bias needs to be explored to ascertain the source for undue negativity, and individual clinicians need to be responsible for ensuring that both their knowledge and biases are reflected upon. In the area studied, this study shows that improved education about prematurity is essential to improve the outcome of vulnerable babies and families. Units who offer intensive care for extremely preterm babies should be aware that accurate knowledge and positivity will improve outcomes. All tertiary hospitals providing neonatal intensive care need to regularly assess the adequacy of knowledge of their staff about extreme prematurity in this era of rapidly improving survival.


Over the past 15 years in Japan, postgraduate mandatory clinical residency training has been introduced and junior residents have been required to learn important clinical skills. As a basic clinical skill, Gram stain for sputum and urine specimens provides clinically meaningful information for pathogen‐directed antibiotic therapy in infectious diseasesTherefore, we conducted a nationwide survey for junior residents about preparation and performance of Gram stain skill. The survey was conducted in January 2020 at the time of the General Medicine In‐Training Examination (GM‐ITE), which has been implemented every year over the last 10 years around the end of academic year. The ethics committee of the Mito Kyodo General Hospital approved the study (No. 18‐37).There were a total of 6869 residents participating in the GM‐ITE . Of these, we analyzed responses from 6164  who agreed to the participation of the survey. Of these 6,164 residents , only 363 (6%) residents reported that they prepared Gram stain for themselves most of the time at diagnosis of respiratory or urinary tract infections  correctly, since it is most difficultIn sputum Gram stain, it is important to identify It is important to master basic clinical skills for junior residents, and it helps them to perform high‐quality clinical care for patients. Gram stain should not be a lost art of future medicine since it is mandatory to care for patients with infectious diseases. Current epidemics of pneumonia by a novel coronavirus infection (COVID‐19) also makes an accurate diagnosis of pneumonia being greater clinical priority. Teaching hospitals and junior residents in Japan should introduce a learning environment for preparation and interpretation of Gram stain with confidence.The authors have stated explicitly that there are no conflicts of interest in connection with this article.


The WHO World Health Assembly established in 2007 a Resolution (WHA60.17) on oral health, which called upon countries to ensure that public health actions for disease prevention and health promotion are established. The objective of the present survey undertaken 10 years later (2017‐2018) was to measure the application of such programmes for key population age groups in low‐, middle‐ and high‐income countries.Oral health focal points of ministries of health worldwide (n = 101) answered a structured questionnaire on existing national oral health systems and the actual public health activities. The response rate was 58.4%. The questionnaire was used to collect information about structural factors, country workforce, financial models, provision of preventive services and promotion for oral health, school health programmes, administration of fluoride, national oral health targets and oral health surveillance. The countries were classified by national income for analysis of data.Coverage of population groups by primary oral health care and emergency care varied by national income. The gap between countries in delivery of preventive care was strong since low‐income countries less often reported preventive activities than middle‐income countries and particularly when compared to high‐income countries. School oral health programmes were less frequent in low‐income than other countries. Moreover, population methods of fluoridation and use of fluoridated toothpaste were unusual in low‐income countries. Health education, mass communication and community events were often essential elements in health promotion. In disease prevention, many countries considered the link between oral health and general health conditions and intervention towards shared risk factors of NCDs. The health concern for the consumption of tobacco, unhealthy diet and sugars was particularly emphasized by high‐income countries but less highlighted by low‐income countries. Finally, while national oral health targets for children and surveillance systems were frequently reported by countries, similar systems for adolescents, adults and older people were rare.The inequities between countries in oral disease prevention and health promotion were substantial. Limited financial resources for preventive care and health promotion; inadequate workforce for oral health, and insufficient coverage in primary health care were observed in low‐resource countries. The results of the survey demonstrate the need for building effective oral health systems oriented towards oral disease prevention and health promotion. They result from the combined efforts of government agencies, institutions and resources, with the aim of improving health of their people. Properly designed health systems should have a strong component of health promotion and disease prevention, provide for early disease detection and health communication and facilitate appropriate intervention.The good news is that the major oral diseases are preventable. Since the 1980s or so, the significance of disease prevention has grown in a number of countries and substantial experience has been gained from implementation of community actions, introduction of preventive activities directed towards population groups and individual preventive care.1.1Oral Health: Action plan for promotion and integrated disease prevention, which stresses the responsibility of countries in developing appropriate public health actions for oral disease prevention and health promotion.Public health intervention against oral diseases of all age groups has gained much concern over the past decades. WHO has given particular emphasis to incorporation of oral health into general health in national NCD prevention programmes and encourages the application of the Common Risk Factors approach.1.2Ten years after the confirmation of the WHA60.17 resolution on oral health, there is a need to ascertain the current application of policy recommendations on the strengthening of population‐oriented oral disease prevention and health promotion. Thus, the objective of this study was to determine the nature and the extent of appropriate oral health intervention activities for children, youth, adults and older people in low‐, middle‐ and high‐income countries.2The survey took place in 2017‐2018. Chief Dental Officers (CDOs) of Ministries of Health were considered relevant informants for the study, as they are responsible for dental public health and supervision of oral healthcare delivery in their country. They were identified in collaboration with the Global Forum for Chief Dental Officers, the Regional Councils of Chief Dental Officers, WHO and WHO Collaborating Centres in Oral Health, and the World Dental Federation (FDI). Some countries did not have a separate office for oral health, and then related NCD staff undertook the work. Alternatively, public health experts with experience in oral health were selected in a few countries. Of the 194 member states of WHO, it was not possible to identify an officer involved with oral health in 23 small countries. Up to four reminders were used for approaching the 173 potential participants of whom 158 were CDOs.The global survey covered 101 countries Figure . The couA structured questionnaire was designed for gathering information on existing national oral health systems and actual preventive activities. The following variables were included: structural factors , percentage of GNP spent on health care and oral health care), country workforce, financial models, organization of work for oral health, school health programmes, community approaches to oral health, fluoride administration, and national oral health targets and surveillance. The questionnaire was pretested by a group of public health administrators from low‐, middle‐ and high‐income countries.Over the years, countries have been encouraged by national and international health authorities like WHO to accumulate quality oral health data regularly in order to undertake oral health surveillance, to facilitate health policy development and to advance building of appropriate oral health systems.t test and ANOVA. The levels of statistical significance of results are presented in the tables.Analysis of data collected by the survey included univariate, bivariate and multivariate frequency distributions, and statistical tests were carried out by chi‐square test, Student's 33.1Table The information about oral health workforce available in countries is shown in Table 3.2Table The involvement of private health insurance in oral health care for population groups is illustrated in Table Table 3.3Table Table Table 3.4Three in four countries had school programmes for oral health though they were less frequent in low‐income countries Table . One thiTable 3.5Table Table 3.6The responses to questions on use of fluoride for prevention of dental caries are summarized in Table One fourth of respondents informed that the political interest in water fluoridation was good or very good; however, the interest in salt fluoridation or milk fluoridation was somewhat lower. The respondents were asked whether essential conditions for population methods of fluoridation  were encountered in their country. Almost half the respondents referred to a high burden of dental caries, about one fourth reported that technical requirements and facilities were available, while nearly one third of respondents declared that the necessary expertise existed in their country. Finally, the respondents were asked whether their country had any plans of introducing population methods fluoridation programmes. Plans about water fluoridation were confirmed by 16.3% of countries, 12.4% for salt fluoridation, and 6.2% of countries mentioned milk fluoridation. Salt fluoridation was considered particularly by low‐income countries.Table 3.7Half the countries reported having oral health targets for children aged 5‐6 and 12 years; meanwhile, targets for oral health of 12‐year‐olds were relatively less frequent among low‐income countries Table . TargetsElements of oral health targets as formulated by countries were measured by an open‐ended question. The major components of oral health targets for children were dental caries prevalence (primary and permanent dentitions); reduction of disease prevalence rates; reducing the unmet need for dental care (d/D index components); prevalence of