Europe has a well‐established network of breeding bird monitoring that is used to produce supranational indices of population trends for many species. However, a comparison of breeding bird censuses with other methods may be beneficial to confirm the validity of such indices. The aim of this study was to assess the value of standardized capture data of migratory birds at migration bottlenecks as an indicator of the effective breeding populations. One limitation to this method is that several populations are co‐occurring at these bottlenecks and their catchment areas need to be clearly identified to allow extrapolation of population indices. Here, we used standardized trends in capture numbers of 30 species on the island of Ponza, a migration bottleneck in the central Mediterranean, and compared them to population trends estimated in the putative catchment breeding areas between 2005 and 2016. The catchment areas were identified through the analysis of ring recoveries during the breeding season of birds passing through Ponza. Our results show an agreement between the population trends observed on Ponza and those in the breeding areas in 15 out of 30 species. The correlations were strongest in species with a more robust definition of the catchment areas, that is, species with more than 10 recoveries, and for which the recoveries were most likely of breeding birds. The main reason for disagreement between the two indices in the remaining species might be related to different intensity of sampling in different areas. This issue can be solved by further developing monitoring projects in underrepresented countries, as well as by intensifying monitoring through ringing, both in the breeding grounds and at migration bottlenecks. These results show that spring migration monitoring at bottlenecks has the potential to provide a valuable complement and an independent control of breeding bird surveys, allowing raising early warnings of population declines and contributing to their conservation. We compared trends of 30 migrant Passerine bird species on passage through a migration hotspot with population trends in their breeding areas. We found good agreement between trends in half of the species. This shows how integrating different count methods could benefit monitoring and conservation. The PECBMS collates national indices obtained from breeding bird censuses to produce supranational and continental trend indices for a large number of common species  located about 50 km off the Western coast of Italy , where we have been monitoring spring migration through capture and ringing since 2002 (www.inanellamentoponza.it). Ponza is located along one of the main Mediterranean migratory routes and attracts large numbers of African‐European migratory landbirds during spring migration  and model  constant throughout the entire study period. Vegetation height was kept constant throughout the entire study period. After being captured, birds were ringed and measured using standard procedures  index (hereafter “Ponza index”), which reflects the year trend proportional to the bird density passing through Ponza. To account for the unbalanced sampling design between years and days (expressed as Julian day) and remove these effects from the nominal CPUE, we used generalized additive models . We extended the database to a longer period of time and to the neighboring islands to ensure a larger sample of recovery data, assuming that the populations of origin would not differ within this small range. We created a database of the Pontine Islands recoveries including all birds ringed on the islands and found elsewhere and foreign birds found on the islands as an ESRI shapefile  and included in the EPE National Database of Italian ISPRA (Phylloscopus trochilus) and Barn Swallow (Hirundo rustica), with sufficient recoveries in the period June‐July , we compared the result obtained with this subset of data with the one obtained with a larger sample  were assigned to the PECBMS region “Central‐East Europe.”We analyzed the geographical distribution of the recoveries at the species level using Directional Distribution Analysis . For species for which the index of one or more PECBMS regions was not available, the weight of the missing region was redistributed between the other regions maintaining the same proportion between the regions with data. Using the weight matrix, an area‐weighted PECBMS index was estimated for every species considered in the analysis. The area‐weighted PECBMS index was then centered subtracting the mean and dividing it by its standard deviation and finally smoothed to extract the long‐term trend. For the Ponza index, we performed LOESS smoothing with the geom_smooth function from the ggplot package in R to extract the long‐term trend and make the two indices comparable. For each species, we analyzed the slopes of the linear correlation between the area‐weighted PECBMS index and the Ponza index in the years between 2005 and 2016, to test the hypothesis that the Ponza index estimated during spring migration is a measure of the size of the bird population estimated by PECBMS in the same year.Species population indices in the PECBMS regions were obtained from the calculation of European species trends and indices for the period 2005–2016 (33.1Sylvia borin) (16) and Whitethroats (Sylvia communis) (13) , followed by “North Europe” 28%) and “West Europe” (24%) (Table 8% and “W3) Table . For 14 3) Table .3.2Phoenicurus ochruros), the correlation was nearly significant with a positive slope. In three species , there was a significant correlation with a negative slope, that is, an increase of birds passing through Ponza Island corresponded to a decrease in their breeding areas in Europe.Area‐weighted PECBMS index and Ponza index for 30 species are shown in Figure When considering only recoveries from June‐July in Willow Warblers and Barn Swallows, our result did not change substantially. The correlation between the two indices remained significantly positive in the Barn Swallow, while there was no significant correlation in the Willow Warbler Table . For bot4https://pecbms.info/country/). Monitoring and conservation of migratory birds in these countries might benefit from trend estimates based on trapping at spring migration bottlenecks, like the ones from small islands in the Mediterranean Sea. Our study identified the catchment areas of birds captured on the island of Ponza to be centered mostly in Central‐Eastern Europe. There was good agreement between population trends in Ponza and those reported from the breeding areas in half of the species. For these species, capture numbers on Ponza can be an ideal complement to breeding bird censuses.Estimating population trends of migratory birds in large areas is challenging and requires a huge effort in terms of observers and time. Several European countries have well‐established protocols for monitoring their breeding bird populations, while others may lack sufficient manpower or funding. This is especially true for countries in the East of Europe , the value of these indices increases considerably when calculated over a long time range.Erithacus rubecula and Common Redstart Phoenicurus phoenicurus) show a mismatch in their population indices between Ponza and the breeding grounds. However, the effects of shifted phenology on breeding bird estimates might become more apparent over a longer study period.While the good association between indices might support the use of either one of the methods for assessing population changes, it is important to note that trends in Ponza did not always match the trends in the breeding areas. However, we found a good match in five of the six species with more than 10 recoveries in the last 30 years. The only exception was the Willow Warbler, for which we did not find a matching trend despite it being the species with most recoveries. One possibility is that some of the recoveries might have been of birds that were still migrating. The Willow Warbler is the species with the northernmost centroid, and Northern populations start breeding later than May 15th, and possibly are on migration again in August  could provide useful data without relying on recaptures, but such a system has not been implemented at a large scale in Europe yet. To increase the number of ring recoveries, the only solution is to increase the effort of bird ringing, especially on the breeding grounds. Standardized projects such as the Constant Effort Site (CES) program . One species was fluctuating without showing a definite trend (Wryneck Jynx torquilla), and 8 species showed decreasing trends . While a discussion of the reasons for these increases or declines is beyond the scope of this study, our results confirm that conservation should be addressed with priority to farmland species and long‐distance migrants ; Writing‐original draft (lead); Writing‐review & editing . Massimiliano Cardinale: Conceptualization ; Data curation (lead); Formal analysis (lead); Investigation (lead); Methodology (lead); Project administration ; Writing‐original draft (supporting); Writing‐review & editing . Andrea Favaretto: Formal analysis (supporting); Visualization (lead); Writing‐original draft (supporting); Writing‐review & editing . Petr Voříšek: Investigation (supporting); Methodology (supporting); Resources (lead); Writing‐review & editing . Fernando Spina: Conceptualization (supporting); Data curation (supporting); Methodology (supporting); Project administration (supporting); Writing‐review & editing . Francesco Maoret: Formal analysis (supporting); Writing‐review & editing . Andrea Ferri: Investigation (supporting); Writing‐review & editing . Sara Riello: Investigation (supporting); Writing‐review & editing . Leonida Fusani: Conceptualization (supporting); Funding acquisition (lead); Project administration (supporting); Supervision (lead); Writing‐review & editing .Fig S1Click here for additional data file.Table S1‐S3Click here for additional data file.Supplementary MaterialClick here for additional data file.


