According to the World Health Organization (WHO), under‐nutrition is associated with 2.7 million child deaths worldwide . This acLack of essential nutrition within an infant's diet can lead to life‐threatening conditions known as moderate‐ and severe‐ acute malnutrition (MAM/SAM). Acute malnutrition is classified as mild, moderate or severe based on anthropometric measurements affecting an estimated 51.5 million children under‐five years of age and being associated with 12.6% of all under five infant mortality . SAM is Maternal breastfeeding and complementary foods have been the most common and accessible modes of providing children with good nutrition during the early years of life. Breast milk has been noted to provide the most optimal nutrition for infants. This is because the components of breast milk including antibodies, hormones and growth factors combined with the nutrition present an optimal diet and protection to the growing child . MoreoveKhichuri ‐ which is a traditional mixed dish of rice, lentils, and vegetables. Commonly supplemented foods include Plumpy'doz, Rice and lentil food supplement and fortified wheat‐soy blend to name a few . Even now in LMICs only 40% of infants 0‐5 months  and 37% It has been reported that a total of 20 countries worldwide have recorded an increase in breastfeeding rates to more than 20% largely through large‐scale implementation of multilevel programs promoting IYCF practices . These i► Providing direct support and education to mothers and families about proper IYCF practices from a variety of healthcare workers  across a range of settings  and throughout different time periods .► Promoting supplementary feeding to prevent moderate and severe acute malnutrition such as endorsing current interventions of preventing SAM/MAM which include tackling the underlying cause of malnutrition through supplementary feeding .► Campaigning programs promoting IYCF through mobile health practices which can include outreach services and peer‐ to‐ peer support groups as reported previously .► Implementing proper training programs to educate healthcare professionals on optimal methods to educate on proper feeding and growth monitoring.Counseling and education on the importance of breastfeeding can be given in a variety of different ways. Counseling can be given in the antenatal and postnatal periods at home, clinics or in outreach setups. This can be done at the individual level or in a group setting. On the other hand, support that involves encouraging mothers and ensuring the benefits of interventions is usually aimed at an individual level. Complementary feeding involves the initiation of food and liquid to meet the nutritional requirement of infants which are no longer met from breast milk alone. Supplementary feeding involves the provision of energy‐dense food  or beverages to prevent under‐nutrition. This may be given at home in a community setting, in school, and daycare.The interventions work to educate mothers and caregivers on infant health and address the barriers that arise in LMICs when engaging in breastfeeding and complementary feeding practices. These interventions aim to promote IYCF guidelines set by the WHO:► Initiation of maternal breastfeeding within an hour post birth and continuation of exclusive breastfeeding practices until 6 months of age (UNICEF 2011) by educating, counselling and through peer‐support. Appropriate introduction of complementary feeding practices in a timely manner from 6 – 24 months of age. Feeding should be adequate to include appropriate frequency, amounts, consistency and with a variety of foods to address all required infant nutritional needs. This includes proper meal preparation with an emphasis on hygiene and food must be appropriately given to satisfy the consistency and texture, an infant is able to consume depending on age.► Continuing frequent on‐demand breastfeeding along with supplemental feeding practices from 6‐24 months of age.The interventions promoting IYCF practices intend to directly increase the number of women and infant caregivers following proper breastfeeding and complementary feeding practices. They also aim to tackle an insufficient diet that may lead to SAM/MAM. This can be implemented through community‐based nutrition education and mobilization programs aiming to increase mother or childcare provider's knowledge in infant feeding practices or with provisional programs to assist families in LMICs with the proper initiation of breastfeeding and complementary feeding practices . Infant In order to maximize developmental potential and health throughout life, it is important to intervene early in life. Appropriate complementary and/or supplementary feeding for less privileged young children can ensure and improve growth and micronutrient status by providing additional energy and required nutrients. Proper nutrition may also improve immunity and reduce the risk of infection  Figure 1Introduction of complementary and supplementary feeding may have some adverse effects as well. The introduction of solids as complementary and supplementary foods means that infants are exposed to microbial contaminated foods . A lowerAccording to the 2013 Lancet series on maternal and child nutrition, implementation of optimal IYCF practices can prevent an estimated 1.4 million deaths annually among children under five years of age , therefore a broader review of available evidence is strongly needed. The proposed review will analyse the effectiveness of interventions on outcomes based on different timings and duration of interventions, setting where interventions were employed, and moderators who provided the intervention, which have not been assessed previously. Some reviews only looked at the impact of education intervention for improving the different types of feedings. Therefore, it is important to perform a comprehensive review on all the past research studying interventions to promote IYCF practices and prevent development of SAM/MAM among children living in LMICs. This review will enable an updated and comprehensive assessment of the effectiveness of these interventions that can promote practices for improving child health and nutrition outcomes. The evidence from this systematic review will be critical to inform policy and programmatic decision‐making in initiating directed programs to increase optimal IYCF practices and decrease the prevalence of MAM/SAM in LMICs. Therefore, we aim to systematically review and assess current evidence on the effectiveness of interventions as well as programmes that have been adapted to manage children with acute malnutrition.The overall objective is to assess the effects of the intervention that aim to promote IYCF practices and their effect on health and nutritional status of children in LMICs. The objectives are to assess the effectiveness of the following on child health and nutritional status:1. Interventions to promote early and exclusive breastfeeding2. Interventions to promote continued breastfeeding3. Interventions to promote appropriate complementary feeding (education or provision of complementary food) during infancy and childhood4. Effectiveness of community‐based interventions to prevent moderate and severe acute malnutritionNote: when completing this section, please refer to the Campbell Collaboration Systematic Reviews: Policies and Guidelines. At a minimum, this section should include the information under each of the sub‐sections below:We will include primary studies, including large‐scale programme evaluations to assess the efficacy and/or effectiveness of interventions using the following study designs:► Randomized controlled trials (RCTs), where participants were randomly assigned, individually or in clusters, to intervention and comparison groups. Cross‐over designs will be eligible for inclusion.► Quasi‐experimental designs, which include:▷ Natural experiments: studies where non‐random assignment is determined by factors that are out of the control of the investigator. One common type includes allocation based on exogenous geographical variation.▷ Controlled before‐after studies (CBA), in which measures were taken of an experimental group and a comparable control group both before and after the intervention. We also require that appropriate methods were used to control for confounding, such as statistical matching  or regression adjustment .▷ Regression discontinuity designs; here, allocation to intervention/control is based upon a cut‐off score.▷ Interrupted time series (ITS) studies, in which outcomes were measured in the intervention group at least three time points before the intervention and after the intervention.without a control group will not be included.Pre‐post studies The target population for the intervention is mothers/caretakers of children under two2 years of age, regardless of health status, living in LMICs. Both women of reproductive age and pregnant women will be included. However, these interventions are aimed to improve child nutrition and health, so we are interested in child outcomes. We will also include studies that only include a subset of eligible participants and have reported outcomes for the eligible participants separately. Where the study includes a subset of ineligible participants we will exclude those studies and report in reasons for exclusion table.The following interventions will be included:► Interventions to promote early initiation and exclusive breastfeeding (such as education and support to mothers to promote early and exclusive breastfeeding)► Interventions to promote continued breastfeeding (such as education and counselling to mothers)► Interventions to promote dietary diversification and appropriate complementary feeding ► Interventions for growth monitoring and promotion . Most growth monitoring programs use weight charts to document child's weight‐for‐age. The promotion interventions can include teaching mothers and guardians, making a contact between mothers and primary health care providers and mobilizing community .