dental erosion; risk factors such as sugars; dental care; treatment services; and school oral health programmes. Additionally, targets for oral health of adults focused on reducing the burden periodontal disease; oral cancer screening; tobacco; quality of care; oral health of pregnant women; oral health awareness of the general population; and improvement of healthy lifestyles. Targets for oral health of older people also included actions for a functional dentition of adults; reducing the prevalence of tooth loss; avoiding root caries, and controlling the need for prosthetic dental care. Reference was often made to WHO indicators; WHO policies on oral health and NCD prevention; health promotion programmes; social determinants of oral health; and primary oral health care.Respondents were enquired about the existence of a national information system for oral health surveillance. Systems for children (58.2%) and youth (50.0%) were fairly often reported, while systems for adults (38.8%) and older people (35.7%) were less frequent. Information systems for children and youth were commonly indicated by high‐income countries.3.8Respondents were asked about the recent country figures on oral health status of the WHO key population groups: children aged 5 or 6 years, 12‐year‐olds, 18‐year‐olds, adults aged 35‐44, and people aged 65‐74/65+. In addition to the participating 101 countries, dental caries data on children aged 12 were supplementary identified from 17 countries as recorded in the WHO Oral Health Data Bank.As shown in Table Information about dentate status and periodontal health conditions was seldom recorded in low‐income countries Table . Worth n3.9Six global maps were prepared to provide a geographical overview of oral health status of the key population age groups. The intention was to portray the oral health situation of countries by prevalence or severity level of disease. Figures Country data were readily available for dental caries among children and adults aged 35‐44 years. In Eastern Europe and parts of Africa and Latin America, most countries had less than 50% of 5‐6‐year‐olds without dental caries. For children aged 12, DMFT values generally were either low or very low. Data on dental caries of older people were scarce and only a limited number of countries had data on dentate status.4Opposite to the existing WHO Global Oral Health Data Bank,In all, an acceptable response rate for the survey was obtained. The response to the survey was approximately the same as obtained in a recent WHO global survey of oral health through schools.Notably, the response rate was high in high‐income countries while the rates were somewhat lower in low‐ and middle‐income countries. Some selection bias might be expected. Participation was very good in countries of Europe and North America. The response of Asian countries was acceptable whereas the response rate was particularly lower in countries of Africa and Latin America. This may be due to several factors; primarily the absence of a country focal point for oral health, the capacity in oral health administration may be limited, or the public health interest of potential informants may be low. The survey indicated that oral health systems and preventive practices were less structured in countries of these geographical areas; consequently, the findings may display a somewhat positive picture as to the existence of programmes in low‐ and middle‐income countries.The questionnaire was formulated in English. Obviously, responders of countries have different languages. The pretest, which took place in a number of low‐, middle‐, and high‐income countries, considered the potential language barrier in understanding the specialized expressions applied. Although language might be a source of information bias, the questionnaire used basic expressions applied in preventive dentistry, which are part of the standard professional terminology across the world.Due to practical and economical concerns, a computer‐assisted questionnaire was used to collect data for the study. The structured questionnaire also included semi‐structured or open‐ended questions. However, compared to personal interviews, self‐completion questionnaire implies some principal disadvantages. There is no one present to help respondents if they have technical difficulties answering questions; there is no opportunity to probe respondents to elaborate an answer; you can never be sure whether the right person has answered the questionnaire; long‐questionnaire is rarely feasible; and there is a greater risk of incomplete answers or missing data. The Chief Dental Officers (CDOs) invited for the study are experienced in questions about oral health administration and supervision of oral healthcare programmes as implemented at national level. Therefore, it is anticipated that the information collected on oral disease prevention and health promotion is fairly accurate and reliable. In addition, CDOs are acquainted with population oral health statistics in their country.4.1Information about economic input and health workforce is essential for understanding the output of oral health systems.The shortage of dentists in low‐ and middle‐income countries is critical to serving the population in oral health care. Particularly, the deficiency in the number of dentists in low‐resource countries is a major barrier to capacity building in public health and the organization of population‐oriented programmes for oral disease prevention and health promotion.4.2Cochrane CollaborationFinancial systems affect the establishment of preventive and restorative services and they may also distress access to oral health care.4.3Major inequities between countries were shown in provision of primary oral health care and accessibility to emergency care. The inadequate number of oral health professionals in low‐ and middle‐income countries implicates incomplete access to primary oral health care.4.4The evidence is strong on various actions of population‐directed oral disease prevention and health promotion.4.5School health education is the most important component of health promotion aiming at improving health and creation of healthy lifestyles. The majority of high‐ and middle‐income countries reported having school oral health programmes, while they were less frequent in low‐income countries. Fluoride mouth rinsing for schoolchildren was indicated by one third of countries while the differences between countries were minor. Country inequities were, however, found for pit and fissure sealants of permanent teeth to schoolchildren as such service was less used in low‐income countries. Many high‐ and middle‐income countries offered dental treatment in schools whereas this was less frequent in low‐income countries, probably because of lack of resources.4.6An increase in the awareness of oral health is a primary effect from use of mass media while improvement of health behaviour is less convincing.High risk of disease is found for certain population groups.4.7The so‐called ‘Common Risk Factors approach’Interestingly, a substantial number of countries replied that they had national NCD programmes, which covered prevention of oral diseases as well. Furthermore, many countries considered the link between compromised nutrition, obesity and dental caries in disease prevention.Several high‐income countries addressed diet and sugars in prevention of NCDs and official recommendation existed often for reducing the intake of sugars. Such recommendation is in agreement with the WHO sugar policy,4.8Administration of fluoride through the environment is a unique opportunity for prevention of dental caries. The fluoridation of drinking water is renowned as an effective, safe and economically beneficial public health measure for disease prevention.About one fifth of people in high‐income countries were exposed to fluoride in drinking water (full or part of population), whereas one tenth of people in middle‐income countries reported the benefit from fluoride in salt; fluoridated milk was seldom. In low‐income countries, it is remarkable that less than one third of people on average benefitted from toothpaste containing fluoride. This was the case for some 50% of people in middle‐income countries but more than 80% in high‐income countries. Toothpaste of low concentration of fluoride (400‐600 ppm F) was used for young children by several countries but the preventive effect of this type is uncertain.The majority of high‐income countries had official recommendations on effective toothpaste containing fluoride; in comparison, nearly half the low‐income countries and one third of middle‐income countries had no such recommendations. It is an important experience that recommendation of fluoridated toothpaste is essential to development of national policies on effective use of fluoride.Significant proportions of respondents informed that population methods of fluoridation existed because of a high burden of dental caries, that technical requirements and facilities were available in the country, and that the necessary country expertise for establishment of population methods of fluoridation was available. These specific conditions were less frequent in low‐ income countries; meanwhile, one fifth of the countries mentioned that they had plans of introducing water or salt fluoridation programmes. In all, the political interest in water fluoridation was higher than for salt or milk fluoridation.4.9WHO, the World Dental Federation (FDI) and the International Association for Dental Research (IADR) published in 2003 a series of global oral health indicators for the measurement of national and global targets by the year 2020.The respondents of this survey were asked if national oral health targets were formulated for key population groups. While nearly half the countries had targets in relation to children aged 5‐6 and 12 years, only 20%‐25% of countries expressed having targets for adolescents and adult age groups. Differences between countries in answers on oral health targets were minor. For countries with expressed targets, components often focused on standards of oral health status to be achieved in the future, oral health systems adjustment and building of adequate primary oral health care, quality of life and health behaviour modification.The oral health indicators suggested by WHOEven with WHO recommendation, statistics on the occurrence of oral disease were limited in low‐income countries; this probably reflects a shortage of public health researchers and a weaker attention by health authorities. The information was considerable in middle‐ and high‐income countries. The survey pointed to a relatively high proportion of caries‐free 5‐ to 6‐year‐olds in high‐income countries while dental caries experience among children aged 12 was prominent in middle‐income countries. This is in agreement with previous reports on global oral health of children.4.10The