The presence or absence of human leukocyte antigen (HLA) antibodies, especially the strength of donor‐specific HLA antibodies (DSAs), has important roles in clinical evaluation and diagnostic decision‐making for solid‐organ transplantation. Dilution patterns help to give a new sight of HLA epitopes. “Epitope matching” is likely to lower the risk of developing DSA and increase the likelihood of matching a compatible donor.We collected data evaluating HLA antibodies with a titration study using mean fluorescence intensity.Diluting the serum of recipients can reduce potential inhibitory effects, accurately evaluate the intensity of donor‐specific HLA antibodies, and guide surgeons to take or not take intervention measures. Dilution patterns also help to give a new sight of HLA epitopes.We believe that from the viewpoint of HLA antibodies, the dilution model can provide new tools and insights for the study of HLA epitopes. This feature may increase the risk of antibody‐mediated rejection and lose valuable opportunities for transplantation, affect graft survival, and even lead to graft failure. A history of sensitization before renal transplantation, including a history of blood transfusion, pregnancy, secondary transplantation, and other risk factors, may lead to the production of donor‐specific HLA antibodies (DSAs) in the body.www.epitopes.net/) has become increasingly popular. HLAMatchmaker identifies the number of differences in amino acid sequences between the donor and recipient HLA antigens, which are called “eplets”. HLAMatchmaker has been used in several studies to calculate “eplet loads” . Tambur and colleagues clearly showed that determination of the eplet load was a powerful tool for risk stratification of patients to understand their likelihood of producing HLA antibodies and to individualize immunosuppression and post‐transplant monitoring.In recent years, computer software called “HLAMatchmaker” (The HLA single‐antigen microbead (SAB) method based on the Luminex platform is the most sensitive method for the detection and semi‐quantitative identification of anti‐HLA antibodies and evaluation of immune risk of organ transplantation.www.epitopes.net/) epitope registration system to screen these mismatched epitopes and recipient HLA alleles corresponding to mismatched epitopes is helpful for clinical selection of suitable donors.This report is aimed at studying HLA epitopes. The specific antibody epitopes can be better identified by the true strength of the antibodies obtained by continuous dilution. The use of “HLAMatchmaker” . Testing of HLA antibody was undertaken using a LifeCodes® Single Antigen kit (Immucor). Both methodologies followed manufacturer guidelines, as described previously.Human leukocyte antigen typing was undertaken using a LABTypeThe MFI of most of the SABs of our patient was >15 000, and the pattern of the cross‐reaction group was disordered , 149A + 150V + 151H and 158V. The epitope symbol is 44KM3, which indicates the possibility of three different epitopes. These epitopes can be predicted as recognition sites of specific antibodies and have been verified by experiments with informative specific antibodies. Each HLA antigen has its own unique combination of epitopes, but at the same time, many of these epitopes are shared with other HLA antigens. Through interpretation of the cross‐reaction group and antibody‐dilution model, the antigenic epitopes described in Table Figure Figure An almost identical dilution pattern indicated that these sites had a common epitope.Figure 3In recent years, explaining what contributes to an HLA B‐cell epitope has proved to be limited and inadequate, as has understanding of the immunogenicity and antigenicity of mismatched HLA epitopes. The concept of epitopes on different HLA antigens has been extended to structural eplets. These epitopes themselves are determined by a small number of adjacent amino acids.We provided data suggesting that MFI values for neat antibody do not always depict antibody strength accurately. A dilution study provides more dynamic monitoring of antibody behavior, and it is a more suitable method for measurement of antibody strength. A dilution method can better describe the antigen‐antibody reaction. Titration studies, on the one hand, show information about current antibody‐naming groups and, on the other hand, provide a tool to decipher antibody reactivity. According to the definition of a dilution pattern, antibodies can provide clues about shared antibody targets, thereby providing clues to physiologic epitopes. Application of an antibody titer for detection of HLA antibodies undoubtedly increases the economic burden of patients. According to the experience of our center, we can choose the original serum plus two additional dilutions  to obtain the trend of the antibody reaction. Dilution methods are recommended to detect HLA antibodies in highly sensitized patients undergoing a secondary transplant.Kosmoliaptsis and colleagues Epitope matching minimizes the risk of developing de novo HLA‐DSAs after transplantation. Therefore, solving the mystery of HLA B‐cell epitopes, understanding HLA epitopes, and deciphering the factors that determine the antigenicity and immunogenicity of epitopes may elicit substantial changes to solid‐organ transplantation. We believe that from the viewpoint of HLA antibodies, the dilution model can provide new tools and insights for the study of HLA epitopes.Written informed consent was obtained from the patient to use his data. Approval from the ethics board of Tianjin First Central Hospital was not required.


The complicated interplay of plant–pathogen interactions occurs on multiple levels as pathogens evolve to constantly evade the immune responses of their hosts. Many economically important crops fall victim to filamentous pathogens that produce small proteins called effectors to manipulate the host and aid infection/colonization. Understanding the effector repertoires of pathogens is facilitating an increased understanding of the molecular mechanisms underlying virulence as well as guiding the development of disease control strategies. The purpose of this review is to give a chronological perspective on the evolution of the methodologies used in effector discovery from physical isolation and in silico predictions, to functional characterization of the effectors of filamentous plant pathogens and identification of their host targets. The chronological progression of methodologies used in effector discovery; from physical isolation and in silico predictions, to functional characterization and host target identification. The detection of the Avr effector by the host carrying the cognate R gene can induce a strong immune response in the plant and inhibit C. fulvum colonization , features that become important in many subsequent effector identification stories  , cinnamomin (P. cinnamomi), and capsicein (P. capsici)  was established following the publication of a white paper ‐mediated defences in planta  experiments , which contains half of all cloned M. oryzae Avrs despite sharing less than 25% sequence identity  and revealed a novel β‐sandwich fold , TMHMM (www.cbs.dtu.dk/services/TMHMM/), and PredGPI (gpcr.biocomp.unibo.it/predgpi/pred.htm), which use neural networks or hidden Markov modelling to recognize motifs within protein sequences associated with these features . This genomic landscape and the presence of TEs serve to drive adaptive evolution  regions of plasticity in the genome  and quantitative trait locus (QTL) mapping can identify loci associated with heritable phenotypic variation, such as virulence, thereby complementing techniques to identify and clone Avr effectors recognized by known host resistance proteins  is another method to disrupt genes and is widely used in plant transformations. ATMT was first used in fungi in budding yeast in 1995 and then the technique was adapted for use in filamentous fungi, including M. oryzae  appears to be the dominant DNA repair pathway over homologous recombination , which is infiltrated with transformed Agrobacterium that delivers the effector gene expressed from an inducible promoter into the plant cell for transient protein production  triggered by detection of the candidate , in rice protoplasts  or luciferase  effectors ATR1 and ATR13 to the N‐terminal secretion‐translocation signals of the P. syringae effectors AvrRpm1 and AvrRps4, the effectors could be secreted into Arabidopsis plant cells and contribute to pathogen virulence. Since then, the T3SS has been used to functionally characterize candidate effectors from multiple oomycetes, including P. infestans and Hyaloperonospora arabidopsidis  and bean rust (Uromyces appendiculatus), this system is rarely used for fungal effector characterization and has limited success on cereals  derived from the tomato bacterial speck pathogen , et al.  to delivArabidopsis that expressed candidate poplar rust fungus (M. larici‐populina) effectors were inoculated with the oomycete pathogen H. arabidopsidis. Eleven of 16 effectors tested supported greater sporulation of this native Arabidopsis pathogen, suggesting that the effectors had the capacity to interfere with processes in a nonhost plant to favour pathogenesis   was first verified as a tool for protein expression when used to overexpress the luciferase reporter gene in protoplast cells and later to express green fluorescent protein (GFP) in planta  has been adapted for use in VOX systems in cereals  may be found. Traditionally in situ hybridization assays were done where antibodies were raised against the effector or an added epitope tag and detected using transmission electron microscopy (TEM). Translocation of fungal effectors into the host cell was first shown using an immunocytochemical approach in rusts. The gold‐ and fluorescence‐labelling of four independently raised antibodies to the RTP1p protein in C. fulvum effectors Avr2, Avr4, Avr9, and Ecp6 were directly isolated from the apoplastic fluid, whereas the P. infestans protease inhibitor EPIC1 was isolated from the apoplast after antibodies were raised  fusion proteins in planta the effectors were shown to accumulate within plant cell nuclei , prior to delivery into the cytoplasm, whereas tagged apoplastic effectors localized to the invasion hyphae  peptide Avitag  of direct contact. The former is very challenging because the effector sequences do not give many clues as to their function(s).5.1et al., Unbiased “forward” screening to find protein–protein interactions (PPI) is a common technique used in many aspects of molecular biology. The yeast two‐hybrid system (Y2H), first developed 30 years ago, allows for the large‐scale screening of cDNA libraries derived from pathogen‐infected plants for effector target identification  effectors were tagged with GFP and expressed in N. benthamiana, five were found to specifically interact with plant proteins by pull‐down assays using anti‐GFP followed by protein purification . Co‐immunoprecipitation is used to screen effector interactors in heterologous systems. When 20 candidate poplar rust fungus  reveals molecular details of the recognition event that leads to HR‐induced cell death   domain of the rice NLR RGA5 was required for effector binding  gene is itself sufficient to induce tan spot symptoms  to molecular plant pathologists as well as the wider life sciences communities.www.phi‐base.org) is a manually curated database comprising over 6,780 genes from 268 pathogens of over 210 hosts (September 2019), of which 60% are plants (Urban et al., Publicly available repositories of curated data regarding proteins with confirmed roles in pathogenicity and virulence are a fundamental tool for effector study. The Pathogen–Host Interactions database (PHI‐base, 8“Would you tell me, please, which way I ought to go from here?”“That depends a good deal on where you want to get to,” said the Cat.Alice in WonderlandLewis Carroll, M. oryzae, we have resolved three‐dimensional protein structures and know interacting surfaces of multiple effectors and their interactors. In other cases, important crop pathogens such as F. graminearum and the newly emerging pathogens Ramularia collo‐cygni and Corynespora cassiicola, although several hundred candidate effectors have been predicted, each lacks functional characterization (McGrann et al., et al., Effectors are the mysterious molecular tools evolved and used by plant pathogens in multiple ways. Effector studies are of vital importance in addressing the global food security challenge, yet the explosion in research efforts aimed at understanding effector biology over the last few decades has left us with a dichotomy in our knowledge. Due to early focus on a small number of pathosystems, whether due to experimental convenience or the economic impact of the disease, for some pathogens, such as et al., The arrival of full genome sequencing almost two decades ago has been a double‐edged sword. Bioinformatic pipelines and the development of prediction software has sped up the refinement of putative effectors whilst simultaneously highlighting the vastness of the gene repertoires to be investigated. For effector characterization, the future efficiency not only depends on the development of ultrahigh‐throughput functional assays but also their use in combination with lower‐throughput novel and well‐established techniques such as QTL mapping and GWAS (Plissonneau Agrobacterium‐mediated transient expression in planta. Armed with only an annotated genome, even understudied conifer‐infecting fungal pathogens can be screened for the presence of putative effector proteins (Raffaello and Asiegbu, et al., et al., Whilst multiple developments in effector discovery have increased our understanding of these enigmatic proteins, arguably the explosion in effector research can be attributed to the development of three approaches: genome sequencing, bespoke bioinformatic pipelines, and Z. tritici and M. oryzae (Singh et al., et al., The genome annotation of multiple isolates through the construction of pathogen pangenomes allows for intraspecific genome analysis and will provide insight into the links between high polymorphisms and host specificity. The use of pangenome analyses has already led to the differentiation between core candidate effectors and novel candidate effectors in


