Background: The relationship of events occurring during pregnancy and perinatal period with attention deficit/hyperactivity disorder (ADHD) is not clear. Thus, the focus of the current study was to examine the effects of events occurring during pregnancy and perinatal period on ADHD.Methods: A two-phase cross-sectional study was performed across 13 schools in Changsha and Yiyang cities from March to December, 2014. We preliminarily screened all students using CBCL and established the diagnosis using Mini International Neuropsychiatric Interview for Children and Adolescents (MINI-KID) and the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV). A total of 3,418 questionnaires were effectively completed in this study.Results: History of threatened abortion (TA) [odds ratio (OR): 1.707 (1.201–2.426)] (vs. No-TA) and neonatal asphyxia (NA) [OR: 2.497(1.225–5.09)]  showed a positive association with ADHD. On subgroup analysis, TA [OR: 2.216 (1.458–3.369)] (vs. No-TA) was a risk factor for ADHD without comorbidity; instrumental delivery [OR: 2.748 (1.057–7.142)]  and NA [OR: 2.789 (1.222–6.361)]  were risk factors for ADHD in the subgroup of ADHD with comorbidity; TA (vs. no-TA) and NA  were risk factors for ADHD among male students , while low birth weight (LBW)  was a risk factor [OR: 2.054 (1.063–3.967)] for ADHD among female students.Conclusion: TA was a risk factor for ADHD in the absence of comorbid conditions; instrumental delivery and NA were risk factors for ADHD in the subgroup of ADHD with comorbidity; TA and NA were risk factors for ADHD among male students. LBW was a risk factor for ADHD among female students. Attention deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental disorder characterized by hyperactivity, impulsive behavior, and poor attention . AccordiHowever, the pathogenic mechanism of ADHD is not clear. Previous studies have found linkage of ADHD with many factors including genetic and environmental factors. In previous studies, some genes related to the dopamine pathway were shown to be associated with ADHD . With reBased on the limitation of prior studies, we hypothesized that certain pregnant and perinatal events were independent risk factors for ADHD. To test this hypothesis, we assessed the relationship between ADHD and events occurring during pregnancy and perinatal period in a large sample after controlling for other possible confounding factors such as parental rearing style and demographic characteristics. In addition, we hypothesized that risk factors for ADHD pertaining to pregnancy and the perinatal period may vary depending on the sex as well as the clinical profile of ADHD . To test this hypothesis, we performed subgroup analysis in subgroups of ADHD disaggregated by comorbidity and sex to further explore the risk factors for ADHD.n = 18,778) in 13 primary and junior high schools of Changsha and Yiyang cities using the CBCL. A total of 17,071 students qualified the selection criteria and were included in the study, while 1707 students were excluded. The reasons for exclusion were: refusal to complete the questionnaire or incomplete questionnaire (n = 782), questionnaire with >10% missing data (n = 522), and age outside the range of 6–16 years (n = 403). If the score of any subscale in CBCL is higher than the norm, it is regarded as CBCL positive. According to the results of CBCL, students who received a positive CBCL score (n = 3465) and matched students who received a negative CBCL score (n = 3465) were selected from the 17,071 students; these students were asked to complete the investigation of parental rearing attitude and behavior and their parents were asked to complete the investigation of events related to pregnancy and the perinatal period of their children. Finally, a total of 3,418 questionnaires were effectively completed for the investigation of events related to pregnancy and the perinatal period and the egma minen bardoms uppfostran (response rate: 49.29%) among students who received a positive CBCL score (n = 3465) and matched students who received a negative CBCL score (n = 3465). Further, students who received a positive CBCL score (n = 3465) and 10% of students who were CBCL negative (randomly sampled) among 17071 students were diagnosed using MINI-KID and DSM-IV; of these, 1,663 students  were confirmed to have a diagnosis of a psychiatric disorder. Among students (n = 3418) for whom the investigation of events related to pregnancy and the perinatal period and the egma minen bardoms uppfostran was effectively completed, 790 students were diagnosed with ADHD , 780 students were diagnosed with mental disorders other than ADHD, and 1,848 students were identified as healthy based on MINI-KID and DSM-IV. The