WHO Global Oral Health Data Bank4.11The first WHO global maps on oral health focused on dental caries (DMFT) in children aged 12 and adults aged 35‐44; they were primarily based on data provided by CDOs or national public health researchers during the late 1990s. Oral health data available were then used to classify countries according to the level of dental caries and they were published by WHO in the World Oral Health Report 2003.The global maps prepared for the present report were based on comprehensive country data reported for the year 2000 and onwards. The respondents were requested information about the national representative values of oral health conditions and they were also asked to provide information about any recent national reports. Such survey information permitted an assessment of potential critical issues of parameters, such as source of statistics, geographical area, socio‐economic status, number of people examined, diagnostic criteria, and number of examiners and training.The classification criteria of the dental caries levels used for the age groups were stable over time. Moreover, the number of maps was extended to cover all the WHO indicators and population groups, that is including the specific oral health conditions of people aged 5 or 6 years, 12 years, 35‐44 years and 65‐74/65+ years.The actual figures indicate that a low level of caries‐free children aged 5‐6 was frequent in certain countries of Eastern Europe, the Middle East, Asia, Africa, and Latin America. North America and Western Europe had moderately high levels of caries‐free children while northern Europe showed the highest scores. When compared to the equivalent maps of the year 2002Traditionally, the severity of dental caries among young adults and older people has been high in many industrialized countries.5The intention of the present global survey was to determine country application of oral disease prevention and health promotion 10 years after the approval of the 2007 WHO World Health Assembly resolution on oral health.Inequities between countries in coverage by primary oral health care and emergency care is substantial. The study indicated that it remains a challenge to especially low‐ and middle‐income countries to realize the WHO policy on Universal Health Coverage as regards oral health and as recommended by the 2019 United Nations Political Declaration on Universal Health Care.A considerable number of countries have established programmes for oral disease prevention and health promotion. Meanwhile, the gap between countries in delivery of preventive services and health promotion is strong, which may relate to health systems factors. In low‐and middle‐income countries, these factors include insufficient financial support to prevention and health promotion; inadequate workforce for oral health; and absence of policies for oral health.The establishment of school‐based oral health promotion varies significantly between countries. Preventive services and health promotion in schools have the potential of breaking existing health inequities in children and adolescents and oral health through schools should be ensured by countries.The use of maternity and child health facilities is occasional in low‐income countries. Such centres are important for health communication, disease prevention and control and should be reinforced.According to the survey, several countries have accepted an integrative approach for oral disease prevention and health promotion. Effective disease prevention links oral health with general health and intervention against NCD risk factors. Intervention against tobacco use and consumption of sugars needs strengthening in low‐income countries. National policies for reduction of the intake of sugars should be encouraged for countries without such authorized recommendations.Most people of high‐income countries benefit from use of toothpaste containing fluoride; however, enhanced use of such toothpaste is desirable in low‐ and middle‐income countries. Fluoridation methods should be considered by countries where people are not exposed adequately to the preventive effect of fluoride. Such initiative may help breaking the inequities in dental caries. Optimal concentration of fluoride in toothpaste is warranted and should be recommended by national health authorities.Formulation of national targets for oral health and establishment of information systems for surveillance of oral health of children, adolescents, adults and older people should be encouraged.All authors have made substantial contributions to the design of the study, data collection, interpretation of data and final approval of the version to be published.Figures S1‐S7Click here for additional data file.Table S1‐S16Click here for additional data file.


Species distribution models (SDMs) are used to test ecological theory and to direct targeted surveys for species of conservation concern. Several studies have tested for an influence of species traits on the predictive accuracy of SDMs. However, most used the same set of environmental predictors for all species and/or did not use truly independent data to test SDM accuracy. We built eight SDMs for each of 24 plant species of conservation concern, varying the environmental predictors included in each SDM version. We then measured the accuracy of each SDM using independent presence and absence data to calculate area under the receiver operating characteristic curve (AUC) and true positive rate (TPR). We used generalized linear mixed models to test for a relationship between species traits and SDM accuracy, while accounting for variation in SDM performance that might be introduced by different predictor sets. All traits affected one or both SDM accuracy measures. Species with lighter seeds, animal‐dispersed seeds, and a higher density of occurrences had higher AUC and TPR than other species, all else being equal. Long‐lived woody species had higher AUC than herbaceous species, but lower TPR. These results support the hypothesis that the strength of species–environment correlations is affected by characteristics of species or their geographic distributions. However, because each species has multiple traits, and because AUC and TPR can be affected differently, there is no straightforward way to determine a priori which species will yield useful SDMs based on their traits. Most species yielded at least one useful SDM. Therefore, it is worthwhile to build and test SDMs for the purpose of finding new populations of plant species of conservation concern, regardless of these species’ traits. We used independent field survey data to assess the accuracy of species distribution models (SDMs) built for 24 plant species of conservation concern. We tested whether traits of species  or characteristics of their distribution  influenced model accuracy. Traits do influence species–environment relationships and therefore SDM accuracy, nevertheless a useful SDM could be built for most species. Many studies evaluating the effect of traits on SDM accuracy have used this method  of the Ontario Ministry of Natural Resources and Forestry (OMNRF). The NHIC maintains records of all species ranked S1, S2, or S3 from herbarium records and confirmed sightings by provincial biologists, other scientists, or members of the public . Our primary set of predictors included 14 variables representing topography, surficial geology, soils, and climate only. SDMs for some species  are more accurate for sites with more contiguous forest surrounding them .We used the independent presence and absence records to calculate the AUC (area under the receiver operating characteristic curve) and the TPR (true positive rate) for each of the eight SDMs for each species. The AUC is a threshold‐independent index that measures the ability of an SDM to correctly discriminate between presences and absences. It ranges between 0 and 1, where values of 0.5 or less indicate that the SDM is worse than a random model would be at predicting presence or absence  with a beta error distribution (“glmmTMB” function). We included SDM model version and regularization factor as random effects to account for potential differences in model performance that might be introduced by SDMs built using different predictors or with different regularization settings. Due to nonlinearity, we fit seed weight as quadratic. We first fit a model including all seven candidate predictor variables. We then used the dredge function of the MuMIn package to create and rank a list of all possible variable combinations based on the lowest AICc. We assessed the relative importance of each of the variables retained in the top ranked model by using a drop1 test to assess the importance of each predictor once all the others were accounted for, based on the difference in AICc with and without the predictor. We report both dAICc and the p‐value of χhielzeth  and repo2 (“r.squaredGLMM” function).Because TPR is a continuous computed rate based on the number of correctly predicted presences (“successes”) out of the total number of independent presences , we analyzed this response variable using a glmm with a binomial error distribution (“glmmTMB” function), including SDM model type and regularization factor as random effects. We assessed model fit and variable importance using the same procedure described above. We assessed overall model fit using RWe chose to use the drop1 test on the top ranked model to assess the relative importance of each predictor rather than taking a model averaging approach and calculating relative importance values  ranged from 0.36 to 0.98, while TPR ranged from 0 to 0.96 Table . The mos2 value of 0.88. The most influential variable was occurrence density—dropping it from the model led to an increase in AICc of 50.3 , which are restricted to the Carolinian zone, may yield more accurate SDMs than species like wild ginseng (Panax quinquefolia), which range widely throughout our study area  and finer resolution, Moore and Elmendorf  and Liparis liliifolia , so any prediction for a particular species based on our glmm might not reflect the true ability of an SDM to predict independent occurrences. This matches the results of McPherson and Jetz ; Data curation (lead); Formal analysis (supporting); Funding acquisition (lead); Investigation (lead); Methodology (lead); Project administration (lead); Writing‐original draft (lead); Writing‐review & editing (lead). Hanna Rosner‐Katz: Data curation (supporting); Formal analysis (supporting); Writing‐review & editing (supporting). Joseph R. Bennett: Funding acquisition (supporting); Project administration (supporting); Supervision (supporting); Writing‐review & editing (supporting). Richard Schuster: Formal analysis (supporting); Writing‐review & editing (supporting). Heather M. Kharouba: Formal analysis (lead); Writing‐review & editing (supporting).Table S1Click here for additional data file.