Identifying how obesity‐related characteristics cluster in populations is important to understand disease risk. Objectives of this study were to identify classes of children based on obesity‐related variables and to evaluate the associations between the identified classes and overweight and obesity.n = 5185) from the TARGet Kids! network (2008–2018). Latent class analysis was used to identify distinct classes of children based on 15 family, metabolic, health behaviours and school‐related variables. Associations between the identified latent classes and overweight and obesity were estimated using multinomial logistic regression.A cross‐sectional study was conducted among children 3–11 years of age , Class 2: ‘Metabolic risk’ (7%), Class 3: ‘High risk’ (6%), Class 4: ‘High triglycerides’ (21%), Class 5: ‘Health risk behaviours and developmental concern’ (22%), and Class 6: ‘Healthy’ (24%). Children in Classes 1–5 had increased odds of both overweight and obesity compared with ‘Healthy’ class. Class 3 'High risk' was most strongly associated with child overweight  and obesity .Distinct classes of children identified based on obesity‐related characteristics were all associated with increased obesity; however, the magnitude of risk varied depending on number of at‐risk characteristics. Understanding the clustering of obesity characteristics in children may inform precision public health and population prevention interventions. Body mass index (BMI)‐defined cut‐points are the most commonly reported measure of overweight and obesity; however, BMI alone may not adequately capture the complex nature of obesity as a disease and may not lead to effective interventions for prevention or treatment.Obesity‐related variables defined broadly to include both risk factors  and complications or consequences of obesity , are generally considered in isolation. A 2014 review paper identified 18 studies on the clustering of diet and physical activity in children, and only three studies used latent class analysis (LCA).Thus, the overall aim of this study was to identify distinct classes of children, as young as 3 years of age, based on a comprehensive range of obesity‐related variables, including family, metabolic, health behaviours and developmental concerns. The primary objective of this study was to identify distinct groups of healthy children 3 to 11 years of age based on obesity‐related variables, overall and stratified by sex. The secondary objective was to evaluate the association between the identified latent classes and BMI categories .22.1A cross‐sectional study was conducted using data from The Applied Research Group for Kids (TARGet Kids). TARGet Kids is a primary care research network in Toronto, Canada. Children <6 years of age were recruited between 2008 and 2018 during well‐child visits at primary care paediatric and family physician practices in the Greater Toronto Area and are followed annually.TARGet Kids! cohort study is registered at www.clinicaltrials.gov (NCT01869530).Trained research assistants recruited study participants and physical measurements, including anthropometrics and blood pressure, blood samples (nonfasted) and parent‐completed nutrition and health questionnaires were collected at each visit.2.2z‐scores (zBMI) were calculated using the World Health Organization (WHO) growth standards for children under five and growth reference for children over five as recommended for the Canadian population.Height and weight anthropometric data were collected by trained research assistants using a precision digital scale  for weight and a stadiometer (seca) for measuring standard height. BMI was calculated by dividing weight (in kilogrammes) and height (in metres squared)2.3Based on previous literature, including the domains identified in the Edmonton Obesity Staging System for Pediatrics,Family characteristics included family history of cardiometabolic disorder defined as ‘yes’ if a mother, father or siblings reported diagnosis of heart disease, hypertension, high cholesterol or diabetes versus ‘no’ if none reported. Parental BMI was calculated based on measured height and weight of either the mother or father collected by the research assistant at the child's primary care visit.Cardiometabolic characteristics for children included high systolic blood pressure (SBP) or diastolic blood pressure (DBP), measured by trained research assistants according to recommendations,Health risk behaviours included parent‐reported physical activity, screen time, sleep and sugar‐containing beverages (SCBs). The measurement of each of these variables is described in more detail elsewhere.Developmental concerns included children that were reported to have been diagnosed with attention deficit hyperactivity disorder, autism, learning problem or developmental delay on the Nutrition and Health Questionnaire (NHQ) that were identified as having developmental concern. Children were identified as having school concerns if parents reported that their child's school expressed concerns about the child relating to the following: speech and language, learning, attention, behaviour, social relationships, physical coordination, fine motor coordination or self‐help skills and independence. Children were identified as requiring extra resources if parents reported the provision of one or more of the following extra resources from their child's school: speech and language therapy, occupational therapy, educational assistance or other extra resources provided at school.2.4Descriptive analyses of covariates including sex, age and weight category were examined for participants, as well as the frequency of responses for each of the 15 latent class indicators across all participants in the sample. For the primary objective, LCA was conducted to identify unobserved (latent) classes based on the categorical indicators described.Analyses were conducted using the LCA procedure in SAS version 9.4 software.Under the assumption that missing data was missing at random (MAR), the model was fit using an Expectation–Maximization (EM) approach, where all available data was used to estimate classes and assign the posterior probabilities of belonging to each class for each individual.33.1A total of 5185 children between 3 and 11 years of age were included in this study; 74% of children were in the 3‐ to <6‐year age range. As described in Table 3.2For the primary analysis, a six‐class model was found to be the optimal model as it had lower cAIC and BIC values compared with the other models (Supporting Information Table S1). Response probabilities for each of the 15 indicators by class are presented graphically in Figure Class 6 was defined as the Healthy class as it had the lowest probability of almost all risk indicators compared with the other classes. Class 1 was defined as Family and health risk behaviours because children in this class were most likely to have parents with overweight or obesity, low neighbourhood household income, and not meet screen time or sugar beverage guidelines. Class 2‐labelled Metabolic risk was defined by a high probability of both high LDL and non‐HDL, in contrast, Class 4 was defined by high triglycerides only. Class 3 was considered a High risk class with high LDL, non‐HDL, triglycerides, screen time, extra school resources and school concern. In comparison, Class 5 was defined as Health risk behaviours and developmental concern only with high screen time, extra school resources and school concern.3.3The distribution of zBMI categories across the six identified latent classes is presented in Figure 3.4Results of the sex‐stratified analyses revealed that a four‐class model fit the data best for the LCA in both males and females separately (model fit indices are provided in Tables S3 and S5). The item response probabilities for each of the 15 indicators by class for males and females are presented graphically in Figure Table 4Results of this study suggest that there are distinct classes of young children defined based on presence of obesity‐related risk factors, and several of these classes were differentially associated with odds of overweight and obesity. A total of six classes were identified, and only one class , which included 24% of children, was defined by the absence of any obesity‐related risk factors . The other five latent classes, which included 76% of the children, all exhibited at least two obesity‐related characteristics. Of concern were the 6% of children identified in Class 3 with multiple obesity‐related characteristics, including family, metabolic, health risk behaviours and developmental concerns.Each of the identified classes, compared with the healthy class as reference, was strongly associated with increased odds of overweight, and obesity in a consistent dose–response manner with much stronger associations for obesity than overweight. However, the magnitude of the strengths of the associations varied by class. Classes 1–3 defined as Family and health risk behaviours, Metabolic risk and High risk were significantly associated with substantial increases in odds of both overweight and obesity, with ORs ranging from 1.6 to 3.3.Sex‐stratified analyses revealed that a LCA model with only four classes fit the data best for both boys and girls, compared with the six‐class model identified overall. Some sex‐differences were observed, but there were also many similarities between the four classes that were identified separately in males and females. Of the six previous LCA studies,Of the 15 indicator variables that were evaluated based on a priori hypothesis, the following five variables had little evidence of an association with any class membership: family history of cardiometabolic disorders, high systolic or diastolic blood pressure, abnormal HDL, sleep duration below age‐specific recommendations and parent‐reported developmental concern. In this study, low physical activity was also not a helpful indicator in determining the classes as it was highly prevalent in all classes ; many children did not meet the recommended physical activity guidelines based on parent‐reported guidelines. This is contradictory to previous studies that found physical activity to be a determining characteristic for particular classes; however, this was in older children greater than 9 years of age.Other studies that have used LCA to evaluate classes in older children, and adolescents did not incorporate metabolic risk factors such as blood pressure and lipid levels and instead focused only on dietary and behavioural risk factors.Strengths of the study include the relatively large sample size and comprehensive data available on family, metabolic, health risk behaviours and developmental concerns that were available for children participating in TARGet Kids!. This is one of the first studies to include such a wide variety of indicators in a cluster analysis, as well children as young as 3 years of age up to age 11. Additional strengths include the use of measured heights and weights and measurement of blood pressure by trained research assistants and blood collection for biomarker analysis. LCA also has several advantages compared with variable‐centred approaches, and it does not require a priori assumptions about the distribution of the data or the number of classes that will emerge.Children in this study were recruited from primary care providers in an urban setting and tended to have relatively high family income that may not be representative of all Canadian children. Missing data was substantial for some indicators because of changes in the study questionnaires over time, age‐specific measures and choice to provide blood samples. In the TARGet Kids cohort, it has previously been shown that characteristics of children who provided blood samples were similar to those who did not on most factors except age.Population‐based interventions for the preventionThis study was supported by funding from the Canadian Institutes of Health Research.JLM received an unrestricted research grant for a completed investigator‐initiated study from the Dairy Farmers of Canada (2011–2012), and Ddrops provided nonfinancial support (vitamin D supplements) for an investigator‐initiated study on vitamin D and respiratory tract infections (2011–2015). No other authors have any conflicts of interest to report.Laura N. Anderson, Cornelia M. Borkhoff, Sarah Carsley, Jonathon L. Maguire and Catherine S. Birken conceived the study and oversaw data collection. Laura N. Anderson, Ravinder Sandhu and Charles D.G. Keown‐Stoneman analysed the data. Laura N. Anderson and Ravinder Sandhu wrote the first draft, and all authors were involved in writing the final paper and approved the submitted version.Table S1: Model fit indices for 1 to 8 class LCA modelsTable S2: Item response probabilities for the six class model overall Table S3: Model fit indices for 1 to 8 class LCA models for males onlyTable S4: Item response probabilities for the four‐class model for males onlyTable S5: Model fit indices for 1 to 8 class LCA models for females onlyTable S6: Item response probabilities for the four‐class model for females onlyClick here for additional data file.