► Interventions to prevent moderate and severe acute malnutrition (education and provision of supplementary food for children suffering from MAM or SAM)Interventions implemented by healthcare workers, community health workers and through mobile technology platforms will be assessed. Interventions will be compared against no intervention or standard of care (whatever is applicable in the setting the study was conducted.Primary outcomes► Rates of early initiation of breastfeeding (within an hour of birth)► Rates of exclusive breastfeeding (at 3 and 6 months of age)► rates of continued breastfeeding (at 12 and 24 months)► Infant growth Both clinical observations and self‐report will be used as methods to assess the rates of breast feeding.Secondary outcomes► Neonatal mortality ► Infant mortality ► Neonatal sepsis ► Incidence of acute respiratory infections► Incidence of diarrhoea► Any adverse events including gastrointestinal symptoms, noncompliance or decreased feeding► Cost‐effectiveness of the intervention (it will be reported in narrative text)► Psychosocial health of the infant Time points will be plotted within same forest plot with different subgroups for growth and psychosocial outcomes.Studies will not be excluded if they have not reported the outcomes mentioned above.Studies from LMICS. Country income will be classified according to the 2018 World Bank List of Country Economies (World Bank 2018). Countries which were in the list of LMIC before 2018 (from 2010) would be treated as LMICs.We will not impose any restrictions, for example language, date publication status, on the literature searches described below. We will also search for any relevant retraction statements and errata for information.Electronic searcheshttps://www.cabi.org/publishing‐products/online‐information‐resources/global‐health/), the WHO nutrition databases (http://www.who.int/nutrition/databases/en/), Popline (https://www.popline.org), Epistemonikos (https://www.epistemonikos.org/en/), Social Science Citation Index, Dissertation Abstracts International, and WHO Global Health Index which covers the WHO Regional journals from Latin America (LILACS), Africa (AFRO) etc. We will also search the websites of selected development agencies or research firms . The trials registry Clinicaltrials.gov and WHO's ICRTP will be searched for ongoing trials. The search will be performed in the following electronic databases: Cochrane Controlled Trials Register (CENTRAL), MEDLINE, Embase, CINAHL, PsycINFO, ERIC, Sociofiles, HMIS , CAB Global Health  flow diagram Two review authors will independently extract data on data extraction sheet. In case of any discrepancies in data entry, we will resolve disagreements by consensus or by involving a third review author. We will use a piloted data collection form for study characteristics and outcome data. If any information from the study is unclear or missing or cannot be obtained from the published papers, we will contact the authors of the original papers for further details. We will not impute missing information and will mention those studies as pending under awaiting classification.Data will be recorded on a data extraction form that will summarise key characteristics of the review/studies including:► methods: study design, study duration; location/setting of study, sample size, dropout/attrition rate► details of study participants including their age, location, sample size, numbers randomised, inclusion and exclusion criteria► interventions: duration of intervention, comparisons, frequency, exposure of intervention, delivery channels for intervention► outcomes and time point► information on sample size, methods of analysis, adjustment of confounders, socioeconomic status, gender, age, race/ethnicity, culture, geo‐political region should be collected specifically for quasi experimental designsIf a study is included with more than two intervention arms, we will only include the arms that meet the eligibility criteria.Two review authors will independently assess the risk of bias for each included study. We will resolve any disagreements by discussion or by involving a third review author.For RCTs, including cluster RCTs, we will use the Cochrane Collaboration Risk of Bias tool . We will► Random sequence generation.► Allocation concealment.► Blinding of participants and personnel.► Blinding of outcome assessment for each outcome.► Incomplete outcome data.► Selective outcome reporting.► Other bias such as the validity of outcome measure and baseline comparability.For non‐randomised controlled trials controlled before‐after studies, and interrupted time series, we will use EPOC methods (EPOC 2017a) to assess the risk of bias according to the following domains. We will justify the categorical risk of bias/study quality judgments  with information directly from the study.► Random sequence generation► Allocation concealment► Baseline outcome measurements► Baseline characteristics Incomplete outcome► Knowledge of the allocated interventions adequately prevented during the study► Protection against contamination► Selective outcome reporting► Other risks of biasWe will upload the outcome data for each study into the data tables in RevMan to calculate the treatment effects (RevMan 2014). We will use the risk ratio (RR) for dichotomous outcomes. We will use the mean difference (MD) for continuous outcomes reported on the same scale, and the standardized mean difference (SMD) for continuous outcomes reporting the same outcome but measured on different scales in different studies included in the same meta‐analysis. We will express the uncertainty with 95% confidence intervals (CIs) for all effect estimates. When means and standard deviations are not reported, we will use other available data  and appropriate methods described in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins(b) 2011) to calculate the means and standard deviations. Where other available data are not sufficient to calculate standard deviations, we will contact the study authors. When we are unable to enter the results in either way, we will describe them in the ‘Characteristics of included studies' tables or enter the data into the ‘Additional tables’ section. We will also consider the possibility and implications of skewed data when analysing continuous outcomes as they can mislead results due to small sample size. We will analyze outcomes from studies with multiple groups in an appropriate way to avoid double counting of participants by adding them to different subgroups within same plot. In such scenario, we will not report the overall pooled estimate and we will only report subgroup pooled estimate.We have a number of different outcomes and outcome subcategories. Conceptually, these subcategories cannot be combined . Therefore, a meta‐analysis will be conducted separately for each outcome. Furthermore, for each outcome, we will separately meta‐analyse different study designs . We will report all the effect sizes for each outcome and will not prioritise any from others.Where trials have used clustered randomizations, we anticipate that study investigators would have presented their results after appropriately controlling for clustering effects . If it is unclear whether a cluster‐ randomised controlled trial has appropriately accounted for clustering, the study investigators will be contacted for further information. Where appropriate controls for clustering were not used, we will request an estimate of the intra‐class correlation coefficient (ICC). Following this, effect sizes and standard errors will be meta‐analysed in RevMan using the generic inverse method (Higgins(b) 2011). They will be combined with estimates from individual‐level trials.We will contact trial authors to verify key study characteristics and obtain missing numerical outcome data where possible (e.g. when we identify a study as an abstract only). If we do not find a full report even after we contact the study authors, we will list such an abstract as a ‘study awaiting classification’. If numerical outcome data are missing, such as standard deviations (SDs) or correlation coefficients, and we cannot obtain these from the study authors, we will calculate them from other available statistics, such as P values, according to the methods described in the Cochrane Handbook for Systematic Reviews of Interventions .2, I2 and significance of the Chi‐square test; we will also assess heterogeneity visually using forest plots. Based on prior theory and clinical knowledge, we expect clinical and methodological heterogeneity in effect sizes in this literature. Therefore, we will attempt to explain any observed statistical heterogeneity using subgroup analysis.Statistical heterogeneity will be assessed using TauIf sufficient studies are found, funnel plots will be drawn to investigate any relationship between effect size and study precision. Ten studies are usually considered sufficient to draw a funnel plot. As a direct test for publication bias, we will compare results extracted from published journal reports with results obtained from other sources (including correspondence). Whilst funnel plot asymmetry may indicate publication bias, this is not inevitably the case, and possible explanations for any asymmetry found will be considered and discussed in the text of the review.We will carry out statistical analysis using RevMan 2014 software.We will prepare a matrix of all studies for each intervention which will outline all the differences in the studies at the intervention, duration, timing etc. and examine how to pool them together. We expect that most of our meta‐analyses will be random‐effects meta‐analyses, given the diverse contexts, participants, interventions etc.For each comparison, we will descriptively summarise the findings from the contextual factors such as setting, timings of intervention, duration of intervention, people delivering interventions etc. to assess their impact on the implementation and effectiveness of each intervention.We will construct ‘Summary of findings’ tables for all of the primary outcomes using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) criteria  which inWe will GRADE and prepare the following summary of findings tablesBreastfeeding► Rates of early initiation of breastfeeding (within an hour of birth)► Rates of exclusive breastfeeding (at 3 and 6 months of age)► rates of continued breastfeeding Complementary feeding► Mean Z score for height‐for‐age► Mean Z score for weight‐for‐height► Mean Z score for weight‐for‐age► Stunting► Wasting► UnderweightSupplementary feeding► Prevalence of malnutrition► Mean Z score for height‐for‐age► Mean Z score for weight‐for‐height► Mean Z score for weight‐for‐age► Stunting► Wasting► UnderweightWe will conduct the following subgroup analyses when there is a sufficient number of studies in each subgroup.► Setting : to see the effectiveness of interventions on the outcomes based on the settings where those were provided► Timing of intervention : to assess the effectiveness of interventions when it was delivered► Duration of intervention: to assess the effectiveness of different durations of intervention► Self‐reported or clinically observed► Recall information from last 24 hours or from birth.► Moderators (People) delivering the interventions and contextual factors▷ Type of health workers , physicians, nurses, midwives, volunteers, counsellors): to assess the most effective mode of intervention distribution▷ Training and supervision of health workers: to assess the most effective mode of intervention distribution.The subgroup analyses will be conducted using Review Manager 5.3 with a test for interaction.Sensitivity analyses will be performed to consider the impact of the following.► Allocation concealment (adequate versus inadequate and/or unclear).► Attrition (< 10% versus ≥ 10%).Imputed inter correlation coefficients that have been derived in different ways.We do not plan to include qualitative research.Lead review author: The lead author is the person who develops and co‐ordinates the review team, discusses and assigns roles for individual members of the review team, liaises with the editorial base and takes responsibility for the on‐going updates of the review.Content: Zohra Lassi, Jai Das, Zulfiqar BhuttaSystematic review methods: Omar Irfan, Zohra Lassi, Jai DasStatistical analysis: Omar Irfan, Zohra Lassi, Jai DasInformation retrieval: Rabia Hadi, Omar Irfan, Zohra LassiFunding for this review came from a grant from the Bill & Melinda Gates Foundation to the Centre for Global Child Health at The Hospital for Sick Children (Grant No. OPP1137750)Rabia Hadi and Omar Irfan: None to declare.Zohra Lassi, Jai Das and Zulfiqar Bhutta have published the following reviews:Lassi ZS, Das JK, Zahid G, Imdad A, Bhutta ZA. Impact of education and provision of complementary feeding on growth and morbidity in children less than 2 years of age in developing countries: a systematic review. BMC Public Health. 