COVID‐19 is a novel disease that mostly affects the respiratory system but as the number of cases is rising significantly around the world, more extra‐respiratory manifestations are being reported among which are various dermatologic manifestations. COVID‐19 is a novel disease that mostly affects the respiratory system but as the number of cases is rising significantly around the world, more extra‐respiratory manifestations are being reported among which are various dermatologic manifestations. It is also associated with extra‐respiratory manifestations including dermatologic symptoms and complications. We report a case of cutaneous leukocytoclastic vasculitis presented with purpuric skin lesions in a patient with COVID‐19 infection.The novel coronavirus disease of 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus (SARS‐CoV‐2) was first identified in Wuhan, Hubei, China in December 2019 and resulted in a pandemic.2A 49‐year‐old otherwise healthy male physician presented to our dermatology department with the chief complaint of skin lesions on his lower limbs. He reported that he had started feeling unwell since April which was followed by aches and profound fatigue and lasted about 10 days. Subsequently, he started developing respiratory symptoms including cough and shortness of breath. He denied having other symptoms such as sore throat, fever, anosmia, or ageusia. On chest computed topography (CT), isolated patchy ground glass opacity accompanied with adjacent nodules with ground glass attenuation in middle zone of right lobe was seen that was suggestive for COVID‐19 Figure A,B,C. He3COVID‐19 presents with many extra‐respiratory manifestations in addition to respiratory symptoms and fever.Also, in our case report, the patient developed skin lesions 2 weeks after the diagnosis of COVID‐19 and they were located on the lower limbs and trunk with histopathologic findings suggesting cutaneous leukocytoclastic vasculitis. Leukocytoclastic vasculitis (LCV) is characterized by leukocytoclasis, which refers to vascular damage caused by infiltration of neutrophils.The pathophysiology of dermatological manifestations of COVID‐19 still remains uncertain, however some theories have been suggested in recent studies.We have no conflicts of interest to declare.All the authors listed in the manuscript have participated actively and equally in presenting the case and providing the final version of the manuscript.This case report was approved by the bioethics committee of Isfahan University of Medical Sciences. Written informed consent was taken from the patient.


Inner strength is a psychological resource that supports people as they move through challenging life events. Understanding how people living with serious illness conceptualize inner strength is key in informing person-centered healthcare. This concept analysis used dimensional analysis methods to explore the nature of inner strength in people aging with serious illness to define a situation specific theory. 1212 abstracts and articles from the published literature were reviewed. 30 articles were included and analyzed as data sources. The resulting explanatory matrix conceptualized inner strength in people as an inward and outward process of “meeting me” – an authentic version of oneself – in the context of shifting health. Of note, while other serious illnesses were represented, no literature focused on dementia. Understanding inner strength in people with dementia is the next step for further inquiry. A pilot study exploring feasibility and informing further research is planned.


When ileal conduit construction is performed for urinary tract drainage during radical cystectomy, the conduit is usually constructed in the right lower abdomen. However, no reports have described ileal conduit construction in the left lower abdomen when it cannot be performed on the right side. In addition, some ingenuity is necessary for construction on the left.A 75‐year‐old woman visited our hospital with chief complaint of gross hematuria. Computed tomography and cystoscopy showed a huge bladder tumor, and blood analysis showed anemia. The patient was treated by radical cystectomy with ileal conduit construction. An ileal conduit was constructed in the left lower abdomen; it was impossible to construct in the right lower abdomen because of the abdominal wall scar hernia due to the past open surgery.We herein reported a patient who underwent ileal conduit for urinary diversion on the left side of low abdominal wall. We reported a patient who underwent radical cystectomy followed by ileal conduit on the left side of low abdominal wall. In this case, no perioperative complications, including ileus, were observed after surgery. We herein reported a patient who underwent ileal conduit on the left lower abdomen and described the surgical technique.Radical cystectomy remains the gold standard procedure for patients with muscle‐invasive bladder cancer.A 75‐year‐old woman visited our hospital with chief complaint of gross hematuria. Computed tomography and cystoscopy revealed a huge bladder tumor. Laboratory evaluation revealed a hemoglobin concentration of 7.2 g/dL . The patient was treated by radical cystectomy and ileal conduit construction. The patient had a history of open surgery for appendicitis and the presence of an abdominal wall hernia in the surgical wound , and written informed consent was obtained from the patient.The authors declare no conflict of interest.