SGLT2 inhibitors (SGLT2i) are glucose‐lowering medications which increase the renal threshold for glucose reabsorption and promote glucosuria. Treatment with these agents raises serum ketone levels, and cases of diabetic ketoacidosis (DKA) during therapy have been reported. The duration of glucosuria and inpatient course of SGLT2i‐related DKA, however, is not well‐characterized. We report 11 inpatient cases of SGLT2i‐related DKA, including a subset of patients who experienced prolonged glucosuria and relapse of DKA during their hospitalization. SGLT2 inhibitors (SGLT2i) are known to increase serum ketones and predispose patients to diabetic ketoacidosis (DKA). The duration of glucosuria after stopping the medication is unknown, and the impact of SGLT2i use on the inpatient course of DKA is not well‐characterized. We identified 11 cases of SGLT2i‐related DKA, a subset of which suggest prolonged glucosuria after discontinuation of the medication. To the EditorSodium‐glucose cotransporter‐2 inhibitors (SGLT2i) have become increasingly popular glucose‐lowering agents, and interest in this class has broadened due to demonstration of cardioprotection in patients with established cardiovascular disease and slowing the progression of renal dysfunction.The Beth Israel Deaconess Medical Center (BIDMC) electronic medical record was systematically queried to identify cases of DKA in patients prescribed SGLT2i. The BIDMC Institutional Review Board exempted this protocol from review. Inclusion criteria were as follows: age 18 or older, admitted between 1 January 2013 and 16 May 2019, prescribed any SGLT2i at any point, and diagnosed with DKA, either by ICD codes or using serum bicarbonate under 20 mEq/L. The records were individually reviewed by a physician to confirm that use of a SGLT2i occurred prior to the admission for DKA. DKA was defined using the American Diabetes Association definition . Venous pH is reported, as arterial pH is not routinely obtained at this institution to diagnose DKA. Serum ketones were not available for most cases. The duration of intravenous insulin infusion, persistence of glucosuria and time to close anion gap (5), defined as time elapsed from the first available serum chemistry to the chemistry measuring an AG ≤ 16, were extracted from medical records.A total of 273 unique patient records were identified and reviewed for inclusion. Reasons for exclusion included: no inpatient or emergency room admission for DKA (249 cases) and not on SGLT2i prior to admission (13 cases). The remaining 11 cases were analysed for this study.Data for the 11 cases are summarized in Table Our study suggests not only that the time to close the anion gap may be longer in patients with SGLT2i‐related DKA, but also that even once the gap has closed, inadequate insulin or carbohydrate delivery can result in recurrent ketosis and risk of DKA relapse due to the ongoing glucosuric effects of the medication. Treatment of DKA relies principally on volume resuscitation and adequate insulin and carbohydrates to suppress ketosis. In classical DKA in patients with type 1 diabetes, this is often accomplished relatively quickly; multiple prior studies have indicated that median time to resolution of classical DKA is approximately 11 hours and total hospital length of stay is 3 days, though patients with classical DKA tend to be younger and with fewer comorbidities compared with those on SGLT2i.This study is the first detailed analysis of the inpatient course of all SGLT2i‐related DKA cases within a single centre and including an evaluation of glucosuria. Prior investigations have focused primarily on defining the risk of developing DKA with SGLT2i use in large populations, indicating an increased risk in someOur study suggests that patients with type 2 diabetes hospitalized with SGLT2i‐related DKA are likely older and have more comorbidities than those with type 1 diabetes, and required a longer period of time on IV insulin and in the hospital. Age and comorbidities certainly may have played a role in their clinical course, and given new SGLT2i indications for prevention of adverse cardiovascular events and progression of renal failure, increasing numbers of chronically ill patients may be started on this therapy. In cases of DKA, particularly when clinical circumstances may require prolonged fasting or oral intake is not adequate due to concomitant medical conditions or treatments, sequential urine glucose measurements may be particularly helpful to assess whether continuous intravenous insulin and dextrose should be extended. In situations where IV insulin is not readily available, frequent scheduled dosing of subcutaneous rapid‐acting insulin every 2‐4 hours with monitoring of blood glucose may be a reasonable alternative.It is essential that inpatient therapy of SGLT2i‐related DKA focuses on providing adequate insulin and dextrose independent of serum glucose concentrations given ongoing glucosuria despite resolution of metabolic acidosis. Monitoring urine glucose concentration may also assist in the risk assessment for DKA relapse in this setting. A larger, prospective multicentre study comparing patients with DKA on SGLT2i to a control group of DKA and no SGLT2i that closely monitors urine glucose concentrations and other relevant clinical factors is necessary to define recommended monitoring and management strategies. Guidelines for treatment of SGLT2i‐related DKA should recognize that patient characteristics and course may differ from that of classical DKA and require vigilance to prevent relapse and extended hospitalization.The authors have no relevant conflicts of interest to disclose.GPW collected and analysed the data and drafted the manuscript. ARS and JM contributed to study design and reviewed/edited the manuscript. FMB conceived of study design, contributed to the data analysis and reviewed/edited the manuscript.


It is important to investigate the current prescription status and clinical outcomes of patients with schizophrenia receiving long‐acting antipsychotic injections. We aimed to determine the prescription proportion of long‐acting antipsychotic injections and hospital readmission proportions of patients with schizophrenia in Japan.An open dataset was created using data from the National Database of Health Insurance Claims and Specific Health Checkups of Japan. Patient records with the term ‘schizophrenia’ were included. In Analysis 1, antipsychotic prescription proportions were determined for outpatients who had visited psychiatric facilities between 1 February 2015 and 31 March 2017. In Analysis 2, patients who had been discharged from a psychiatric facility and had received a long‐acting antipsychotic injection prescription within 90 days after initial discharge were selected; then, their readmission proportion was examined for 365 days after the initial discharge.The long‐acting antipsychotic injection prescription proportion was 3.5% for outpatients with schizophrenia receiving antipsychotics. The readmission proportion was 41.0% in the entire patient population, 36.2% in patients receiving typical long‐acting antipsychotic injections alone, and 23.5% in patients receiving atypical long‐acting antipsychotic injections alone.Long‐acting antipsychotic injections are not yet widely used in Japan. The readmission proportion was lower in the patients receiving atypical than typical long‐acting antipsychotics injections. The results may provide important basic information to develop new future research questions but should be interpreted with caution because generalizability may be limited by the use of aggregated data and the data structure of the database used. Schizophrenia is a chronic disease, and most patients experience relapses even when symptoms have previously been alleviated by antipsychotic treatment.Long‐acting antipsychotics injections (LAIs) have become widely used around the world.Prevention of relapse decreases readmission proportions and therefore decreases time spent in a psychiatric facility. The Ministry of Health, Labour and Welfare (MHLW) of Japan has produced publications entitled ‘Vision for Reform of Mental Health Care and Welfare’LAIs may help prevent readmission and extend time spent in the community, and were found to be marked superior to oral antipsychotics (OAPs) in preventing hospitalization according to a meta‐analysis of mirror‐image studies.et al. reported that LAIs were prescribed in 36% of 1616 assertive outreach team patients with schizophrenia in the UK and Humberstone et al. reported that LAIs were prescribed in 23.3% of 3178 outpatients who received antipsychotics in Auckland, New Zealand.Several studies have investigated LAI usage in Japan, and found that LAIs have not been widely adopted by Japanese psychiatrists. Tanaka and Fujii reported that the LAI prescription proportion at Yamanashi Prefectural Kita Hospital was 15.6%.et al. analyzed antipsychotics prescriptions among outpatients with schizophrenia spectrum disorders in Connecticut, USA and reported an LAI prescription proportion of 28%.It is also clinically important to determine regional variations in the LAI prescriptions proportion. For example, Covell To realize the MHLW's goal of early discharge and community integration for inpatients with acute schizophrenia, it is important to understand the conditions that lead to re‐hospitalization. Cheung and colleagues studied re‐hospitalization and emergency department visit rates using the Diagnostic Procedure Combination database. They found that both of these rates decreased more with LAIs prescriptions than with OAP prescriptions. They also found that re‐hospitalization and emergency department visit rates decreased more with typical and atypical LAI prescriptions than with OAP prescriptions, but this decrease was not statistically significant.Against this background, we conducted this nationwide survey investigating LAI prescriptions and readmission proportions among outpatients with schizophrenia in Japan—both nationwide and in individual prefectures—by applying the same analytical approach to data collected during the same period. The purpose of this study was to investigate the current status of LAI prescriptions for outpatients with schizophrenia in Japan and to investigate the readmission proportion and the current status of LAI prescriptions for outpatients with schizophrenia after psychiatric hospitalization.The National Database of Health Insurance Claims and Specific Health Check‐ups of Japan (NDB) is operated by the MHLW.The policy research group started in 2016 to establish various datasets useful for policy making and was supported by an MHLW research grant,https://www.ncnp.go.jp/nimh/seisaku/study/analysis/assets/0002.xlsx.The MHLW has approved public access to our dataset, which is