2013;13 Suppl 3:S13.Imdad A, Yakoob MY, Bhutta ZA. Impact of maternal education about complementary feeding and provision of complementary foods on child growth in developing countries. BMC Public Health2011;11 (Suppl 3) :S25Imdad A, Yakoob MY, Bhutta ZA. Effect of breastfeeding promotion interventions on breastfeeding rates, with special focus on developing countries. BMC Public Health2011; 11 (Suppl 3) :S24Haroon S, Das JK, Salam RA, Imdad A, Bhutta ZA. Breastfeeding promotion interventions and breastfeeding practices: a systematic review. BMC Public Health201313 (Suppl 3) :S20Approximate date for submission of the systematic review: July 2019Please note this should be no longer than two years after protocol approval. If the review is not submitted by then, the review area may be opened up for other authors.Reviews should include in the protocol specifications for how the review, once completed, will be updated. This should include, at a minimum, information on who will be responsible and the frequency with which updates can be expected.Zohra S Lassi will be responsible for updating this review and the review will be update every two years after publication date.By completing this form, you accept responsibility for preparing, maintaining and updating the review in accordance with Campbell Collaboration policy. Campbell will provide as much support as possible to assist with the preparation of the review.A draft review must be submitted to the relevant Coordinating Group within two years of protocol publication. If drafts are not submitted before the agreed deadlines, or if we are unable to contact you for an extended period, the relevant Coordinating Group has the right to de‐register the title or transfer the title to alternative authors. The Coordinating Group also has the right to de‐register or transfer the title if it does not meet the standards of the Coordinating Group and/or Campbell.You accept responsibility for maintaining the review in light of new evidence, comments and criticisms, and other developments, and updating the review at least once every five years, or, if requested, transferring responsibility for maintaining the review to others as agreed with the Coordinating Group.The support of the Coordinating Group in preparing your review is conditional upon your agreement to publish the protocol, finished review, and subsequent updates in the Campbell Library. Campbell places no restrictions on publication of the findings of a Campbell systematic review in a more abbreviated form as a journal article either before or after the publication of the monograph version in Campbell Systematic Reviews. Some journals, however, have restrictions that preclude publication of findings that have been, or will be, reported elsewhere and authors considering publication in such a journal should be aware of possible conflict with publication of the monograph version in Campbell Systematic Reviews. Publication in a journal after publication or in press status in Campbell Systematic Reviews should acknowledge the Campbell version and include a citation to it. Note that systematic reviews published in Campbell Systematic Reviews and co‐registered with Cochrane may have additional requirements or restrictions for co‐publication. 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In patients with cancer of unknown primary (CUP), the efficiency of reexamination in the improvement of the prognosis has not been demonstrated yet. In the present case, ampullary adenocarcinoma, initially diagnosed as CUP, was revealed by endoscopic forceps biopsy for the ampullary lesion progressing over time. Reexamination of the primary site in patients with CUP could contribute to better treatment options and improvement in the prognosis. A woman in her 60s with fatigue and weight loss was referred to our hospital because of increased fluorodeoxyglucose uptake detected in the pancreatic head upon positron emission tomography‐computed tomography (PET/CT) scan. Laboratory data revealed no abnormality, including tumor markers. Contrast‐enhanced CT revealed an 18‐mm enlarged lymph node, adjacent to the pancreatic head Fig. , while nCUP is pathologically diagnosed as metastatic carcinoma in which the obvious anatomical primary site is not identified after adequate diagnostic work‐up and occupies 2–5% of all diagnosed cancers.Written informed consent was obtained from the patient for the publication of this case report and accompanying images.


Vipera berus). During the winter, adders hibernate underground, buffered against subzero temperatures, and re‐emerge in the spring to reproduce. We derived annual spring‐emergence trends between 1983 and 2017 from historical observations in Cornwall, UK, and related these trends to the microclimatic conditions that adders experienced. Using a mechanistic microclimate model, we computed below‐ and near‐ground temperatures to derive accumulated degree‐hour and absolute temperature thresholds that predicted annual spring‐emergence timing. Trends in annual‐emergence timing and subsequent exposure to ground frost were then quantified. We found that adders have advanced their phenology toward earlier emergence. Earlier emergence was associated with increased exposure to ground frost and, contradicting the expected effects of macroclimate warming, increased post‐emergence exposure to ground frost at some locations. The susceptibility of adders to this “climate trap” was related to the rate at which frost risk diminishes relative to advancement in phenology, which depends on the seasonality of climate. We emphasize the need to consider exposure to changing microclimatic conditions when forecasting biological impacts of climate change.Climate change can not only increase the exposure of organisms to higher temperatures but can also drive phenological shifts that alter their susceptibility to conditions at the onset of breeding cycles. Organisms rely on climatic cues to time annual life cycle events, but the extent to which climate change has altered cue reliability remains unclear. Here, we examined the risk of a “climate trap”—a climatically driven desynchronization of the cues that determine life cycle events and fitness later in the season in a temperate reptile, the European adder ( In this study, we use novel microclimatic modeling techniques to assess whether advancement in the spring‐emergence phenology of the European adder has resulted in an increased exposure to frost, despite an overall reduction in frost risk under macrowarming. This study is among the first to demonstrate the existence of a climate trap, conceptually analogous to an ecological trap, and challenges traditional assumptions that macroclimatic warming will benefit species that require warmer conditions. ConventWhile there are high‐profile examples of climate change leading to biotic asynchrony  observations per year, with most observations (64%) occurring between 2010 and 2019.Historical records of adder sightings in the form of occurrence data were obtained from three sources: (1) The Environmental Records Centre for Cornwall and the Isles of Scilly ERCCIS, , (2) the2.3We predicted spatiotemporal patterns of emergence as follows. First, for each spring adder location in each year, we computed the temperatures of adder hibernacula at hourly intervals using a microclimate model. Second, we assumed that emergence is related to an accumulation of degree‐hours. Third, we related adder observations in the spring to accumulated degree‐hours to compute the threshold at which emergence occurs, but because each observation occurs at some unknown time period after emergence, we computed the cumulated degree‐hours at the time for sighting separately, ranked each sighting by their cumulative degree‐hour value, and selected the 5th percentile value as the trigger of emergence. Lastly, to calculate the spatiotemporal trends in emergence, we ran the microclimate model separately for each adder sighting location in the dataset and year to calculate the date on which the cumulative degree‐hour threshold for emergence was reached. Additionally, we explored the sensitivity of our results to assumptions made about the climatic drivers of emergence and percentile values triggering emergence see .In the absence of radiative heat provided by the sun, body temperatures closely match surrounding air temperature , within years with which suitable climate data were available 1983–2017), the cumulative degree‐hours prior to the sighting were computed. Since all sightings relate to individuals that had already emerged from hibernation, but emergence may have occurred at some unknown period prior to the sighting, we ranked each sighting by their cumulative degree‐hour and maximal soil temperature values and selected the 5, the cum2.4To estimate frost risk, we computed ground surface temperatures at hourly intervals at each sighting location in each year using the microclimate modeling procedures described above. Frost exposure was considered to occur if ground temperatures were ≤0°C after emergence. The relative exposure to ground frost was then quantified as the total number of hours after the emergence in which ground temperatures were ≤0°C between January and June. To assess inter‐annual variation in conditions and adder phenology, trends in the rates of predicted overall spring ground frost, emergence, and post‐emergence ground frost exposure were computed, respectively, using linear mixed models with year as a fixed factor. To account for variation between locations, we included each sighting location as a random factor.2.5To test for potential climate traps, we pooled all emergence and ground frost exposure data and used a Pearson product–moment correlation to assess the relationship between emergence timing and subsequent exposure to ground frost at each site. We also assessed trends in the risk of experiencing a climate trap across sites over the study period. To do so, site‐specific trends in emergence timing and exposure to ground frost after emergence were calculated for each location using linear models. The model coefficients were then plotted on a map to depict the magnitude and direction of long‐term trends in exposure to ground frost after emergence at each site. To illustrate the mechanism underpinning climate trap formation, plots for one inland and one coastal site with divergent post‐emergence ground frost exposure trends were generated for a typical cold (1987) and warm (1995) year. We defined coastal sites as those which situated within 3km of the coastline. To examine the differences in the types of sites where climate traps were arising, we performed a logistic regression, which regressed the number of post‐emergence ground frost hours against the site location . The analyses described above were performed in R programming language version 4.1.2. . Adder emergence was predicted to occur when accumulated degree‐hours above 7°C reached 21. Over the duration of the study, macroclimate warming appeared to have resulted in hibernacula warming. The timing of predicted emergence advanced by 28 (± 0.8) days  compared with coastal sites. Coastal sites in general also showed greater variability in the magnitude of change in adder post‐emergence exposure to ground frost. While ground frost appears to have reduced at most sites (64%), there were a clear trend toward increased exposure at southern coastal sites, and a general trend toward reduced exposure at inland sites  as rates of post‐emergence ground frost exposure were significantly reduced at inland sites  over a 25‐year time period ; Data curation ; Formal analysis ; Methodology ; Writing – original draft (lead); Writing – review & editing . Ilya M. D. Maclean: Conceptualization ; Data curation ; Formal analysis ; Methodology ; Writing – review & editing .Supplementary MaterialClick here for additional data file.