These multimodal agents comprise a fluorescent dye for OI and the chelator diacetic acid‐1‐glutaric acid) (NODA‐GA) for PET radiometal isotope labelling. Special focus was put on the influence of the used dyes on the properties of the whole bioconjugates. For this, several compounds with different fluorescent dyes and non‐dye carrying subunits were synthesized and investigated. As fluorescent dyes, dansyl, NBD, derivatives of fluorescein, coumarin and rhodamine as well as three pyrilium‐based dyes were employed. Considerable influence of the charge of the colored unit on hydrophilicity as well as in vitro target receptor binding was observed and classified. High radiochemical yields and purities were found during radiolabeling of the multimodal imaging subunits as well as their GRPR‐specific bioconjugates with 68Ga. Examinations of the photophysical properties of both molecule species displayed no loss or alteration of fluorescence characteristics.We describe multimodal imaging probes for gastrin‐releasing peptide receptor (GRPR)‐specific targeting suited for positron emission tomography and optical imaging (PET/OI), consisting of PESIN (PEG Multimodal imaging: Different PESIN‐homodimers, suited for positron emission tomography and optical imaging, were synthesized. Special focus was set on the influence of different dyes on biomedical‐related properties. Multimodal imaging, based on molecular probes, becomes an important and growing field in medical diagnostics.Each modality brings its own specific advantages and can be sorted in one of two subgroups:At first glance, the PET/OI combination does not seem to offer such favorable complementary information as both are found in the imaging subgroup of “high sensitivity and little morphological information provided”.5d, 6 18F]FDG and other PET nuclide‐labeled radiotracers demonstrate daily.Furthermore, the combination of PET/OI features certain important advantages over other imaging techniques. In fact, PET/OI offers the possibility of performing pre‐ and intra‐operative imaging.5a, 5bTo be able to obtain optimal imaging results by PET/OI, suitable combined imaging agents have to be available. Such multimodal agents possess the advantage of co‐localization of both signaling units as the use of a single probe results in the same pharmacokinetics for both the fluorescent dye and the radioactive nuclide.Designing multimodal imaging agents for PET/OI, the different building blocks which need to be integrated to obtain a fluorescent, positron‐emitting and target tissue‐specific multimodal agent have to be chosen carefully:+‐emitters are suitable for medical imaging.+‐emitter being required for radiolabel introduction into the molecular probe. Due to the short half‐life of most diagnostic β+‐emitters and the common‐sense avoidance of every unnecessary radioactivity handling, the radiolabeling will always be the last synthesis step for preparing a multimodal PET/OI agent. Macrocyclic chelators and their well‐established coordination chemistry show the advantages to selectively trap metal ions fast and under mild conditions and thus have found widespread use in clinically relevant radiotracers. The macrocycle is usually introduced into the biomolecule used as targeting vector either via click chemistry or even more simple, by acid‐amide coupling.One is the positron‐emitting nuclide to be applied as several βAlso, an appropriate targeting vector has to be chosen and adequately functionalized to be able to be used in the context of a multimodal imaging agent. For the target‐specific uptake of imaging agents into malignancies, certain receptors which are overexpressed on a specific tumor entity are in general addressed. This can be achieved by using receptor‐specific biomolecules and their analogs, serving as tumor‐targeting vectors. For this purpose, different biomolecule classes can be used: antibodies, specifically binding their antigen with very high affinity and specificity,Finally, also the fluorescent dye to be used for OI during combined PET/OI and its conjugation chemistry have to be chosen. A huge library of dye molecules with very variable fluorescent characteristics exists.16, 183‐BBN7‐14) backbone to address the gastrin‐releasing peptide receptor (GRPR) which is overexpressed on different cancers.‐Tyr4‐bombesin as competitor. All bioconjugates showed IC50 affinity data in the range of 15.51±0.76 nm to 62.07±3.87 nm  All bioconjugates showed a decreased GRPR binding affinity compared to the endogenous reference BBN, ii) Interestingly, the bioconjugates 6, 7, 8 and 13 (containing pyridinium dyes and rhodamine 101) demonstrated GRPR‐affinities  being very similar to that of the reference 14 (18.67±1.37 nm), lacking both chelator and fluorescent dye. In these bioconjugates, the applied fluorescent dyes exhibit a positive charge. iii) Correspondingly, the group of bioconjugates carrying an overall uncharged fluorescent dye  also showed similar, slightly decreased GRPR affinity values of 26.20±0.89 nm, 25.19±2.82 nm and 20.91±1.67 nm, respectively, iv) The only bioconjugate carrying a twice negatively charged fluorescein dye (11) shows a considerably decreased GRPR affinity .Endogenous bombesin (BBN) was used as internal reference and showed an IC14, lacking both chelator and dye showed a GRPR‐affinity comparable to the multimodal imaging agents 6, 7, 8 and 13 comprising positively charged dyes, thus indicating that neither the introduction of the chelator or fluorescent dye per se result in a loss of receptor affinity but seem to have—if at all—only a minor influence on the resulting receptor binding characteristics. In contrast, with increasing number of negative charges in the dye molecule, the binding affinity considerably decreases, indicating a much higher influence of charge than of molecular size and composition of the MIUs on the receptor affinities of the corresponding bioconjugates.Taken together, the charge of the fluorescent dye introduced into the MIUs and in the following also into the corresponding bioconjugates seems to be the factor mainly influencing the biological receptor binding properties of the resulting multimodal imaging agents. The reference compound CC1–CC3 and the reference compounds CC4 and CC5CC1–CC3, a strong correlation between the number of negative charges of the respective dye molecule and GRPR affinity decrease was observed, going from CC1  over CC2  to CC3 . The binding data obtained for the reference compounds CC4  and CC5  support the conclusions drawn before.This conclusion is supported by the GRPR affinity data that were obtained for the ICG‐comprising bioconjugates This indicates a clear influence of the sum net charge of the introduced fluorescent dye on the GRPR binding capability and no or only an insignificant impact of the spatial demand of the chelator, dye or dye‐chelator‐complex. The choice of a suitably charged dye thus offers another possibility to tailor the characteristics of hybrid multimodal imaging agents regarding target receptor binding and thus in vivo pharmacokinetics.After studying the chemical and biological properties of the MIUs and their corresponding multimodal imaging agents, the photophysical properties of the substances were finally under investigation. Here, we found no significant decrease or shift of the optical and fluorescent characteristics of the bioconjugates compared to the MIUs Table .6, 7 and 8 as they were shown to exhibit large Stokes Shifts of 130 nm for 6, 140 nm for 7 and even 230 nm for 8, respectively.[6, initial confocal microscopy experiments were performed on GRPR‐positive PC‐3 cancer cells and revealed—as expected—a cytoplasmic localization of the agent  the GRPR binding capability of the bioconjugates is not influenced by the introduction of the NODA‐GA chelator or a fluorescent dye per se, but strongly depends on the sum of the net charge of the applied dye. We found a strong correlation between the number of negative charges and the decrease in GRP receptor affinity. iv) no significant decrease or alteration of the photophysical properties for neither the MIUs nor the bioconjugates was observed.We aimed here to design multimodal imaging units being composed of a chelator for PET radiometal nuclide labeling, a fluorescent dye and a chemoselectively and efficiently reacting functional group. These were introduced into a symmetrically branched PESIN homodimer serving as target‐specific vector, resulting in GRPR‐specific multimodal imaging agents. In the following, we determined how the different fluorescent dyes introduced influence chemical, biological and photophysical parameters of the final bioconjugates and found that: i) the lipophilicity of the MIUs as well as their bioconjugates strongly depends on the solubility properties of the respective fluorescent dye and only to a minor extent on the other molecular building blocks (chelator and peptide dimer), ii) the radiolabeling properties with the β68Ga‐radiolabeling nor the photophysical properties of the described complex systems are influenced by any of the molecular units forming together the multimodal imaging agents. In contrast, logD values and GRP receptor binding characteristics strongly depend on the choice of the respective fluorescent dye which gives a perfect tool in hand to tailor the properties of hybrid multimodal imaging agents on the basis of PESIN homodimers regarding their intended application for multimodal PET/OI imaging of malignant tissue.Taken together, neither the Experimental details, synthetic procedures, analytical data and photophysical investigations are provided in the supporting information.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Endoscopic retrograde cholangiopancreatography (ERCP) in patients with surgically altered gastrointestinal anatomy has been reported to be useful. However, selective biliary cannulation through the papilla is difficult in cases with surgically altered gastrointestinal anatomy. Herein, we report a successful biliary cannulation using a pancreatic duct (PD) stent in patients with Roux‐en‐Y anatomy. A 70‐year‐old man who underwent total gastrectomy with Roux‐en‐Y anatomy was admitted to our hospital with jaundice due to recurrence of gastric cancer. ERCP was performed for biliary drainage. We approached the papilla using a short‐type single‐balloon enteroscope . Because the papilla was positioned tangentially, it was difficult to adjust the catheter in the direction of the bile duct. As only a PD could be cannulated, we placed a guidewire in the PD. Although we attempted the double‐guidewire technique using a guidewire placed in PD, selective biliary cannulation was difficult. Therefore, we placed a PD stent 5Fr‐5cm  to assist biliary cannulation. We inserted a catheter crossing the PD stent. With this, selective biliary cannulation was successful. We successfully performed selective biliary cannulation using the PD stent as we were able to fix the papilla, straighten the common channel and the axis of the bile duct, and not restrict scope movement by not using the PD guidewire placement method. This novel technique using a PD stent appears to be useful in patients with surgically altered gastrointestinal anatomy. This study presents the case of a 70‐year‐old man who underwent total gastrectomy with Roux‐en‐Y anatomy. It was difficult to adjust the catheter in the direction of the bile duct despite using double‐guidewire technique. We placed a 5Fr‐5cm pancreatic duct (PD) stent to assist biliary cannulation. We inserted a catheter crossing the PD stent. By doing so, selective biliary cannulation was successful. When performing endoscopic retrograde cholangiopancreatography (ERCP) in patients with surgically altered gastrointestinal anatomy, postoperative adhesion and special anatomical characteristics, such as distance or strong curvature of the intestine, increase the difficulty of reaching the papilla or choledochojejunal anastomosis using a conventional endoscope. Similarly, using a duodenoscope and performing bile duct cannulation and subsequent treatments are technically more difficult in patients with altered anatomy than in those with normal anatomy. A short‐type single‐balloon enteroscope, with a working length of 152 cm and working channel of 3.2‐mm, was developed to increase the number of devices that can be used during ERCP in patients with surgically altered gastrointestinal anatomy and was reported to be useful.A 70‐year‐old man who underwent total gastrectomy with Roux‐en‐Y anatomy was admitted to our hospital with jaundice due to the recurrence of gastric cancer. The stricture was located at the hilar bile duct. ERCP was performed for biliary drainage  and PD stent placement methods are useful to fix a papilla and straighten the common channel and the axis of the bile duct. We believe that the PD stent placement method is useful in patients with surgically altered gastrointestinal anatomy because, in patients with normal anatomy, the guidewire placed in the PD acts in a direction away from the bile duct due to gravity or scope, such that the bile duct and PD are separated. On the other hand, in patients with surgically altered gastrointestinal anatomy, as the guidewire placed in the PD acts the same direction as the bile duct, the bile duct and PD cannot be properly separated Fig. g,h. FurtWe successfully performed selective biliary cannulation using the PD stent as we were able to fix the papilla, straighten the common channel and the axis of the bile duct, and not restrict scope movement by not using the PD guidewire placement method. This novel technique using a PD stent appears to be useful in patients with surgically altered gastrointestinal anatomy.Video Clip S1 As the papilla was positioned tangentially, it was difficult to adjust the catheter in the direction of the bile duct despite using the double‐guidewire technique. We placed a pancreatic duct (PD) stent to assist biliary cannulation. The catheter was inserted crossing the PD stent. Fluoroscopy demonstrated selective biliary cannulation using the PD stent.Click here for additional data file.