available at In this study, an NDB‐derived dataset was created to investigate the LAI prescription status in outpatients with schizophrenia and to determine the readmission proportions of previously discharged patients according to whether they were prescribed typical or atypical LAI treatment.The study period is illustrated in Figure This study involved two analyses of outpatients with schizophrenia who were prescribed antipsychotic drugs. Analysis 1 examined the LAI prescription proportion, and Analysis 2 examined the readmission proportion after discharge. The index date for inclusion in Analysis 1 was the date of the first antipsychotic prescription during the evaluation period. For Analysis 2, the index date for inclusion was the date of the first LAI prescription within 90 days after discharge from a psychiatric facility.Patients who met the inclusion criteria were included in the study. We did not set the exclusion sequences, enrollment windows, and enrollment gaps for the dataset, which were decided by the policy research group. The inclusion and exclusion windows for the present study's dataset were defined as between 1 February 2015 and 29 June 2016.http://www.iryohoken.go.jp/shinryohoshu/downloadMenu), which is provided by the Health Insurance Bureau of the MHLW. This list was used to identify antipsychotics in the policy research group's dataset. Antipsychotics were classified as typical or atypical based on atypical antipsychotics listed as additional items and as LAIs or non‐LAIs based on the dosage form  Prescribed antipsychotics based on diagnosis code F20–F29 according to the International Classification of Diseases, 10th Revision  prescribed only typical LAI, (2) prescribed only atypical LAI, (3) prescribed typical and atypical LAI, (4) prescribed typical LAI and non‐LAI, (5) prescribed atypical LAI and non‐LAI, and (6) prescribed typical LAI, atypical LAI, and non‐LAI.Furthermore, in Analysis 2, patients who received LAI prescriptions within 90 days after discharge from a psychiatric facility met the following criteria: (i) hospitalized for schizophrenia and discharged from a psychiatric facility between 1 February 2015 and 31 March 2016 and before discharge received treatment based on diagnosis code F20–F29 according to ICD‐10 Table , Code C7Subjects were outpatients in both Analysis 1 and Analysis 2. For Analysis 2, exposure was defined as LAI prescription within the first 90 days after discharge from a psychiatric facility and the follow‐up period was 365 days after discharge. It was necessary to include patients who received LAIs during continuous medical treatment after discharge. The Japanese national health insurance system regards such patients as new patients if they have not visited a hospital within 90 days after discharge. Therefore, we regarded patients with an LAI prescription within 90 days after discharge as receiving continuous medical treatment from discharge and included them in our study.The 29 antipsychotics that are currently used to treat schizophrenia patients in Japan were selected and classified as either typical or atypical antipsychotics and as either LAI or non‐LAI according to the formulation described in the claims database Table .In Analysis 1, the event date was defined as occurring between 1 February 2015 and 31 March 2016. The proportion of patients who had received LAIs was defined as the proportion of outpatients who had been assigned a drug code corresponding to LAIs and who met the inclusion criteria Table , Code D4In Analysis 2, the readmission date was defined as the date of the first psychiatric hospitalization within 365 days after discharge from a psychiatric facility Table , Code F1Furthermore, in Analysis 2, the readmission proportion was defined for each prescription group as the number of patients readmitted within 365 days after discharge out of the total number of patients in the group Table , Code F2Informed consent could be waived because only anonymous and aggregated data were used. Furthermore, according to the Japanese Ethical Guidelines for Medical and Health Research Involving Human Subjects, the institutional review board did not need to be informed of our research because all data were based solely on summary tables (i.e. secondary use of a database). In addition, our study was approved in advance by the committee for commissioned joint research of the National Center of Neurology and Psychiatry in Japan.2 test was performed with one degree of freedom, an upper limit of statistical significance of 5%, and a critical value of 3.84 for the readmission proportions of the typical LAI alone and atypical LAI alone groups. The policy research group used SPSS modeler 18.1, Microsoft Excel 2016, and Python3 in PyCharm for data analysis. We used the CHISQ.DIST.RT function in Microsoft Excel 2016 to calculate P‐values.In Analysis 1, summary tables from the policy research group were used to obtain the number of patients who had received antipsychotics. In Analysis 2, summary tables from the policy research group were used to obtain the number of patients in each prescription group. The number was not published, so we estimated the number of patients readmitted 365 days after discharge using the number of readmissions in each prescription group multiplied by the readmission proportion. In addition, we assessed the statistical significance of differences between the typical LAI group and the atypical LAI group. The χThe data selection flow diagrams for Analysis 1 and Analysis 2 are shown in Figure n = 7907), 36.2% among patients prescribed only typical LAI (n = 210), 23.5% among patients prescribed only atypical LAI (n = 947), 43.9% among patients prescribed typical LAI and non‐LAI (n = 2496), 42.1% among patients prescribed atypical LAI and non‐LAI (n = 3946), and 59.6% among patients prescribed typical LAI, atypical LAI, and non‐LAI (n = 287) .A total of 7907 patients (3975 men and 3932 women) met all criteria for Analysis 2: 71 were aged <20 years; 2456 were aged 20–39 years; 4505 were aged 40–64 years; 741 were aged 65–74 years; and 134 were aged ≥75 years. Among those who received an LAI prescription after discharge from a psychiatric facility, the readmission proportion within 365 days after discharge was as follows: 41.0% among all patients prescribed an LAI after discharge  among prefectures, ranging from 2.0% to 6.8%. This difference may have been due to (i) the prescribing trends of physicians in certain hospitals, which affects the region's prescribing trends and (ii) socioeconomic regional characteristics. LAIs are more likely to be prescribed in areas with poor access to health care than in those with good access to health care.Few studies have compared prescription proportions between first‐generation  LAIs and second‐generation  LAIs. In this study, the difference in LAI prescription proportions was small between conventional typical LAIs and the newer atypical LAIs, suggesting that in the short period since their introduction to Japan, atypical LAIs have been readily adopted in the Japanese clinical setting by physicians who already have experience with LAIs.Analysis 2 determined the readmission proportions of patients with schizophrenia receiving LAIs after discharge from psychiatric facilities. The readmission proportion increased over time, reaching 41% at 365 days after discharge  was lower in patients prescribed atypical LAI alone than that in those prescribed typical LAI alone. This difference was significant and may reflect physicians' drug selection behaviors being influenced by the following factors: (i) atypical LAIs were introduced later than typical LAIs, so more physicians are likely to administer them in patients with a relatively short history of schizophrenia, (ii) atypical LAIs cause fewer side effects such as malaise than typical LAIs, leading to the higher continuation rate of atypical LAIs. We also found that the readmission proportion was the highest in patients who had been prescribed all three types of treatment . This suggests that the physicians had been struggling to determine the best treatment for the patient and that atypical LAIs did not seem to be effective. Kishimoto and colleagues showed in their recent meta‐analysis of cohort studies that illness severity and/or chronicity was significantly greater in patients prescribed LAIs than in those prescribed OAPs.n = 6750) received antipsychotics from multiple categories. This may be related to Japan's polypharmacy problem. For example, a 2016 REAP (Research on Asian Prescription Pattern) study examined the use of antipsychotics in patients with schizophrenia in Asian countries and found that the proportion of patients on a combination of two or more antipsychotics in Japan was 55.0%, higher than the proportions in other countries.Of the patients who received LAIs within 90 days after discharge, 85.6% , though a large majority of patients receive clinical care under the health insurance system. Third, the primary aim of this study was to investigate the current status of LAI prescriptions, so we did not adopt a new‐user design or adjust for regional variations. Finally, the dataset we used from the policy research group was limited to the period between 1 February 2015 and 31 March 2017. There may be selection bias and prevalent user bias, but our discussion was based on the notion that these biases did not critically influence our investigation of the current status of LAI prescriptions, which was the aim of this study.In Japan, where most of the population is covered by universal health insurance, we used NDB data to conduct this comprehensive study on the current status of LAI prescriptions and the outcomes of patients with LAI prescriptions. The study revealed that LAIs have not been widely adopted in Japan compared with other countries. The readmission proportion to psychiatric facilities was lower for patients with an atypical LAI prescription than with a typical LAI prescription.This study was a collaboration between the National Institute of Mental Health, National Center of Neurology and Psychiatry, and Otsuka Pharmaceutical Co., Ltd. We thank Otsuka Pharmaceutical Co., Ltd. for their generous financial support. MU, RO, and YY have no conflicts of interests to report. YK is a full‐time employee of Otsuka Pharmaceutical Co., Ltd.All authors conceived and designed the study. M.U. and Y.Y. analyzed the data. M.U. and R.O. drafted the article and wrote the manuscript. All authors critically reviewed the manuscript and approved the final version of this manuscript.Table S1. Supplementary table prepared in compliance with the International Society for Pharmaceutical Engineering guidelines.Click here for additional data file.Table S2. List of 29 antipsychotic drugs.Click here for additional data file.