The health sector plays an important role in the delivery of high‐quality nutrition interventions to women and children in low‐ and middle‐income countries (LMICs). However, there are no standardized approaches to defining and measuring nutrition service quality in these contexts. This study aims to systematically develop quality of care indices for direct health systems nutrition interventions using a five‐step process: (1) identify recommended interventions for inclusion in indices, (2) extract service readiness, provision of care, and experience of care items from intervention‐specific clinical guidelines, (3) map items to data available in global health facility surveys, (4) conduct an expert survey to prioritize interventions and items, and (5) use findings from previous steps to propose quality of care metrics. Thirty‐two recommended interventions were identified, for which the guidelines review yielded 763 unique items that were reviewed by experts. The proposed nutrition quality of care indices for pregnant women reflects eight interventions and the indices for children under 5 reflects six interventions. The indices provide a standardized measure for nutrition intervention quality and can be operationalized using existing health facility assessment data, facilitating their use by LMIC decision makers for planning and resource allocation. Achieving meaningful reductions in the burden of maternal and child malnutrition across low‐ and middle‐income countries requires both increasing the coverage of heath sector nutrition interventions and ensuring high‐quality delivery of these services. However, many countries lack information on nutrition intervention quality.Standardized indices for nutrition quality of care can help facilitate consistent measurement of nutrition service quality and support global monitoring and country planning processes.This study proposes a set of nutrition quality of care indices for pregnant women and children, which is an important step towards improving availability of data on nutrition intervention quality that will enable LMIC governments to prioritize and allocate resources towards more effective services.Revision of existing health facility assessments to better support quality of care measurement for nutrition interventions as well as further refinement of global implementation guidance for nutrition interventions to facilitate translation of research to practice would bolster efforts to improve quality of nutrition services delivered through the health system in LMICs.1Maternal and child malnutrition remains a prominent issue in low‐ and middle‐income countries (LMICs) where there remains a high prevalence of undernutrition and a growing prevalence of overweight and obesity  to reaching population health goals, there is a shifting focus on improving and therefore measuring and monitoring service quality  and sick and well child visits has facilitated delivery of nutrition interventions. However, this integration presents challenges to clearly delineating nutrition services from other interventions and in turn to developing nutrition QoC indicators. Nutrition interventions are typically integrated across multiple broader service area guidelines  and not necessarily distinguished as nutrition‐focused  and Service Availability and Readiness Assessment (SARA), will support feasibility of country operationalization ICF, , n.d.‐b.22.1To define QoC measures for direct health systems nutrition interventions, we followed a five‐step process: (1) identify globally recommended interventions for pregnant women and children in LMICs delivered through the health sector for inclusion in indices; (2) extract service readiness, provision of care, and experience of care items from intervention‐specific clinical and service implementation guidelines; (3) map the identified items from the guidance documents to available data in global health facility surveys; (4) conduct an expert survey to prioritize interventions and items; and (5) develop QoC metrics informed by QoC frameworks, clinical guidelines, and expert survey results.2.2We conducted a structured literature review  provision of care, (2) experience of care, and (3) service readiness . Nonmatches were items for which there was no appropriate match within the SPA and SARA questionnaires . Partial matches were subsequently divided into high and low partial matches based on the specificity of the SPA and SARA item compared to the guidance document. For example, specific intervention guidelines  were considered a high partial match to broader service areas guidelines that include that intervention . Staff trained in a specific nutrition intervention  were considered a low partial match to SPA and SARA indicator of staff trained in a broader service area package  because it is not clear whether the specific intervention was included in training.2.5The criteria for participation in the expert survey was self‐reported (1) knowledge of nutrition interventions delivered through the health system in LMICs, (2) clinical knowledge of protocols, and physical and human resources requirements for high‐quality delivery of nutrition interventions through LMIC health systems, and (3) expertise in nutrition for children or pregnant women. The recruitment of survey participants began by reaching out to known nutrition experts through a global nutrition network to identify a pool of qualified respondents. This process resulted in the identification of 92 nutrition experts meeting the criteria that were sent the expert survey.The survey design was informed by prior research with similar aims of developing QoC metrics . Next, respondents were presented with all the items they categorized as “essential” within a dimension and asked to rank order these items according to their relative importance for inclusion in a summary measure of nutrition service quality. Finally, an open‐ended question asked respondents to identify interventions and/or items they thought were missing from the survey. The survey was administered using Qualtrics software  and the essential items required to deliver those interventions. Prioritization of items was important to ensure the QoC indices focused on nutrition interventions specifically. We also aimed to limit indices to a reasonable number of items so they were feasible to collect and calculate and in turn more likely to be used.Within each of the dimensions , items that fell in the bottom tertile of the range of Sutrop scores were excluded. We arrived at this exclusion cut off by comparing several thresholds. As a sensitivity analysis, we examined other statistics including the mean Likert score, the frequency of essential responses within the Likert score, a product of the rank score and the number deeming the item essential, and the Smith index, an alternative salience measure to the Sutrop score  and included both items in the QoC indices to ensure a consistent measure. For example, based on expert survey results alone, oral rehydration salts (ORS) was included in the readiness dimension, but the provision of oral rehydration therapy (ORT) was excluded in the provision of care dimension. This was resolved by adding ORT provision back to the provision of care dimension. Finally, we examined the remaining items to identify interventions that were completely excluded from the QoC indices. Interventions excluded based on survey cut‐off criteria but identified by the research team as important for maternal or child nutritional status were added back into the index if health facility assessment data for related items was available.Finally, we assessed possible methods for combining the QoC index items into a score for readiness and a score for provision and experience of care for pregnant women and children under 5, respectively. We considered the distribution of items within dimensions to decide on a simple or weighted average approach to calculating the index scores. If the number of items was similar in each dimension, then a simple average would be used, if not then a dimension weighted average would be used.2.8The institutional review board at the Johns Hopkins Bloomberg School of Public health deemed this non‐human subjects research.33.1A total of 71 interventions were identified through the literature and document review and 28 interventions met the inclusion criteria for guideline review and extraction Table . Many of3.2Table Table Partial and nonmatches reflected a number of common limitations in the SPA and SARA specific to nutrition services. There was a lack of items on specific nutrition training topics and a lack of specificity about guideline content for nutrition services. No dosage information is captured in SPA and SARA for micronutrient supplements, but this information is required to confirm readiness for specific interventions . The SPA and SARA counselling items were also too broad to capture nutrition intervention specific messaging.There were also several nutrition interventions that were not included within the current SPA and SARA, as evidenced by a lack of data on service readiness (commodities that would be required for the interventions) and provision of care (observations of what was provided). Both the SPA and SARA lack essential core items for the following nutrition interventions: calcium supplementation, balanced energy and protein supplementation, multiple micronutrient supplementation, treatment of noncomplicated severe acute malnutrition, inpatient treatment of complicated severe acute malnutrition, and multiple micronutrient powder supplementation.3.3n = 9) and sub‐Saharan Africa (n = 13). Respondents were from a variety of organizations including academic institutions (n = 10), donor agencies (n = 5), UN agencies (n = 3), Ministry of Health (n = 1), and nongovernmental organizations headquarters (n = 2) and in‐country offices (n = 6).Of the 92 individuals who received the survey, 30 fully completed it and three partially completed it. The section on nutrition for pregnant women was completed by 24 respondents, whereas 25 respondents completed the section on nutrition for children under 5. Respondents were from 12 countries, with the majority based in North America (n = 22) to 5% (n = 1). In the dimension of service readiness, guidance documents and staff training items had the highest number of “essential” responses. Within the provision of care dimension, items from the nutrition assessment domain were deemed essential by the most respondents. Looking at the prioritization by intervention, across both service readiness and provision of care, items relating to IFA supplementation, assessment and treatment of anaemia, and deworming were the interventions that received the most “essential” responses. Experience of care items that touched upon the communication between the care provider and the client, such as feeling comfortable discussing problems or concerns with the provider, being satisfied with how the staff treated them, and being satisfied with the amount of explanation received about the problem for treatment had the highest number of “essential” responses.Likert scores for each item for pregnant women are presented in Figure n = 22) to 0% (n = 0). Similar to nutrition for pregnant women, items in the guidance documents and staff training domains had the highest number of “essential” responses. By intervention, service readiness items related to growth monitoring and promotion, and complementary feeding received a high number of “essential” responses whereas provision of care items related to breastfeeding and infant and young child feeding (IYCF) counselling, assessment of illness and feeding during illness, and growth monitoring and promotion received a high number of “essential” responses. The same experience of care items that had the highest number of “essential” responses for pregnant women also had the highest number of “essential” responses for children under 5.Likert scores for each item for children under 5 are presented in Figure The Sutrop scores presented in Table For both pregnant women and children under 5, the service readiness items show little trend