Cassava brown streak virus, Ugandan cassava brown streak virus, and Euphorbia ringspot virus are unusual in encoding an enzyme not yet found in any other virus, the “house‐cleaning” enzyme inosine triphosphatase. Inosine triphosphatases (ITPases) are highly conserved enzymes that occur in all kingdoms of life and perform a house‐cleaning function by hydrolysing the noncanonical nucleotide inosine triphosphate to inosine monophosphate. The ITPases encoded by cassava brown streak virus and Ugandan cassava brown streak virus have been characterized biochemically and are shown to have typical ITPase activity. However, their biological role in virus infection has yet to be elucidated. Here we review what is known of viral‐encoded ITPases and speculate on potential roles in infection with the aim of generating a greater understanding of cassava brown streak viruses, a group of the world's most devastating viruses.Plant viruses typically have highly condensed genomes, yet the plant‐pathogenic viruses  What we know and do not know about the inosine triphosphatase Ham1 found in the viruses causing cassava brown streak disease, namely cassava brown streak virus and Ugandan cassava brown streak virus. The viral‐encoded Ham1 shows sequence motifs typical of ITPase enzymes involved in dephosphorylation of noncanonical nucleotides, atypical nucleotides that arise from oxidative damage or stresses within the purine salvage pathway.2Escherichia coli and purification of the ITPases encoded by CBSV and UCBSV has shown that both are indeed functional ITPase enzymes with typical activity towards deoxyinosine triphosphate (dITP) and xanthosine triphosphate (XTP)  can build in the cells through normal metabolic processes or via exposure to external mutagens that result in modification of canonical nucleotides. If incorporated into DNA or RNA, noncanonical nucleotides can result in nonspecific base pairing and random mutagenesis or lead to chromosomal aberrations through error‐prone repair mechanisms. A number of different enzymes function in house‐cleaning roles to reduce the pool of noncanonical NTPs by hydrolysing pyrophosphate to give nucleotide monophosphates. There are four structurally distinct families each defined by a strictly conserved substrate specificity domain that is essential to exclude canonical nucleotides. The families are Nudix hydrolases, dUTPase, all‐α NTP pyrophosphatases (MazG), and ITPase (Maf/Ham1) , human (ITPA), and yeast (Ham1). The yeast ITPase is named for its mutant phenotype that shows sensitivity to the mutagenic purine analog, 6‐N‐hydroxylaminopurine (HAP). This mutant phenotype was also seen in ITPA knockdown HeLa cells and E. coli ‐causal viruses. These studies showed no change in expression of either ITPase‐like gene in response to UCBSV CBSV by cassava whiteflies and EuRSV by aphids. Despite the similarities between the viruses, the genes encoding ITPase are not considered to be homologs. Although it is likely that the genes encoding ITPase in CBSV and UCBSV are evolutionarily related due to their close phylogenetic relationship, the EuRSV Ham1 is distinct. The latter, when coupled with the absence of Ham1 in other members of the It is possible that the viruses acquired the ITPase gene from a host plant by horizontal gene transfer (HGT). HGT between host and virus is predicted to have protective functions through either production of a viral antigen by the host or mimicry of a component of the host immune signalling pathways by the virus CBSV infection with increased expression of genes belonging to the pathogenesis‐related (PR) gene family  has been shown to increase expression of PR genes in 7.3E. coli ITPase, YjjX, has been shown to interact with the GTPase, elongation factor Tu (EF Tu), which is involved in peptide elongation CBSV and EuRSV: prevention of mutagenic effects of an increased pool of ITP, disruption of plant signalling following infection, and disruption of other potential cellular roles of ITP. Further research should target these as well as the function and expression profiles of plant‐encoded ITPase, particularly in the host plants for (U)CBSV and EuRSV. It is unclear if viral ITPase expression is important as a result of reduced expression of endogenous ITPase, potentially resulting in increased pools of cellular ITP. Viral sequences do not appear to show increased levels of mutation; however, this has yet to be shown in the host plant. Potentially, these viruses, which infect members of the Euphorbiaceae family, have acquired ITPase in response to a host‐specific regulation of ITP pools. A greater understanding of the host's maintenance of cellular ITP will help to elucidate a role for viral Ham1.