Lasers based on biological materials are attracting an increasing interest in view of their use in integrated and transient photonics. Deoxyribonucleic acid (DNA) as optical biopolymer in combination with highly emissive dyes has been reported to have excellent potential in this respect. However, achieving miniaturized lasing systems based on solid‐state DNA shaped in different geometries to confine and enhance emission is still a challenge, and the physicochemical mechanisms originating fluorescence enhancement are not fully understood. Herein, a class of wavelength‐tunable lasers based on DNA nanofibers is demonstrated, for which optical properties are highly controlled through the system morphology. A synergistic effect is highlighted at the basis of lasing action. Through a quantum chemical investigation, it is shown that the interaction of DNA with the encapsulated dye leads to hindered twisting and suppressed channels for the nonradiative decay. This is combined with effective waveguiding, optical gain, and tailored mode confinement to promote morphologically controlled lasing in DNA‐based nanofibers. The results establish design rules for the development of bright and tunable nanolasers and optical networks based on DNA nanostructures. Miniaturized, wavelength‐tunable lasers based on deoxyribonucleic acid (DNA) nanofibers might combine enhanced stability of embedded dyes and tailored emission through morphology control. Here, these systems are studied by a quantum chemical investigation of the DNA–dye interactions, and elucidated in their waveguiding and mode confinement properties that lead to morphologically tailored lasing from the doped DNA structures. This concept has been gaining a continuously increasing interest for the generation of multicomponent systems for photonics and optoelectronics. Applications demonstrated to date include using DNA as electron blocking layer in organic light‐emitting devices, and as template for nonlinear optical materials. Furthermore, the use of DNA in organic micro‐ and nanolasers is especially appealing, since embedded chromophores might show enhanced performance compared to blends with other synthetic polymers. Such systems, primarily if equipped with effective chemostructural tools for spectral control, can lead to the development of optical communication chips and high‐throughput chemical and biological sensors. The encapsulation in confining structures or matrices suitable to conveniently tailor the energy levels of lasing dyes can be highly relevant in this respect, as demonstrated in shape‐engineered microlaser based on metal–organic frameworks. In addition, DNA is available as a waste product of the fishing industry, thereby potentially embodying a sustainable building block for realizing transient photonic components.Supramolecular chemistry and nanotechnology approaches based on deoxyribonucleic acid (DNA)1 are  and hemicyanine show improved fluorescence in DNA, mechanisms at the base of these properties remain elusive due to the concomitant effects from intercalation or groove‐binding as well as from microstructure cavity morphologies. Among other solid‐state structures, DNA‐based micro‐ and nanofibers might offer significant advantages in this respect, including highly effective control of sustained optical modes in the fibrous geometry. Light scattering and confinement in the complex microstructure of the biopolymer filaments, together with potential dye stabilization resulting in the significant reduction of nonradiative decay channels for excitations, would make light‐emitting DNA‐based fibers ideal systems for lasing action. So far, dye‐doped fibers of DNA complexed with the cationic surfactant cetyltrimethylammonium chloride (CTMA) have been reported as fluorescent material, as white emitters upon donor–acceptor resonance energy transfer, and as all‐optical switches through incorporation of a push–pull, π‐conjugated pyrazoline derivative featuring intramolecular charge transfer, but laser systems based on such class of nanofibers are still unexplored.While a few studies reported that dyes such as sulforhodamine5b andH‐pyrazole] (DCNP), and the behavior of optical modes due to confinement in the assembled fibrous architecture. Time‐dependent density functional theory (TD‐DFT) calculations are also performed for DCNP in gas‐phase, in a polar solvent and when intercalated in a DNA fragment. The found synergistic effects lead to measure for the first time lasing action from the DNA‐based fibers, with remarkable wavelength tunability and emission regime controllably switchable from multimodal to broadband lasing.In this work, we clarify the dual aspects of fluorescence enhancement due to DNA interaction for the lasing dye, , with V = (πdF/λp) × (nF2 − 1)1/2 where λp is the emission wavelength and nF is the refractive index of DNA (≅1.5). A transition from multimode to broad, single‐band lasing is found for the DCNP/DNA‐CTMA fibers upon increasing the fiber diameter above λp, which corresponds to an increase of the confinement factor that closely approaches unity. Overall, a remarkable wavelength tunability is achieved, namely, a lasing wavelength varying from 630 nm to about 655 nm depending on the fiber morphology. The observed red‐shift of the lasing wavelength upon increasing the transversal size of the fibers is related to the degree of overlap of the guided modes with the gain medium. For larger fibers, the guided mode is effectively contained in the fiber (η > 99%), which occurs for all the wavelengths in the spectral interval of the DCNP optical gain (620–660 nm). This condition clearly leads to lasing closely to the wavelength of maximum optical gain. Upon decreasing the fiber size, the fraction of the mode that is effectively overlapped with the gain medium is decreased as well, and this effect is more critical for longer wavelengths. For instance, for 400 nm fibers, η decreases from 0.80 to 0.77 upon increasing the wavelength in the range of the DCNP optical gain. Therefore, lasing is correspondingly favored at shorter wavelengths. The relative intensity of individual narrow peaks can change due to mode competition for lasing . The guided mode dispersion lies in between the light line in air (left dashed line) and in the polymer (right dashed line), i.e., they cannot propagate in air as their momentum exceeds the maximum in air. The transverse intensity profiles of the first ten modes is displayed in Figure To rationalize the special lasing behavior of the DCNP/DNA‐CTMA ribbon‐shaped nanofibers, we also calculate numerically the profile of sustained optical modes. In fact, the modes of an individual fiber can be calculated analytically for a cylindrical geometry,15 whe3In summary, we have reported that lasers with remarkable wavelength tunability, and with regime varying between multimodal and broadband action can be realized based on DNA fibers incorporating a DCNP dye. With the help of quantum chemical calculations, we have shown that the DCNP interaction with DNA effectively hinders the excited state geometrical distortions responsible to nonradiative decay channels thus leading to enhanced light emission. The finding, that upon suppressed twisting the nonradiative decay is disfavored as well, has general validity once an enough viscous environment or steric hindrance are provided in the host micro‐ or nanostructures. This makes DCNP/DNA‐CTMA fibers particularly promising for enhancing light emission as both constraining conditions are in place for the dye. On top of this, the effective waveguiding, optical gain, and tailored mode confinement in DNA nanofibers work as supporting mechanisms to promote lasing action with morphology‐controlled properties. These results represent an important step toward the design of miniaturized, highly controlled lasers based on DNA nanostructures, as well as toward the rationalization of the various effects promoting enhanced emission in the same.4−1 , a distance between the needle and collector surface of 20 cm, and an applied voltage of 10 kV. To generate aligned fibers, a rotating cylinder was used as collector. The morphology of the doped fibers was investigated by scanning electron microscopy  with an accelerating voltage of 3 kV and an aperture size of 30 µm, following thermal deposition of 5 nm of Cr . The average diameter of the fibers was calculated from the SEM micrographs using an imaging software. Atomic force microscopy on individual DCNP/DNA‐CTMA fibers was performed by a Multimode system (Veeco) equipped with a Nanoscope IIIa controller and working in tapping mode.DNA  functionalized with the CTMA surfactant (Sigma‐Aldrich) and doped with DCNP  was used. For realizing DCNP/DNA‐CTMA fibers, the DNA‐CTMA complex was dissolved in a mixture of ethanol and chloroform (3:1 v/v), with a polymer to solvent weight ratio varying in the range 2–4% (w/w), and the obtained solution was stirred for 24 h. A 1% concentration of DCNP was used in the dry mass of biopolymer. Solutions were electrospun using a syringe with stainless steel needle and a feeding rate of 0.5 mL hConfocal fluorescence micrographs were obtained by exciting the sample with a diode laser (λ = 405 nm), focused through an objective lens . The emission from the fibers was then collected by the same objective and analyzed by a multianode photomultiplier. A spectrophotometer  was used for transmittance measurements. Lasing was achieved by pumping samples with the third harmonic (λ = 355 nm) of a Nd:YAG laser beam , focused in a rectangular stripe (400 µm × 1 cm). The emission was then collected from the sample edge, coupled to an optical fiber and analyzed by a monochromator  equipped with a CCD detector . Lasing thresholds were measured by systematically varying the excitation fluence. The waveguiding properties of individual DCNP/DNA‐CTMA fibers were analyzed by using a microphotoluminescence (µ‐PL) setup, based on an inverted microscope  equipped with a 60× oil immersion objective  and a CCD camera. The PL was excited by the diode laser coupled to the microscope through a dichroic mirror and focused on the sample by the objective. Part of the light emitted by the DCNP chromophore in the DNA‐based fiber, excited by the tightly focused laser spot, was coupled into the fiber and waveguided. The optical losses coefficient was then measured upon collecting images of the intensity of emission diffused by the fiber surface, and by analyzing the spatial decay of emission as a function of the distance from the exciting laser spot.14a version of the PCM (IEF‐PCM). All calculations of DCNP intercalated in DNA were performed using a simplified model that limits the interactions between DCNP and DNA to the two pairs of adjacent bases (GC‐AT), the deoxyribose sugars and the phosphate groups that connect these bases. The hydrogens of the OH present in the sugar were replaced with neutral groups –CH3. The complex formed by DCNP and the DNA fragment was embedded in a cavity of a dielectric medium to mimic the effect of the solvent. Different intercalation modes were generated for DCNP using the criterium of maximizing its stacking and electrostatic interactions with DNA for both sandwich‐like and rotated configurations. These 20 initial structures were subjected to a geometry optimization with the semiempirical PM6 method and finally compared to eliminate eventual replicas. The remaining 12 structures are shown in Figure S5 in the Supporting Information. The two structures showing the lowest energy for the sandwich‐like and for the rotated configuration (reported in the two boxes) were finally reoptimized at the same DFT description used for the solvated system (B3LYP/6‐31G(d)) but with the addition of empirical dispersion corrections. In the optimization of these intercalated systems, the geometry of the DNA pocket was kept frozen and described using a 6‐31G basis set. For both solvated and intercalated systems, excited state calculations were performed at TD‐DFT level using the CAM‐B3LYP functional in combination with 6‐31+G(d) basis set: this functional is in fact known to properly describe charge‐transfer states. Relaxed excited state potential energy surfaces (PESs) were obtained assuming a complete relaxation of the solvent polarization. To account for a proper response of the solvent, a state‐specific correction was introduced for each point of the PESs through the corrected linear response formulation. All calculations were done with Gaussian '16 software.Calculations were performed with DFT and its TD extension for excited states. For the isolated and the solvated DCNP, geometry optimizations in the ground state were performed at B3LYP/6‐31G(d) level. Solvent effects were included through the integral equation formalism11 verr1 and r2, respectively. The calculations were performed for a constant ellipticity r2/r1 = 0.5. Intensity profiles for typical modes were calculated for fibers with r1 = 0.25 µm and r2 = 0.5 µm. From the numerical simulations, the electric field E was obtained in the fiber and its wavevector k′, E = E × exp(−ik′z), from which the gain was estimated as [Im(k′)/Im(k)]2, where k is the wavevector in the bulk biopolymer.The optical modes supported in the fibers were calculated using a commercial mode solver as provided by Lumerical. This is a numerical solution of the Maxwell's equations in the fiber geometry, to find the eigenmodes of the structure. The fiber structures simulated were elliptical in their cross‐section, with semiminor and semimajor axes The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