in prioritization by domain; however, the provision of care items consistently show a low prioritization of documentation items. The same experience of care item, the client's ability to discuss problems or concerns with the provider, had the highest Sutrop score of all experience of care items for both pregnant women (0.6465) and children under 5 (0.5148). For pregnant women, readiness and provision of care items related to IFA supplementation and anaemia assessment and treatment were among the highest Likert and Sutrop scores. For childhood interventions, readiness items related to growth monitoring and promotion and treatment of diarrhoea with both ORS and zinc were among the highest Sutrop scores while the provision of care items related to breastfeeding and IYCF counselling, assessment of illness and feeding during illness, and growth monitoring and promotion had the highest Sutrop scores.3.4After excluding items that fell in the bottom tertile of the range of Sutrop scores, the maternal QoC indices had 40 items and the child QoC indices had 31 items. For the maternal QoC indices, all type 1 items were excluded, all type 4 items were included, and the majority of the type 3 items were included. In addition, only one type 2 item was included as it was deemed essential by few respondents but very highly ranked. For the child QoC indices, all type 1 and type 2 items were excluded, whereas the majority of type 4 and some type 3 items were included.To ensure consistency between readiness and provision of care items, we added two provision of care items back into the pregnancy index . Similarly, four provision of care items were added back into the child index .Finally, the research team identified one pregnancy intervention (breastfeeding promotion) and two child health interventions (assessment and treatment of anaemia and deworming) that were excluded from the QoC scores as key interventions for child nutritional status. Therefore two items for breastfeeding promotion (“discussed exclusive breastfeeding” and “discussed early initiation and prolonged breastfeeding”), two items related to assessment and treatment of anemia , two items related to deworming for children , and one item related to counselling on feeding for diarrhoea (“told the caregiver to continue feeding the child during illness”) were added back into the QoC indices.Table Pregnancy interventionsAssessment and treatment of anaemiaBlood glucose testingCalcium supplementationDaily IFAIntermittent IFANutrition education and counselling during pregnancyIntermittent preventive treatment for malaria in pregnant women (IPTp)DewormingChildren under 5 interventionsPostnatal breastfeeding counselling (for early and exclusive breastfeeding and PMTCT)Complementary feeding counsellingAssessment and treatment of anaemiaDeworming in children 12–59 monthsGrowth monitoring and promotionScreening for acute malnutritionVitamin A supplementation in children 6–59 monthsTreatment of non‐complicated SAMCounselling on feeding for diarrhoeaORS during diarrhoeaZinc treatment for diarrhoea3.5For both pregnant women and child nutrition indices, we found that items were relatively evenly distributed across domains. Therefore, we recommend a simple additive approach for combining items into a single index score as it is easier for countries to operationalize and it is consistent with other studies which have taken a similar approach for developing QoC indices for antenatal care and child health . Our findings highlight the gaps in existing facility‐based surveys to fully capture the availability, readiness, and provision of key nutrition interventions. While investments in delivery of nutrition interventions to key populations grows, many LMICs have insufficient data to monitor and evaluate their implementation  (López‐Ejeda et al., 5The development of the nutrition QoC indices for pregnant women and children under 5 that reflect global guidelines and expert opinion is an important step towards improving availability of data on nutrition intervention quality that will enable LMIC governments to prioritize and allocate resources towards more effective services. Future research is needed to apply these indices to country data, operationalize the nutrition QoC indices for pregnant women and children under 5, and evaluate the proposed indices for construct validity and reliability. Efforts towards applying findings to guide evidence‐informed decision making must also be prioritized. Improving the QoC of facility‐based nutrition interventions is vital to ensuring the health sector is effectively contributing to a multisectoral approach of reducing malnutrition in LMICs.The authors declare that they have no conflicts of interest.AS, MKM, and RH contributed to conceptualizing the paper and analysis. SK and YYX conducted the analysis. SK and AS prepared the manuscript. MKM, RH, SW, and YYX critically reviewed and revised the manuscript.Figure S1: Nutrition intervention selection processTable S1: Nutrition interventions identified through literature reviewTable S2: Items identified within implementation guidanceTable S3: Tabulation of item matching from intervention guidelines to SPA/SARA itemsClick here for additional data file.Data S1. Supporting InformationClick here for additional data file.


The detection and quantification of apoptotic cells is a key process in cancer research, particularly during the screening of anticancer therapeutics and in mechanistic studies using preclinical models. Intravital optical imaging enables high‐resolution visualisation of cellular events in live organisms; however, there are few fluorescent probes that can reliably provide functional readouts in situ without interference from tissue autofluorescence. We report the design and optimisation of the fluorogenic probe Apotracker Red for real‐time detection of cancer cell death. The strong fluorogenic behaviour, high selectivity, and excellent stability of Apotracker Red make it a reliable optical reporter for the characterisation of the effects of anticancer drugs in cells in vitro and for direct imaging of chemotherapy‐induced apoptosis in vivo in mouse models of breast cancer. We describe Apotracker Red as a single fluorescent activatable probe for robust quantification of cancer cell death in fluorescence‐based assays and in vivo using multiphoton intravital imaging in mouse models of breast cancer. Cancer research is rapidly evolving and new therapeutic approaches—including immunotherapies and combination therapies—are in high demand. In line with this progress, technologies that can reliably determine and quantify the efficacy of anticancer drugs across platforms and in multiple biological assays are urgently needed.Anticancer therapeutic strategies aiming at the full eradication of tumour cells are continuously under development, and the extent of cancer cell death is one of the most reliable indicators of drug efficacy.Fluorescent peptides are versatile structures for imaging of biological events with high spatial and temporal resolution in a broad range of experimental conditions.Our group has previously reported green fluorescent probes for imaging of apoptotic bodies and apoptotic cells by incorporating the Trp‐BODIPY fluorophore (emission maximum ≈520 nm) into different cyclic peptide structures.0 complexes without affecting the BODIPY core . Notably, this procedure resulted in a significant improvement of the purity of the crude peptides prior to HPLC purification  and the overall recovery yields (22 % yield for Apotracker Red). The simplicity of this methodology and compatibility with commercially available building blocks  makes it an efficient approach for the scalable SPPS of BODIPY‐labelled fluorescent peptides, overcoming cumbersome purification steps and low yields .[We designed the chemical synthesis of Apotracker Red in two main steps: 1) solid‐phase peptide synthesis (SPPS) of the protected linear sequencexamples).16aNext, we examined the optical properties of Apotracker Red in different microenvironments mimicking those found in apoptotic cancer cells. First, we recorded the fluorescence emission of Apotracker Red in suspensions of viable as well as apoptotic cancer Jurkat cells. As shown in Figure Apotracker Red displayed significantly brighter fluorescence after interaction with PtdSer when compared to other phospholipids  in 96‐well plates (10–20×103 cells/well) for high‐throughput flow cytometry. After drug treatment , Apotracker Red was incubated for 10 min and fluorescence readings were directly acquired without any additional processing steps ,s Figure . The lows Figure .First, all drugs were tested in the 8 different cell lines to determine the percentages of Apotracker Red‐stained apoptotic cells compared to no treatment Figure . Cisplat−1). Previous studies have reported strong chemotherapeutic action for cisplatin in vivo after 3 days;Optical intravital imaging is increasingly used for the preclinical evaluation of anticancer drugs in vivo. Although imaging modalities like computed tomography, positron emission tomography and magnetic resonance imaging can help to visualise whole intact organs in a non‐invasive manner,We acquired time‐lapse microscopy images of both non‐treated and cisplatin‐treated mice that had received the same amount of Apotracker Red intravenously. In cisplatin‐treated mice, we observed strong red fluorescent signals indicating cancer cell death induced by the drug, whereas untreated mice were devoid of Apotracker Red fluorescence Figure . NotablyFinally, we assessed the in vivo selectivity of Apotracker Red in harvested tumours from mice expressing colony‐forming stimulating factor (cfms)‐EGFP macrophages, which had been treated with cisplatin and Apotracker Red. First, we did not observe changes in the number of macrophages found in both tumours . Moreover, fluorescence microscopy revealed little co‐localisation between the signals of Apotracker Red and EGFP‐positive macrophages Figure .This result suggests that 1) Apotracker Red is not largely taken up non‐specifically by phagocytic cells, and 2) the extent of efferocytosis  during the imaging procedure is limited; hence, the majority of the Apotracker Red signals can be attributed to the presence of apoptotic cells in tumours. Finally, we performed intravital imaging under spinning disk fluorescence microscopy where we could follow the interactions between individual EGFP‐positive macrophages and Apotracker Red‐stained cancer cells over time . These results confirm the utility of Apotracker Red to study dynamic cellular events in the tumour microenvironment in vivo with high spatiotemporal resolution.2+ independence, two‐photon excitation, high chemical stability, and compatibility with wash‐free protocols. Furthermore, unlike protein‐based reagents, Apotracker Red can be scaled‐up in batches (>50 mg) to enable large in vitro screening assays and in vivo experiments. We have demonstrated the preferential binding of Apotracker Red to PtdSer‐expressing apoptotic cells and its application to high‐throughput screens using a broad range of cancer cell lines and FDA‐approved drugs. Furthermore, the suitability of Apotracker Red for intravital imaging can provide additional insights into the mode of action of anticancer therapeutics in vivo and to study the dynamics of drug action with high spatiotemporal resolution. All these features make Apotracker Red an optimal chemical tool to accelerate the identification and preclinical validation of anticancer therapeutics.In summary, we describe Apotracker Red as a new fluorescent probe for rapid and robust detection of apoptosis in vitro and in vivo. We have optimised a methodology for the SPPS of BODIPY‐labelled peptides using orthogonal Alloc protecting groups with improved recovery yields (>20 %) and minimum purification steps. Apotracker Red is the first fluorogenic probe to allow robust quantification of apoptotic cancer cells both in drug screens and in vivo in live tumours of breast cancer mouse models. Apotracker Red overcomes some limitations of existing probes  given its CaThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.