In this review, we explore various aspects of SL function and highlight distinct areas of agriculture that may benefit from the use of synthetic SL analogues, and we identify possible bottlenecks. Our objective is to identify where the contributions of science and stakeholders are still needed to achieve harnessing the benefits of SLs for a sustainable agriculture of the near future.Strigolactones (SLs) represent a class of plant hormones that regulate developmental processes and play a role in the response of plants to various biotic and abiotic stresses. Both  Some of the synthetic SLs that have been produced so far include GR24 , which is mediated by the receptors D14 and KAI2, respectively, is dependent on the structural variations in the chiral carbon orientations at the junction of the BC and D‐rings  complex and targets the downstream repressors of karrikin and SLs signalling for degradation by ubiquitination  using SL‐biosynthesis mutants  treatment suggests that SL induces expression changes in fungal genes involved in respiration, production of chitin oligosaccharides, and transcriptional reprograming in the fungus . There are reports of increased nodulation of alfalfa inoculated with Sinorhizobium meliloti following SL (GR24) treatment , in principle, independent of auxin signals . There are also reports of their involvement in moderating auxin canalization from buds to the main stem through the internalization of the auxin export protein (PIN1), thereby maintaining apical dominance , probably due to the high cost of SL synthesis and the knowledge gap on any potential off‐target environmental risks and/or side‐effects from SL degradation products. Targeted research is therefore needed to understand the specificity of various SLs, the importance of time of application, the possible dose‐dependent environmental risks above or belowground, SL effects on soil microbiota at the community level beyond the effects on a single species or genus, and so on. Importantly, SLs should be properly acknowledged and emphasized in studies in which the effects of SLs are evident, just like with other hormones. Also, collaboration among all stakeholders  is inevitable for the eventual large‐scale application of SL products. Finally, regulatory advocacy and public awareness may be necessary to provide law makers, farmers, and the general public with the correct information about SLs on which to base their judgements.et al., et al., Striga‐resistant cv SRN‐39, which exudes a high orobanchol to 5‐deoxystrigol (5‐DS) ratio, recruits a different bacterial community than high 5‐DS/orobanchol‐exuding genotypes  using CRISPR/Cas9‐targeted mutation of the CCD8 SL‐biosynthesis gene  showed distinctive and differential gene‐expression levels in root tissues, and in response to exogenous auxin, respectively, suggesting that SL biosynthesis and function may be altered at the allelic level  has been used to demonstrate SL effects on plant–microbial interactions at the community level organisms at both the individual and community levels , CLA was reportedly converted directly to orobanchol through the action of an as‐yet‐unknown enzyme(s) , oxidation, epoxidation, ring cleavage, and lactonization steps have been postulated to result in the unique SL zealactone from CLA  influence the SL profile produced  and root‐knot nematode (Meloidogyne incognita), based on its cross‐talk with other hormones, like jasmonic acid, salicylic acid, and abscisic acid (ABA)  the soil microbiome by plants, other beneficial agricultural implementations of SLs in the soil are the control of parasitic weeds and defence against diseases. Parasitic weeds pose a serious threat to agriculture, considering the long viability of their seeds in the soil and their spread to other agricultural fields , Ha et al.  in Kalliola et al. (d10 and d17) and insensitive (d3) mutants had a high ABA accumulation in the shoot, resulting in drought tolerance, whereas the mutant, d27, was deficient in ABA and susceptible to drought  and its high expression in the SL‐deficient and insensitive mutants suggest that D27 may be involved in SL–ABA crosstalk. A further investigation of the aforementioned contrast using Arabidopsis single and double mutants of SL signalling (max2), ABA biosynthesis (aba2), and ABA guard cell signalling (ost1) in Kalliola et al. (MAX2‐mediated stress response. This ABA‐independent (MAX2‐mediated) stress response, however, may be a concerted role of the strigolactones–karrikins paralogous pathways, since both molecules signal through MAX2. Also, microarray analysis of the max2 mutant in Ha et al.  resulted in a transcriptional downregulation of SL biosynthesis genes. Such ABA regulation of SL biosynthesis may depend on the direction of the reversible conversion of all‐trans‐β‐carotene to 9‐cis‐β‐carotene by DWARF27, since both hormones share all‐trans‐β‐carotene as a common precursor  suggest that mutations in SL biosynthesis genes may contribute to ABA (in)sensitivity  and a three‐point deletion (NtCCD8B), and the exogenous ABA treatment of these mutants resulted in a three‐fold boost in the gene expression of NtCCD8B but not NtCCD8A. Interestingly, however, the gene expression of NtCCD8A under phosphate starvation (nutrient stress) was six‐fold higher than that of NtCCD8B, suggesting that allelic variations in SL biosynthesis genes may also influence response mechanisms to different stresses.Other reports re‐emphasize that the basis of the aforementioned ABA–SL crosstalk may be upstream of their respective biosynthesis pathways. Wang et al.  demonstr, et al.  differedet al., et al., et al. (WT) and SL‐depleted (SCSL) scions on to wild‐type (RSWT) and SL‐depleted (RSSL) root stocks to investigate drought responses  induced the expression of SL biosynthetic genes in the shoot, which was similarly observed in the SL‐depleted root stock grafted on wild‐type scion (SCWT/RSSL) under irrigated conditions. The graft combinations with wild‐type scions were more drought tolerant than the graft with SLs‐depleted scion. This suggests that local SLs availability in the shoot may be critical for drought tolerance response. In fact, Visentin et al. ‐overexpressing lines in Petunia hybrida, it was shown that an optimization of SL transport could release the feedback inhibition on SL biosynthesis and boost plant rooting properties  suppressed shoot branching in max2  locus, a maize homologue of BRC1  and tb1 mutant and the double mutant (tb1‐Zmccd8) revealed that although SLs may not completely regulate the TB1 gene in maize, they have significant additive effects on shoot branching and plant height under limited TB1 allele dosage , may be of higher agronomic value, and tiller number may be associated with the production of less SLs  to delay anthocyanin accumulation and ripening  between plant species may differentially affect microbial communities. Research efforts to identify the respective SL biosynthesis pathways in distinct plant species would aid our understanding of such effects. It is essential to understand the roles that plant developmental stage, root architecture, environmental conditions, soil types, and so on play in the interactions between SLs and microbial communities. For this, the fundamental biology of SLs needs to be understood; for instance, the downstream signalling components in plants and perception in microbes, and the mechanisms of SL molecular and physiological interactions. The use of targeted mutation techniques (like CRISPR/Cas9) and population‐based studies could unlock the genetic basis of SL effects and potential for heritability. Key for this improved understanding is the use of pure SLs instead of racemic mixtures in SL studies, as different isomers may signal through different pathways (Scaffidi In conclusion, many of the beneficial effects described herein are highly aligned with the sustainability criteria; for example: the better use of natural resources, such as soil nutrients; and the increase of crop resilience, particularly in relation to climate change threats. New SL‐based technologies will represent a substantial paradigm shift in the area of crop protection by moving from a classic pest/disease control approach to more of a crop enhancement effect that harnesses the soil potential.HJB, ADM and CS initiated the idea of the paper. EBA, TM and HJB reviewed the scientific literature. CS and ADM contributed on the industrial aspect of the manuscript.