To assess needs for a gender‐sensitive perimarital counselling services in Iran.A descriptive cross‐sectional study.This was a study on all 236 premarital counselling providers in 37 health centres in Shiraz‐Iran. The tools for data collection included the following: (a) a demographic information questionnaire and; (b) a valid and reliable Needs Assessment questionnaire for Gender‐Sensitive Perimarital Counseling Services (GSPCS) in 3 sections of needs for process, structure and policy making of the perimarital counselling services. Data were analysed using SPSS 21.SD 5.35) years participated in the study. Results demonstrated highest scores for needs related to facilities as the structure of the services (90.09 SD 13.70 per cent) and community empowerment (89.50 SD 16.67 per cent) as the necessary policy for the gender‐sensitive services. We concluded that providing gender‐sensitive perimarital counselling healthcare services needs to reform the process, structure and policies of the services.All health providers with average working experience of 8.63 ( The scores of items were summed up for each sub scale and total scale and then converted to per cent (0–100).GSPCS was developed by authors of the present study through a qualitative study by content analysis approach and an extensive literature review (inductive‐deductive method for the tool development) in Persian language. Then Content validity index and content validity ratio of the GSPCS were calculated 0.99 and 0.95, respectively. Reliability of GSPCS is also confirmed by alpha Cronbach 0.96 for internal consistency and intra‐cluster correlation coefficient 0.804 of test retest for stability. Data were analysed using SPSS 21.3.4Two hundred and thirty‐six counselling providers with at least two years’ experience in premarital counselling from all 37 health centres in Shiraz agreed to participate completed the questionnaires. The questionnaires were self‐completion forms. The questionnaires were completed by responders anonymously. The responders were ensured about the confidentiality of their personal information, by providing explanation about anonymous responses and keeping and using information just for the study's objectives. All participants contributed to the research after giving the informed consent. All 236 counselling providers accept to complete the paper questionnaires, and all completed the questionnaires completely and so no drops were happened. The questionnaires were collected, and the data were analysed.3.5Data were analysed using SPSS 21 and descriptive analytical methods such as calculating frequency and percentage and sorting results for prioritizing of the needs.3.6The study was approved by the Ethical committee of Shahid Beheshti University of Medical Sciences. All participants of the study gave a written consent for their participation in the study.4SD 6.48) years and with working experience duration of 8.63 (SD 5.35) years participated in the study. Majority of the counselling providers were female and midwife. Table Two hundred and thirty‐six premarital counselling provides with average age of 32.90 (5This was the first study to assess needs for providing a gender‐based perimarital counselling service in Iran. Findings of present study showed the highest priorities for reforms in the structure especially facilities of the pre‐marriage services, and policy making by community empowerment through providing opportunities for the education before choosing a spouse at the community level.The most important care was demonstrated to be “Postmarriage continuous care and counselling for sexual reproductive health problems” and then “Providing possibilities for examination and screening of male's and female's reproductive system disorders.” This study demonstrated a lack of continuity in marriage counselling services in the present health system. Although the present counselling services provide a good opportunity to increase couples’ knowledge about sexual health and rights but it is not responder to their postmarriage problems, questions and needs such as about their reproductive system (Bostani Khalesi & Simbar, Findings showed two priorities in the educational needs including “Education about hymen and correcting of myth about that” and “Couple's education about interpersonal skills based on reproductive rights.” Lack of knowledge about anatomic condition of hymen, the myth of “intact hymen means chastity” and the rejection of girls from family for hymen injuries following prohibited sexual relations are from important issue in relation to inequality imposed on girls (Robatjazi et al., Results demonstrated highest scores for the service structural reform through improving facilities including “Providing facilities for diagnosis, treatment and follow‐up of STIs for men” and “Professional counselling based on clients’ age.” Reform in all areas of reproductive health services is important as men are usually deprived of the services because of the traditional focus of clinics on serving women and children (Narges Eskandari et al., The priorities in the dimension of necessary human resources for gender‐sensitive premarital counselling services were “Employing trained personnel for sexual health counselling” and “Educating personnel on skills for couples’ counselling.” The most important reason for failure of providers in sexual health counselling is lack of education in the field of sexual counselling. Sexual issues are from private area of individuals and counsellor should be trained and skilful to communicate with clients effectively to talk about sexual health. However, many sexual health providers are even not willing to consult and discuss in this area (Bostani khalesi et al., Findings showed the highest scores for the needs for “Management of comprehensive services for premarital counselling and female adolescents’ health” and “providing a gender‐based educational content and counselling approach,” respectively. Female adolescents are vulnerable to reproductive problems and need high‐quality counselling services, because of low experience, female adolescents are not able to reject high‐risk relationships (Alimoradi et al., Concerning community empowerment in the promotion of pre‐marriage counselling services, "providing opportunities for education before choosing a spouse at the community level," and then "providing counselling without discrimination in relation to sexual desire in society," showed highest scores. Taboo of talking about reproductive issues cause to reduce effective communication between health system, schools and even parents with their children, while supportive family and youth friendly services (Shahhosseini et al., Finding demonstrated the two top priorities in the intersectional cooperation dimension of the policy section including "Teaching essential points before choosing a spouse in school health" and “Planning for comprehensible sexual health education in health services." Since reproductive behaviours and marriage are influenced by social, cultural and individual conditions, successful interventions for premarital counselling should be planned based on adolescents’ perspectives and experiences on a specific social context (Chandra‐Mouli et al., In the section of supportive policies, the needs for “Planning a STIs risk assessment programme in counselling process " and “Planning for individuals’ education on negotiation skills for sexual health” took the highest scores from the point of view of the participants. In the cultures that men and women suffer from sexually transmitted diseases that leads to violence, isolation and social stigmatization for women, a risk assessment system should be designed for overcoming the barriers of detected high‐risk behaviours and using experience of other countries (Khalesi et al., 5.1The taboo for discussing about sexual and gender issues was the main limitation of this study, which was partially controlled by keeping confidentiality of the names of individuals, explaining about goals and intimate encounters with the participants. Healthcare providers who recruited as the participants of the present study live in our traditional society and they may follow the same myth and traditions of the community. However, they receive a comprehensive sexual education in academic courses but gender‐based effective factors on couples’ health during marital life are not considered in the courses. Besides, it is shown that talking about sex topic is hard for counselling, especially when providing sexual counselling for clients of opposite sex is necessary (Pourmohsen et al., 6Perimarital counselling services can be improved by paying attention to gender‐sensitive needs. The top priorities for reforming the present perimarital counselling are as follows: community empowerment through providing opportunities for education before choosing a spouse; the advocating policies for planning a STIs risk assessment programme in counselling process; improving facilities for counselling preventing, diagnosis, treatment and follow‐up of men's sexually transmitted diseases; employing trained personnel for sexual health counselling; continuity of counselling and care to postmarriage sexual and reproductive health problems; and education about hymen and correcting of the myth.Otherwise, the findings of this study suggest that cultural structure need to reform at the community level to correct some of misconceptions such as “considering the hymen examination as a virginity test" but also structural reforms are necessary in the health management section such as the employment of trained male personnel to provide premarital sexual reproductive health counselling, and providing space and facilities for providing sexual health counselling services for both women and men. In addition, appropriate educational design and content for counselling at clinical level are essential with considering cultural factors related to gender affecting the couple's sexual reproductive health and relationship after marriage.6.1World Health Organization emphasizes on improving quality of reproductive health services including premarital counselling services by providing gender‐sensitive services. Improving quality of services in all 3 dimensions of healthcare services including structure, procedure and outcome is essential. The present study assessed the needs for providing a gender‐sensitive premarital care services in all 3 dimensions using a standard questionnaire and showed the gaps and needs of the system to reform. The process of the present study and its needs assessment tool can be applicable in similar services for needs assessment and showing the gaps of the system which needs intervention and reform for providing quality gender‐sensitive services. The research shows gender‐based educational planning are necessary to integrate in present programmes to improve quality of perimarital care and counselling programmes. Therefore, further research is suggested to find other culturally gender‐based barriers and facilitators to improve quality of care in other aspects of sexual reproductive health such as in the area of family planning and adolescents health.The authors declare they have no conflict of interest.All authors: substantial contribution to and agreement on the final version of the manuscript.