Neuropathic pain is a major type of chronic pain caused by the disease or injury of the somatosensory nervous system. It afflicts about 10% of the general population with a significant proportion of patients’ refractory to conventional medical treatment. This highlights the importance of a better understanding of the molecular pathogenesis of neuropathic pain so as to drive the development of novel mechanism‐driven therapy. Circular RNAs (circRNAs) are a type of non‐coding, regulatory RNAs that exhibit tissue‐ and disease‐specific expression. An increasing number of studies reported that circRNAs may play pivotal roles in the development of neuropathic pain. In this review, we first summarize circRNA expression profiling studies on neuropathic pain. We also highlight the molecular mechanisms of specific circRNAs  that play key functional roles in the pathogenesis of neuropathic pain and discuss their potential diagnostic, prognostic, and therapeutic utilization in the clinical management of neuropathic pain. CircRNAs‐regulated genes expression via sponging miRNAs and played crucial roles in excitability and inflammation in neuropathic pain. The biological functions and molecular mechanisms of specific circRNAs involved in the pathogenesis of neuropathic pain are also discussed in relation to their diagnostic, prognostic and therapeutic potentials in clinical settings.2Microarray is an efficient tool for circRNA profiling. Cao and colleagues inflicted chronic constriction injury (CCI) to the sciatic nerve of rats to induce neuropathic pain.With the advancement of linear RNA depletion and bioinformatic workflow, circRNAs have been increasingly profiled by RNA sequencing. Zhou and colleagues performed RNA sequencing to profile the expression levels of non‐coding RNAs (ncRNAs) in the spinal cord after spared nerve injury (SNI)‐induced neuropathic pain.Recently, He et al. performed RNA sequencing to study the expression patterns of circRNAs, lncRNAs and miRNAs in the spinal cord of streptozotocin‐induced diabetic neuropathic pain (DNP) mice.33.1CircHIPK3 is a circRNA that has been shown to function as a tumour suppressor gene or oncogene in a context‐dependent manner to modulate tumour progression through sponging miRNAs.3.2Vegfb. Moreover, cytoplasmic circAnks1a acted as a miRNA sponge to repress miR‐324‐3p to disinhibit VEGFB. Overexpression of VEGFB contributed to the increased excitability of the dorsal horn neurons and SNL‐induced pain. These data suggested that the circAnks1a‐miR‐324‐3p‐VEGFB axis is a novel therapeutic target in neuropathic pain.Zhang and colleagues identified the aberrant upregulation of circAnks1a in the rat spinal dorsal horn after SNL by RNA sequencing.3.3The circRNA ciRS‐7 has been found to take part in the development of different diseases.3.4Wei and colleagues studied the expression and functional role of miR‐24‐3p in the development of neuropathic pain in the CCI rat models.3.5Li and colleagues demonstrated that the expression of miR‐22‐3p was downregulated in the dorsal spinal cord of CCI rats at the postoperative day 0, 3, 7, 10 and 14 as compared to the sham‐operated rats.3.6circ_0005075 deregulation has been implicated in cancer development.4Neuropathic pain is a serious public health issue that is poorly tackled by medical treatment, representing an unmet medical need. Emerging molecular studies have shed new light on the mechanisms of peripheral and central sensitization in neuropathic pain.The increasing number of studies have suggested that circRNAs play crucial roles in the development of neuropathic pain through neuroinflammation in both the dorsal root ganglia and spinal cord dorsal horns. From the mechanistic point of view, circRNAs may regulate glial activation and expression of the pro‐inflammatory genes by sponging pain‐related miRNAs . These studies have also supported the potential clinical utility of circRNAs and their downstream signalling mediators as therapeutic targets. In this connection, different approaches could be used to target pain‐related circRNAs—(1) CRISPR/Cas9‐mediated ablation; (2) antisense oligonucleotides or small interfering RNAs‐mediated knockdown; and (3) steric blockade of circRNA‐miRNA interactions by morpholinos. Through circRNA microarray and RNA sequencing, a growing number of deregulated circRNAs are expected to be identified in neuropathic pain. The potential implications in the clinical diagnosis and prognostication of circRNAs in neuropathic pain will be achieved. Then, it needs to measure these deregulated circRNAs in large samples of neuropathic pain. However, unlike other ncRNAs, such as long non‐coding RNAs (lncRNA), the correlation between pain scores of patients and circRNA levels in the plasma has not yet to be demonstrated. Further research in this direction will help identify novel biomarkers for monitoring patients with neuropathic pain. However, more in‐depth functional and mechanistic investigations on pain‐related circRNAs are warranted. It is hopeful that, with more translational studies, circRNAs will one day be utilized for the clinical management of patients with neuropathic pain.The authors declare that they have no competing interests.DRX, XXM, CS, JL H, CLZ: drafted and wrote the manuscript. MTVC and WKKW: revised the manuscript. DRX and CLZ: participated in the design of the review. All authors read and approved the final manuscript.


Cytauxzoon felis is a life‐threatening protozoan disease of cats. Identification of schizont‐laden macrophages is a point‐of‐care diagnostic test for acute cytauxzoonosis.The primary objective determined cytologic agreement between sample types to diagnose acute cytauxzoonosis. The secondary objective evaluated novices' ability to identify cytauxzoon organisms in blood films and tissue aspirates.Thirty‐eight cats with suspected acute cytauxzoonosis and 5 controls examined postmortem.Cases were prospectively submitted and collected. Blood film, lymph node, and splenic aspirates were blindly reviewed for sample quality, presence of schizont‐laden macrophages, and agreement between sample types. A subset of cases and controls were evaluated by 12 blinded novice observers to determine sensitivity and specificity for identifying organisms in various sample types.P = .03) or blood film . Novice observers were more likely to agree with experts when identifying schizont‐laden macrophages in splenic aspirates  versus lymph node aspirates  or blood films .Acute cytauxzoonosis diagnosis was made on at least 1 sample type in 28/38 cats. Schizont‐laden macrophages were seen on 33% (10/30) of blood films, 56% (19/34) lymph node aspirates, 77% (26/34) splenic aspirates. Schizont‐laden macrophages were more likely seen on splenic than lymph node aspirates (McNemar's, Schizont‐laden macrophages are most frequently identified in spleen, even by novice observers. If the diagnosis of acute cytauxzoonosis cannot be confirmed via blood film, then splenic, followed by peripheral lymph node aspirates can be considered. BCPboard certified clinical pathologisthpfhigh powered fieldIMRinternal medicine residentlpflow powered field1Cytauxzoon felis that is transmitted by the Amblyomma americanum tick.Lynx rufus), are the natural reservoirs and domestic cats that survive infection could become persistently infected.Cytauxzoonosis is an acute, life‐threatening illness in domestic cats caused by the apicomplexan protozoan parasite Acute cytauxzoonosis occurs when schizont‐laden macrophages become distended and lodge in small blood vessels, resulting in multiorgan failure and often death of the infected cat. Clinical signs of schizogony include high fever, lethargy, inappetence, icterus, peripheral lymphadenomegaly, and hepatosplenomegaly. As the disease progresses, schizont‐laden macrophages rupture and release merozoites that are taken up by red blood cells through endocytosis. Although anemia often accompanies initial erythrocyte infection, cats that survive the schizogenous stage of infection often maintain low levels of intraerythrocytic merozoites (piroplasms) indefinitely without apparent illness and become persistently infected.A commercial PCR test, sensitive and specific for disease detection preceding cytologically detectable parasitemia, is available. Previous PCR tests detecting 18S ribosomal RNA did not differentiate between cats with acute cytauxzoonosis and the chronic carrier state.Delay in diagnosis and treatment of cytauxzoonosis can prove fatal.Unlike the limitations of both the PCR test and the identification of piroplasms, identification of the schizont‐laden macrophages would provide a practitioner an immediate diagnosis of acute cytauxzoonosis allowing them to feel more confident when talking with the owner regarding the potential overall cost of therapy and prognosis. The primary objective of this study was to determine the agreement between the evaluation of blood films, splenic aspirate, and lymph node aspirate samples to support a diagnosis of acute cytauxzoonosis. The secondary objective was to evaluate a novice's ability, when compared to clinicians with more advanced training in clinical pathology, to identify piroplasms or schizont‐laden macrophages in blood films and tissue aspirates collected from domestic cats with a high clinical suspicion for acute cytauxzoonosis.2C. felis research were contacted to participate in this study. Clinicians were asked to collect 3 sample types from cats suspected to have acute cytauxzoonosis and that died or were euthanized on the day of presentation. Samples requested included 2 blood films, aspirates from any lymph node , and splenic aspirates. Written informed client consent was granted before collection of tissue and blood samples. As disseminated intravascular coagulation (DIC) is common in cats with cytauxzoonosis, we requested only postmortem samples to avoid iatrogenic bleeding from tissue aspirates.General practitioners in Oklahoma, Missouri, Tennessee, and Arkansas who previously participated in In order to address the primary objective—to determine the agreement between blood film evaluation, splenic aspirate, and lymph node aspirate for diagnosis of acute cytauxzoonosis—all slides were evaluated by 2 observers with more advanced training in clinical pathology and interobserver agreement was calculated (see statistics section below). The 