Maize yields have significantly increased over the past half‐century owing to advances in breeding and agronomic practices. Plants have been grown in increasingly higher densities due to changes in plant architecture resulting in plants with more upright leaves, which allows more efficient light interception for photosynthesis. Natural variation for leaf angle has been identified in maize and sorghum using multiple mapping populations. However, conventional phenotyping techniques for leaf angle are low throughput and labor intensive, and therefore hinder a mechanistic understanding of how the leaf angle of individual leaves changes over time in response to the environment.Zea mays subsp. mays) and sorghum (Sorghum bicolor) were obtained using battery‐powered time‐lapse cameras. A MATLAB‐based image processing framework, Leaf Angle eXtractor (LAX), was developed to extract and quantify leaf angles from images of maize and sorghum plants under drought conditions.High‐throughput time series image data from water‐deprived maize (Leaf angle measurements showed differences in leaf responses to drought in maize and sorghum. Tracking leaf angle changes at intervals as short as one minute enabled distinguishing leaves that showed signs of wilting under water deprivation from other leaves on the same plant that did not show wilting during the same time period.Automating leaf angle measurements using LAX makes it feasible to perform large‐scale experiments to evaluate, understand, and exploit the spatial and temporal variations in plant response to water limitations. Over the past century, improvements in plant architecture and adaptation to higher planting densities have significantly increased maize grain yield. During this period, the average plant density per hectare has more than doubled due to changes in leaf angle that allow more efficient light capture Duvick, . Leaf anAdvances in breeding and agronomic practices have led to steady growth in agricultural productivity, but increased weather variability threatens food security , was used to analyze data from two sets of experiments employing maize and sorghum plants exposed to water deficit stress. While the framework can, in principle, be applied to individual images, it provides the greatest time savings when employed for the rapid analysis of time‐lapse data with minimum additional user input. LAX made it possible to track leaf angle changes in individual plants at intervals as short as one minute under increasingly intense drought stress conditions. Given these results, LAX has the potential to serve as a valuable framework to analyze multiple genotypes for variations in leaf angle and to measure their responses to drought stress, and is particularly adept at tracking individual plants over time.Three sets of plants, either maize or sorghum, were used to generate images for this study. The first set of plants (Set 1) comprised maize plants of the inbred line W22, grown in the greenhouse at the Donald Danforth Plant Science Center by the authors between 7 October 2013 and 20 November 2013. The greenhouse target conditions were 32/22°C day/night temperatures, 30% relative humidity, and a 16 : 8‐h photoperiod using supplemental metal halide lighting. The second set of plants (Set 2) were also maize (inbred line B73) grown at the University of Nebraska–Lincoln’s (UNL) automated phenotyping greenhouse under conditions described in Ge et al. . The thiImages for Set 1 were captured 44 days after planting using a first‐generation Raspberry Pi camera , and the image frames used for the initial development of the LAX framework were extracted from a compressed video file Video S1) generated from the original series of still images collected by the Raspberry Pi camera. Set 2 plants were imaged using the automated greenhouse imaging system and RGB camera described in Ge et al. . Set 3 p generateAn image processing framework was implemented in MATLAB R2014a  using Graphical User Interface Development Environment (GUIDE), a built‐in GUI editing utility  and background (everything else) using an intensity threshold. Otsu’s method Otsu,  was usedUser input is required to identify the plant stalk and leaves for leaf angle measurements. The user selects the stalk by placing a dot on the bottom center of the stalk, and then selects each leaf by placing a rectangular box close to the leaf–stalk junction without touching the stalk. The rectangular boxes are auto‐propagated to all the images in the series and will be considered for leaf angle measurements. These inputs help avoid background noise due to overlapping leaves that affect leaf angle measurements Appendix A. The frSelected images are converted into grayscale images and sharpened using the ‘imsharpen’ function in MATLAB, which uses unsharp masking where an image is sharpened through subtraction of a blurred version of the image from itself. The image is complemented using ‘imcomplement’ to reverse the black and white pixels within the image to make the plant an active object. The ‘imcomplement’ function subtracts each pixel value from the maximum pixel value supported by the class and outputs an image based on the difference in pixel values. The modified grayscale image is converted to binary using ‘im2bw’, which replaces all pixels with the value ‘1’ if the luminance is greater than the provided level, and replaces all other pixels with the value ‘0’ Appendix B–E. MATLTo make this framework more accessible to researchers, a GUI was built into the application allowing for an intuitive interaction between the user and the software. The GUI instructs the user on each step of the operation and supplies confirmation of successful operations, thereby separating the end‐user from the underlying math, image processing, and computational processes.Validation was performed through a comparison of the angle measurements generated by the image processing software described above and manual measurements of the same images. Ten images were selected randomly for each of six plants, and for each plant the angles of the same three to five leaves were manually measured using a ruler and protractor across all 10 images, resulting in a total of 240 manual measurements of leaf angle.We developed a MATLAB software package for measuring leaf angles from large stacks of time‐series images collected from a single viewpoint using maize plants from Set 1 (see Methods). The basic organizational structure of the approach is illustrated in Fig. Previously published image data from a maize drought stress experiment conducted in an automated phenotyping greenhouse using the LemnaTec conveyor belt imaging system  were used to capture leaf angle changes  were obtained from six plants.r values) for LAX measurements and ground truth data for sorghum and maize were 0.82 (P < 0.001) and 0.83 (P < 0.001), respectively  under water‐stressed conditions were used to track changes in leaf angle over the course of the experiment. Consistent with previous reports, under non‐stress conditions sorghum leaves exhibited more erect leaf angles than maize Appendix  Flénet . AlthougUnderstanding the molecular mechanisms underlying complex traits such as drought response is challenging due to the manifestation of a multitude of physiological changes. Some of these stress phenotypes are easily measured, and their molecular mechanisms are better understood, while more complex phenotypes such as leaf angle measurements require advances in phenotyping techniques and data extraction to link phenotypes to genetic variation. This study reports the development of a MATLAB‐based framework (LAX) and its application to efficiently extract leaf angle measurements from time‐course image data showing leaf wilting during drought stress in maize and sorghum using both an automated greenhouse imaging system and the more portable and much lower‐cost system based on Bushnell 6‐MP Trophy Cams.Dynamic traits such as stress responses change with time and environment, thus requiring repeated measurements over the course of an experiment  Image showing the actual 6‐MP camera setup at the Beadle Greenhouses, University of Nebraska–Lincoln. (B) Illustration showing the distance between the camera and the pots, and the perpendicular angle made by the axis of the leaf phyllotaxy with the face of the camera.Click here for additional data file.APPENDIX S2. Stepwise progression of image processing to obtain leaf angle measurements from plant images. (A) Individual plant selected for leaf angle analysis. (B) Image converted to grayscale . (C) Enhancing gradient. (D) Inverting color of the picture. (E) Image converted to binary. (F) Image thickened and stalk of the plant emphasized. (G) Unconnected components are bridged, small discontinuities corrected, and image blobs removed. (H) Skeletonized image. (I) Branches are pruned. (J) Determination of leaf angle from the processed image. Click here for additional data file.APPENDIX S3. Average difference between leaf angle measured using LAX and ground truth measurements.Click here for additional data file.APPENDIX S4. Differential phenotypic response to drought stress in sorghum (A–C) and maize (D–F). (A) Six‐week‐old sorghum plant (Btx623). (B) Sorghum plant nine days after water deprivation. (C) Sorghum plant showing leaf rolling symptoms 11 days after water deprivation. (D) Six‐week‐old maize plant (B73). (E) Maize plant nine days after water deprivation showing signs of leaf rolling. (F) Maize plant showing leaf wilting after 11 days of water deprivation.Click here for additional data file.APPENDIX S5. Consistent wilting of the upper leaf with clearly visible collar across replicates. (A–C) Maize plant 9‐M2, showing wilting of leaf after day 11 of water deprivation. (D–F) Maize plant 10‐M3, showing wilting of the same leaf after day 11 of water deprivation. The blue arrow indicates the leaf that showed wilting in both maize replicate through the time‐course images.Click here for additional data file.APPENDIX S6. Sample screens showing LAX framework usage. (A) Graphical user interface (GUI) welcome screen for the LAX framework obtained after running new_wilt_gui.m function in MATLAB. (B) Selection of the first image in the series. (All images belonging to a series stored in a folder can be loaded at once.) (C) Selecting the stalk of the plant by clicking the cursor at the center of the plant stalk. (D) Adjusting the width and the height of the plant image suitable for leaf angle measurements. (E) Selection of leaves for leaf angle measurement. Rectangles must be drawn close to the leaf–stalk junction without touching the stalk. Care must be taken while drawing the rectangle so that the entire change in the leaf angle can be captured in the drawn rectangle (as shown in H). (F) Leaf angle measurements will be recorded for as many leaves as selected by the user. (G) Thresholding can be adjusted by moving the slider or inputting threshold values. (H) When thresholding is completed for the last image in the series, the screen shows both the first and the last image and the rectangles drawn to select the leaves. (I) Clicking ‘Start analysis’ begins the analysis of each leaf for angle measurements. (J) The final screen shows the plant image with rectangle boxes and leaf number. Clicking the ‘Export Data’ icon at the bottom outputs leaf angle measurements for the selected leaves as a Click here for additional data file.VIDEO S1. Time‐lapse video showing the drop of maize leaves in response to water deficit stress over a single day. This video is also available at https://vimeo.com/256137800.Click here for additional data file.