Aging adults from low‐income households are individuals with a high risk of cognitive decline who incorporate ulam consumption and the working memory and cognitive flexibility among aging adults from low‐income households who are more susceptible to cognitive decline.To examine relationship between Cross‐sectional.Thirty‐two aging adults (45–75 years old).1‐weighted anatomical images, T2*‐weighted imaging data.Task‐based fMRI, 3.0T, Tulam consumption were assessed using the respective validated Dietary History and semiquantitative Food Frequency questionnaires. Working memory and cognitive flexibility were evaluated by using neuropsychological batteries  and task‐based fMRI (N‐back and Stroop Color Word Test [SCWT]). Brodmann's areas 9 and 46 were the regions of interest (ROIs) of DLPFC activation.The dietary and ulam consumption and the working memory and cognitive flexibility, while analysis of covariance (ANCOVA) was used to compare the difference of working memory and cognitive flexibility among four percentiles of ulam consumption, after age, gender, and education years adjustments. Significance was decided by two‐sided, P < 0.0042 and P < 0.05.Multiple linear regression used to understand the relationship between ulam consumption was positively associated with the Digit Span , right DLPFC activation (1‐back) , left DLPFC activation (SCWT‐1) , and left DLPFC activation (SCWT‐2) . The ulam consumption at the 75th and 100th percentile from the ANCOVA analysis had shown a better working memory and cognitive flexibility as compared with those of the 25th and 50th percentiles (P < 0.05).The multiple linear regression revealed that ulam consumption was related to a high intensity of brain activation in DLPFC; however, the elucidation of the neuroprotective properties of ulam have yet to be established from clinical trial studies.This study found that high Level of Evidence: 2Technical Efficacy: Stage 4J. Magn. Reson. Imaging 2020;51:1142–1153. COGNITIVE DECLINE is an indication of mental health and can lead to irreversible dementia if left untreated.ulam, which is a type of traditional salad that is normally consumed in raw form by the Asian populations  with rice or fermented sauces. ulam was also reported in a review as attenuating the oxidative stress that consequently prevents cognitive decline.Polygonum minus supplementsCentella asiaticaOenanthe javanicaMany Asian countries have conducted a series of studies on the prevention of neurodegenerative diseases through food‐based recommendation such as those of ulam consumption among the population with a high risk of cognitive decline. For this reason, this study aimed to examine the association between ulam consumption and the working memory and cognitive flexibility of the aging adults from low‐income households through the use of fMRI.Functional MRI had been used in several studies for determining the relationship between dietary nutrients or vegetables consumption and brain activationZα = confidence interval 95% = 1.96; Zβ = 80% power = 0.842; C = 0.5*ln[(1 + r)/(1‐r)] = 0.61; r = correlation coefficientA cross‐sectional study was conducted on 32 subjects (aged 45–75 years old) that were recruited from the low‐income residential areas of Klang Valley, Malaysia, and were screened earlier for eligibility by basing on the inclusion and exclusion criteria. In this study, the inclusion criteria consisted of adults between the age of 45–75 who are able to speak Malay or English languages, while the exclusion criteria were a history of mental health illness , physical disabilities, chronic kidney disease, undergoing dialysis, alcohol and drug users, being claustrophobic, and having internal metallic or electronic implants. The basic health profiles and medical history were obtained by referring to a personal medical book and self‐reported by the subjects. The sample size was calculated using the formulaulam consumption by the subjects was validated using the vegetables and fruits Food Frequency Questionnaire (FFQ) of 58 vegetables, 24 ulam, and 26 fruitsThis study was conducted according to the guidelines as stated in the Declaration of Helsinki 1964. A detailed information sheet that consisted of the purpose of the study, procedures, instructions on completing a required task, and risks was then distributed and explained to the subjects before the attainment of an informed consent. Apart from using the surveys to gauge the sociodemographic background, the trained fieldworkers had also obtained the dietary intake information of the subjects such as the type, amount, cooking method, and the frequency of the common food consumption in the past 7 days through the use of a validated Dietary History Questionnaire (DHQ).This study utilized three validated neuropsychological batteries , while those of the N‐back task and SCWT were conducted via the T2*‐weighted imaging data .Single‐shot spin‐echo echoplanar imaging (EPI) was acquired with the fMRI data and the fMRI images were performed on a 3.0T magnetic resonance (MR) scanner  with each of the subjects being subjected to high‐resolution TResponse timeCorrect response was used to analyze the behavioral performance of data.The percentage of accuracy and the mean response time (RT) on the N‐back and SCWT task of each subjects were then recorded in the calculation of the average data. Correct response (CR) is the percentage of correct responses from total response performed by each subject. A new index 1‐weighted image and estimated against a standardized Montreal Neurological Institute (MNI) stereotaxic space, where the spatial normalization procedure would involve a six‐parameter affine transformation with a spatial transformation matrix. After undergoing the normalization process, all of the functional volumes were then subjected to spatial smoothing with a 6‐mm full‐width half‐maximum of isotropic Gaussian kernel as a way of increasing the signal‐to‐noise level through the removal of the high‐frequency information and the reduction of its intersubject variability.A quality control test for the MRI machine was done routinely by the professional engineer to make sure that the quality of the functional images was good. The preprocessing and data analysis stage utilized the Statistical Parametric Mapping software implemented in MatLab 9.4.0 R2018a . The first ten volumes were discarded because of equilibration effects. Functional images were first slice time‐corrected and subjected to a realignment of motion correction according to the mean image of the series. These functional images would then be coregistered to the subject's mean TP < 0.05, familywise error [FWE]‐corrected), the primary outcome of the ROI brain activation would then be represented by the respective subject's percent of signal change.The DLPFC that was selected had been defined by the WFU PickAtlas with Brodmann's areas 9 and 46, since prior studies had identified this ROI as being responsible for generating the working memory and cognitive flexibility of the human brain.P < 0.05. Apart from utilizing Shapiro–Wilk for testing the normality of the data (P > 0.05), the demographic data of all the respondents were also presented in the form of percentages or mean and standard deviation. Pearson correlation was used to analyze the relationship between behavioral performance index and brain activation. While the association between ulam consumption and working memory and cognitive flexibility  had been established from the use of a simple linear regression, the adjusted age, gender, and years of formal education were then conducted via the multiple linear regression. β represents the correlation value of the analysis. To control for the inflated FWE rates that results from performing multiple tests on the same data, the significance of these multiple linear regression at a Bonferroni‐adjusted alpha level was performed. To make adjustment, the family‐wise alpha level (0.05) was divided by the total numbers of dependent variables using the formula:All of the statistical analyses were performed using the Statistical Package for the Social Sciences  v. 23.0 software with a significance level of ulam consumption as controlled by the age, gender, and years of formal education would be further determined by a univariate analysis of covariance (ANCOVA). The magnitude of ulam was taken daily in order to have better working memory and cognitive flexibility that could be obtained from ANCOVA analysis.The difference in the neuropsychological test scores and DLPFC activation among the four percentiles of ulam consumption was found to be 31.7g/day (~0.5 servings/day), while the average of reaction accuracy and reaction time was 58.7% (N‐back), 70.6% (SCWT), and 2295 (N‐back) and 2495 (SCWT) msec.As shown in Table r = –0.456, P = 0.009), 1‐back , and left DLPFC activation while subjects performed SCWT‐1  and SCWT‐2 .Table ulam consumption and the Digit Span , the univariate analysis also showed that ulam consumption was correlated with the right DLPFC activation (0‐back) , right DLPFC activation (1‐back) , left DLPFC activation (SCWT‐1)  and the left of DLPFC activation (SCWT‐2)  , right DLPFC activation (1‐back) , left DLPFC activation (SCWT‐1) , and left DLPFC activation (SCWT‐2)  after the age, gender, and years of formal education adjustments  and Rey auditory verbal learning test (RAVLT)   than those of the 25th percentile's, while the ulam consumption at the 100th and 75th percentile demonstrated a significantly higher right DLPFC activation for the 0‐back condition , 1‐back condition , SCWT‐1 condition , and the SCWT‐2 condition  as compared with the 25th and 50th percentiles.A further analysis from the univariate ANCOVA is shown in Table P < 0.05, FWE‐corrected) as depicted in Table P < 0.05, FWE‐corrected) and the other activated regions were observed in the superior frontal gyrus, inferior frontal gyrus, and precentral gyrus.Meanwhile, the activated brain region in DLPFC (Brodmann's areas 9 and 46) when performing the N‐back task .ulam, Centella asiatica, was also reported in Thailand, where the herb was not only discovered for improving working memory and alertness, but also in relieving anger as well.Our findings demonstrated that high ulamulam metabolites with the improvement of working memory, however, is yet to be elucidated by future research.The high level of antioxidants and polyphenols in ulam consumption resulted in the better cognitive processing strategies, adaptation and flexibility,ulam had entered a specific brain area  and led to the improved cognitive flexibility, further randomized control trials and pharmacological studies would still be required to validate this presumption. The effect size of the ANCOVA analysis from this study was also considerably large,ulam consumption (at least ½ servings per day) had shown substantially higher Digit Span and RAVLT test scores and greater DLPFC activation than those with lower percentiles of ulam consumption.This study had indicated that a high ulam consumption as demonstrating a greater right‐hemispheric dominance during the visual‐spatial processing stage as compared with lower ulam consumption.Another main finding of this study was the highest DLPFC activation that was found in the middle frontal gyrus during the fMRI scan as well as the other activated regions that had appeared in the precentral, superior frontal, and inferior frontal gyrus. While the attention, working memory, and executive functioning are controlled by the prefrontal cortex that is located at the frontal part of the brain,ulam consumption as demonstrating better left DLPFC activation for both of the SCWT tasks, with the left hemisphere displaying more of the brain activity during the assigned word recognition task. The latest evidence therefore reported the left hemisphere of the human brain as being more affected by the interference than the right hemisphere when naming the color of a word that conflicted with the displayed word . Since the left DLPFC had been selectively activated during the high conflict of the SCWT trials,ulam consumption.ulam consumption, its activation could have been due to the macro‐adjustments of the cognitive control, which minimized the subsequent attentional conflict during the frequent congruent and incongruent SCWT.As the involvement of the middle frontal gyrus while performing the SCWT had been proven from a previous study,ulam consumption and brain activation had been successfully conducted through the use of the fMRI; hence, corresponding to several previous studies that were conducted on the nutrients intake and brain activation with a small sample (n = 15–30).The sample size of this study was also calculated and justified sufficiently, where the investigation on the association between ulam consumption and brain activation from a cross‐sectional study design, randomized control trials through the use of fMRI, and the metabolomic approach are therefore recommended to further determine the efficacy of ulam for improving brain activation as well as to gauge a better understanding of its neuroprotective properties. In addition, we analyzed the brain activation using ROI analysis ; however, whole‐brain analysis is recommended to investigate the ulam consumption related to other activated brain regions in future studies.The main limitation of this study was its inability for elucidating the cause–effect relationship between ulam consumption was related to a high intensity of brain activation in DLPFC using the fMRI technique. However, there is a need to adopt whole‐brain analysis and randomized controlled trials to investigate the role of ulam as a neuroprotective agent affirmatively.In conclusion, this study found that high