2 observers were a board certified clinical pathologist (BCP) and a third‐year internal medicine resident (IMR). There was no time limit for scanning the slides for any observer. Observers were blinded to each other's results. Blood films were evaluated for all stages of disease . The monolayer was defined as an area of the blood films in which the red blood cells were evenly distributed with infrequent touching and no large spaces without cells. The monolayer was evaluated at high power using the 100× objective lens for the presence of piroplasms. If piroplasms were visualized, an average number per high powered field (hpf) was determined by counting the number of organisms/hpf for 10 fields. Blood films were evaluated for schizont‐laden macrophages at low power using the 10× objective lens. The entire slide was evaluated for schizont‐laden macrophages. Slides prepared from the splenic and lymph node aspirates were evaluated for cellularity and the severity of peripheral blood contamination; a scoring system Table  was adapAfter completion of individual review of samples by BCP and IMR for the purpose of determining interobserver agreement, samples for which disagreement was present were reviewed by both observers simultaneously to reach a consensus. If a consensus could not be reached a third expert, also a boarded clinical pathologist, evaluated the slide independently. Interobserver agreement was determined by comparison of identification of all stages of disease; however, the diagnosis of acute cytauxzoonosis was based on the presence of schizont‐laden macrophages in any tissue type. Expert observer consensus was then used to make comparisons between sample types for the detection of schizont‐laden macrophages.C. felis negative). The 10 cases were then organized using a random number generator for each novice. Novices were given a brief training on general cytology techniques and organism identification by BCP. Along with the training, the novices were provided with positive control cytology samples from each sample site and textbook resources for reference. No time limit for evaluation of the samples was implemented for the novices. Novice agreements (to identify piroplasms and schizont‐laden macrophage in blood film and schizont‐laden macrophages only in tissue aspirates), sensitivity, and specificity in comparison to the consensus data of BCP and IMR were determined.To address the secondary aim of this study—to evaluate a novice's ability to identify schizont‐laden macrophages as well as piroplasms—12 additional novice observers, all veterinary professional students, reviewed 10 complete slide sets for the presence or absence of organisms. Five cases were selected randomly from all study cases, regardless of presence or absence of schizont‐laden macrophages, and 5 cases were selected from historical submissions to the University of Minnesota's clinical pathology service to serve as negative controls. Negative control cytology and blood films came from cats residing outside of endemic regions that had no travel history  and the expert consensus data was used as the gold standard for comparison. The 95% confidence intervals were calculated for each of those proportions using a bootstrap method. R version 3.6.1 was used for all computations.To assess agreement of the 2 experienced readers, both overall agreement and Cohen's kappa were computed. A 33.1Diagnostic samples were submitted from 38 cats ranging in age from 8 weeks to 15 years. Though cat signalment data was not available for all submissions, both male (5 neutered and 2 intact) and female (1 spayed and 3 intact) cats were included. Submissions originated from Missouri (n = 15), Tennessee (5), Oklahoma (6), Arkansas (3), or unknown (9). The 3 requested sample types  were not available for all submissions. Complete sample sets  were available for 26 cases. A total of 99 total cytology sites  submitted for evaluation Table .3.2C. felis for all sample sites  evaluated was 94.9% (kappa = .88). IMR and BCP disagreed on 5/98 samples, including 2 splenic aspirates, 2 lymph node aspirates, and 1 blood film. There was disagreement regarding the presence of a single organism on 1 blood film; IMR observed the single piroplasm that BCP did not. A second expert (BCP), who was not blinded to the previous results, was consulted to reach a consensus and a single piroplasm was identified on the entire blood film. For the remainder of samples in which there was disagreement BCP saw the organisms and IMR did not. When those samples were reviewed together a consensus was reached that the organism was truly present. When individual sample types were compared, the overall agreement between IMR and BCP for the blood film, lymph node aspirates, and splenic aspirates was 96.7%, 94.1%, and 94.1%, respectively , the overall agreement on the presence or absence of 3.3P = .04); increased nucleated cells on a slide increased the likelihood of observing schizont‐laden macrophages in splenic aspirates. In other words, the more cellular the aspirate the more likely schizont‐laden macrophages were observed.Using the consensus data, a Kendall's rank correlation coefficient was used to determine if the degree of cellularity and blood contamination correlated with the presence of organism on the slide. Only the degree of cellularity of the splenic samples was statistically significant , 19/34 (56%) lymph node aspirates and 26/34 (77%) splenic aspirates. On all blood films in which schizont‐laden macrophages were observed, piroplasms were also observed. For 8 of these blood films in which the diagnosis of acute cytauxzoonosis could be made, both lymph node and splenic aspirates were also available; schizont‐laden macrophages were present on both lymph node and splenic aspirates of 6 cases. Of the 2 remaining cases schizont‐laden macrophages were present on the splenic aspirates but not the lymph node aspirates.Of the 20 blood films in which the diagnosis of acute cytauxzoonosis was not able to be made, 18 had both lymph node and splenic aspirates available for review. In 14 of these 18 the diagnosis of acute cytauxzoonosis could be made. Schizont‐laden macrophages were present in both lymph node and splenic aspirates in 11/18 cases and present in splenic aspirates only in 3/18. The remaining 4/18 cases with complete slide sets were negative for schizonts‐laden macrophages in all samples. Of the 2 cases in which there were not complete data sets, a lymph node aspirate (n = 1) was submitted and was negative for shizont‐laden macrophages, or neither were submitted (n = 1).3.5P = .06)  Table .P = .03).When lymph nodes and splenic aspirates were compared, 6 cats had schizont‐laden macrophages that were observed in the splenic aspirate but not the lymph node. There were no instances in which there were schizont‐laden macrophages detected in the lymph node and not in the spleen. Schizont‐laden macrophages were more likely to be found in splenic aspirates than lymph node aspirates .Finally, when splenic aspirates and blood films were compared for schizont‐laden macrophages there were 4 cats in which schizont‐laden macrophages were absent in both blood and spleen and there were 8 cats in which they were present in both sample sources. There were 14 cases in which the schizont‐laden macrophages were present in the spleen and absent in the blood film and there were no cats in which schizont‐laden macrophages were present on the blood film and absent in splenic aspirates. Schizont‐laden macrophages were more likely to be found in splenic aspirates than on blood film review  was higher than their sensitivity to detect schizont‐laden macrophages in the blood (41.7%). The sensitivity of the novice observers identifying the presence of schizont‐laden macrophages in lymph node aspirates was 52.8% and 77.1% in splenic aspirates. Overall, the novices' were more likely to identify schizont‐laden macrophages in the spleen compared to any other sample type.4Acute cytauxzoonosis is the result of the schizogenous replication of the parasite in mononuclear cells and therefore illness can occur before piroplasms are identifiable within the red blood cells.C. felis organisms in any sample precludes us from determining sensitivity and specificity of sample types for diagnosis of acute cytauxzoonosis. Schizont‐laden macrophages were seen on 33 (10/30) of all available blood films. The identification of these schizont‐laden macrophages on the blood film allows for a rapid diagnosis of acute cytauxzoonosis and allows the practitioner to forgo potentially more invasive diagnostics such as splenic or lymph node aspirates. When individual sample sources were compared using McNemar's test, schizont‐laden macrophages were more likely to be found in splenic aspirates than lymph node aspirates. In cases in which schizont‐laden macrophages cannot be identified using blood film alone, based on these results, splenic aspirates followed by peripheral lymph node aspirates could be used by practitioners to identify the schizont‐laden macrophages.The first objective of this study was to determine the agreement between various sample sources; blood film, splenic and lymph node aspirates for the diagnosis of acute cytauxzoonosis. The lack of confirmatory molecular testing and inclusion of cats without cytologic evidence of Antemortem aspiration of the spleen is still an uncommon procedure in most primary care clinics but is becoming a more common practice as the cost of ultrasound units continues to decrease, and teleradiology and mobile diagnostic imaging services become more widely available in both urban and rural settings. This change will likely result in an increased comfort level and frequency of acquiring splenic aspirates in cats with suspected acute cytauxzoonosis. It should be noted that acquisition of splenic aspirates is not without risk. Disseminated intravascular coagulation is common in cats with naturally occurring cytauxzoonosis.The secondary aim of the study was to evaluate a novice's ability to identify piroplasms or schizont‐laden macrophages in blood films and tissue aspirates from cats with high index of suspicion for acute cytauxzoonosis. The novice observers identified the organisms with relatively high frequency and relatively high agreement with the experts. The specificity of the novice's ability to identify schizont‐laden macrophages on blood film and in lymph nodes and splenic aspirates was slightly higher than their ability to identify piroplasms on blood films. This was not a surprising finding, as piroplasms are significantly smaller (1‐2 μm) in size than the intact schizont‐laden macrophages  observed in the splenic and lymph node aspirates. Piroplasms can be easily mistaken for stain precipitant, Howell‐Jolly bodies, artifacts, or potentially overlooked with low parasitemia. The sensitivity of the novices' identification of schizont‐laden macrophages was highest in splenic aspirates compared to lymph node and blood films. When schizont‐laden macrophages were identified on blood film, they were typically found along the feathered edge and were sometimes broken, making them more challenging to diagnose. Additional training on identification of schizont‐laden macrophages in blood films and the knowledge that they can be identified in some cases of acute cytauxzoonosis could be helpful to diagnose these cases with the least invasive means necessary.C. felis by a combination of PCR, cytology, and postmortem examination was not performed. Samples were collected based upon a strong clinical suspicion for acute cytauxzoonosis by general practitioners in endemic regions. However, cytologic evaluation failed to identify C. felis organisms in one quarter (10/38) of the cats in this study, and so it is possible that these cats were suffering from diseases other than acute cytauxzoonosis. The lack of molecular diagnostic and postmortem confirmation of infection in all patients precluded our ability to determine the sensitivity and specificity of the various sample sources to rapidly diagnose acute cytauxzoonosis. Future studies evaluating cytologic diagnosis of acute cytauxzoonosis would benefit from concurrent PCR testing of collected blood or tissues, as well as postmortem histologic evaluation of formalin‐fixed tissues, including spleen, lymph node, liver, and lung.There are several limitations of this small study. First, an important limitation to this study is that definitive diagnosis of A second limitation of the study is that although acute cytauxzoonosis is a systemic disease affecting multiple organs, clinicians were not asked to collect samples from visceral organs aside from the spleen, nor were they asked to collect samples from a consistent anatomic lymph node . In this study, 12 of the 38 cats lacked sampling of all 3 evaluated tissues. For future studies and in clinical work up of cats suspected to have acute cytauxzoonosis, we recommend sampling and evaluation of blood, spleen, and lymph node, as well as liver, if possible. This approach would allow for the most thorough cytologic assessment. Future studies could also investigate whether the anatomical location of the lymph node(s) sampled or the degree of lymphadenomegaly impacts the likelihood of seeing schizont‐laden macrophages in aspirates.A third limitation was that the sample size was relatively low and might have been inadequate to identify differences in the presence of schizont‐laden macrophages between blood smears and lymph node aspirates. Additionally, the novice observers only evaluated a small number of cases. Further studies are needed in which novice observers evaluate a larger number of cases to determine sensitivity and specificity for identification of schizont‐laden macrophages in various sample sources.In conclusion, if schizont‐laden macrophages are not identified in blood films, splenic then lymph node aspiration should be considered as the next point‐of‐care diagnostic step for the diagnosis of acute cytauxzoonosis. Novice observers were able to identify schizont‐laden macrophages most readily in splenic aspirates, followed by lymph node aspirates and blood films.Authors declare no conflict of interest.Authors declare no off‐label use of antimicrobials.Informed consent was obtained from the owner or legal custodian of all animals included of all animal(s) described in this work for the procedure(s) undertaken.Authors declare human ethics approval was not needed for this study.Table S1 Scoring system for cellularity and blood contamination of splenic and lymph node aspirates.Click here for additional data file.Table S2 Summary of the BCP and IMR consensus data on the presence or absence of organisms in various sample types.Click here for additional data file.


Breath holding divers display extraordinary voluntary control over involuntary reactions during apneic episodes. After an initial easy phase to the breath hold, this voluntary control is applied against the increasing involuntary effort to inspire. We quantified an electromyographic (EMG) signal associated with respiratory movements derived from broad bandpass ECG recordings taken from experienced breath holding divers during prolonged dry breath holds. We sought to define their relationship to involuntary respiratory movements and compare these signals with what is known to occur in obstructive sleep apnea (OSA) and epileptic seizures. ECG and inductance plethysmography records from 14 competitive apneists  were analyzed. ECG records were analyzed for intervals and the EMG signal was extracted from a re‐filtered version of the original broad bandpass signal and ultimately enveloped with a Hilbert transform. EMG burst magnitude, quantified as an area measure, increased over the course of the struggle phase, correlated with inductance plethysmography measures, and corresponded to significant variance in heart rate variability. We conclude that an EMG signal extracted from the ECG can complement plethysmography during breath holds and may help quantify involuntary effort, as reported previously for obstructive sleep apnea. Further, given the resemblance between cardiac and respiratory features of the breath hold struggle phase to obstructive apnea that can occur during sleep or in association with epileptic seizure activity, the struggle phase may be a useful simulation of obstructive apnea for controlled experimentation that can help clarify aspects of acute and chronic apnea‐associated physiology. Breath holding divers display extraordinary voluntary control over involuntary reactions during apneic episodes. We show that an EMG signal extracted from the ECG can complement plethysmography as a measure of breath hold respiratory function and may help quantify involuntary effort, as reported previously for obstructive sleep apnea. Further, given the resemblance between cardiac and respiratory features of the breath hold struggle phase to obstructive apnea that can occur during sleep or in association with epileptic seizure activity, the struggle phase may be a useful simulation of obstructive apnea for controlled experimentation that can help clarify aspects of acute and chronic apnea‐associated physiology. The breath hold, which can last many minutes, is comprised of an early quiet or easy phase and a later struggle phase. The prolonged breath hold is one of the best examples of voluntary control over involuntary physiology and breath hold phases have been studied to identify contributors to the diver's voluntary or involuntary decision to break the breath hold . Severe OSA is defined by ≥30 apneic or hypopneic events per hour AASM,  and indiAccordingly, we sought to quantify the EMG events occurring in the struggle phase of a breath hold in elite apneists, to define their relationship to involuntary respiratory movements, and compare these signals with what is known to occur in OSA and epileptic seizures. Validation of the inspiratory effort‐associated EMG signal under different clinical apneic conditions also establishes its use in settings where plethysmography is not available.22.1Recordings from fourteen competitive apneists  were analyzed. Average years of competition was 4 years .Divers performed breath holds as part of a previous IRB‐approved study with a separate a priori question where subject specifications are detailed . A low‐pass filter was applied with auto adjusted transition width, which was sampled at 1 kHz using LabChart7 Pro . Fast Fourier transform analysis of recorded signals showed that high frequency components existed in the recorded data out to nearly 500 Hz. Inductance plethysmography was performed using a calibrated custom‐built strain gauge strap placed around the midline of the chest and integrated into LabChart7 Pro.2.3EMG associated with respiratory effort has been shown to mix with the ECG or EEG , which eliminated essentially all of the ECG signal in most cases. The resulting filtered signal was squared as a method to full‐wave rectify the signal and enhance the signal‐to‐noise ratio, smoothed , and then enveloped with a Hilbert transform  Microsoft Excel , and Graph Pad Prism software . Means are plotted with 95% confidence intervals and comparisons across dive phases were made with mixed effects or repeated measures analyses of variance with Dunnett's multiple comparisons tests for post hoc corrections. Statistical significance was considered for 33.1A resting breathing recording was taken from each diver .A single breath hold from each diver was studied. During a voluntary breath hold, the diver can voluntarily suppress respiratory movements with relatively less effort (the quiet phase), but eventually, strong involuntary attempts to draw a breath must be met with additional voluntary effort to keep from breathing (the struggle phase)  and obstructive apnea (which resembles the struggle phase of a breath hold).p = 0.0050; p = 0.0006 for rest versus breath hold after post hoc correction. For SDNN  that characterize the struggle phase of the breath hold. Upon quantification with Hilbert transforms, we found that there was a magnitude increase in EMG bursts over the course of the struggle phase that correlated better with the slope of the inductance plethysmogram inspiratory phase than the peak‐to‐peak plethysmogram amplitudes. An increase in heart rate variability and a shift of RR interval minima from expiration at rest  to inspiration during the struggle phase  are similar to the features of obstructive apnea , Formal Analysis, Writing––Original Draft, Writing Review and Editing. Anthony Bain ‐ Conceptualization, Methodology (collecting data from divers), Formal Analysis, Writing—Original Draft, Writing Review and Editing.