Ethics Committee at the Second Xiangya Hospital approved this study. All parents or guardians and students in this study gave written informed consent.This study used data from the epidemiological investigation of Hunan children and adolescent psychiatry disorders (age range of subjects: 6–16 years) from March to December, 2014 . This stDemographic variables include age of students, gender of students , place of residence (city/semi-urban areas/village/migrants from countryside residing in city), single-child (yes/no), father’s education level and mother’s education level . Events related to pregnancy and the perinatal period (from parent reports) included maternal health status during pregnancy , threatened abortion (no/yes), mode of delivery , newborn health , birth weight , and feeding patterns .In this study, the section of the child behavior checklist (CBCL)  pertaining to behavioral problems was used to quantify behavioral problems  of children in the age-group of 6–16 years. Each item in this section is scored as 0, 1 or 2. The Chinese version of the CBCL has been validated with satisfactory psychometric properties  .The MINI-KID, a structured interview instrument to diagnose psychiatric disorders among subjects in the age-group of 6–16 years, was designed according to DSM-IV and the 10th revision of the International Statistical Classification of Diseases and Related Health Problems (ICD-10). Both the Chinese parent and child versions of the MINI-KID have been shown to have satisfactory validity , 2011. IThe Egma Minnen Barndoms Uppfostran (EMBU) is an 81-item self-reported scale that is widely used to assess the subject’s memories of parental rearing attitude and behavior. The items are rated on a four-point scale ranging from 1  to 4  for the father and the mother separately. The EMBU consists of 11 factors including father rearing attitude and behavior (6 factors) and mother rearing attitude and behavior (5 factors). The Chinese version of EMBU used in this study has been validated in Chinese adolescents with correlation coefficient between factors and total scale scores ranging from 0.34 to 0.65; the Cronbach’s alpha ranges from 0.65 to 0.86 .M) ± standard deviation (SD), and categorical variables are presented as frequency (%). The association between each research variable and ADHD was tested individually using univariate analyses  and odds ratios (OR) with 95% confidence intervals (CIs) calculated. We also performed multivariate analysis  by including variables that qualified the following criteria: variables that showed a significant association with ADHD (p < 0.1) in univariate analyses; variables meeting clinical plausibility despite not showing a significant association in univariate analyses. For multivariate analysis, we employed two models. In the first model, we included events occurring during pregnancy and perinatal period in the logistic regression model. In the second model, we included demographic variables and parental rearing style on the basis of the first model. In addition, subgroup analysis was also performed by disaggregating subjects with ADHD by comorbidity and gender to explore the association of different subgroups with events occurring during pregnancy and perinatal period in the above two models. P-values in all analyses were two sided, with a significance level of 0.05.Data analysis was conducted using SPSS version 21 . Continuous variables are presented as mean . Further, variables such as migrants from countryside living in cities (vs. city), father punishment, father interference, father rejection, father overprotection, mother emotional warmth, mother interference, mother rejection, mother punishment, single child (vs. non-single child), hypertension of pregnancy , threatened abortion (TA) (vs. no-TA), cesarean delivery , instrumental delivery , neonatal asphyxia (NA) , and low birth weight (LBW)  were found to be risk factors for ADHD  (The demographic characteristics are summarized in  < 0.05) .We included all maternal events occurring during pregnancy and perinatal period in the logistic regression model 1; the results showed that TA [OR: 1.922 (1.399–2.64)] (vs. no-TA), instrumental delivery [OR: 2.238 (1.087–4.606)]  and NA [OR: 2.34 (1.234–4.436)]  were risk factors for ADHD . In modeIn subgroup analysis, we employed univariate analysis and multivariate analysis in the first model –9. In thMultivariate analysis in the subgroup of ADHD without/with comorbidity and gender were adjusted for demographic variables, and parental rearing style. The results of final models showed that TA [OR: 2.216 (1.458–3.369)] (vs. no-TA) was a risk factor for ADHD without comorbidity; instrumental delivery [OR: 2.748 (1.057–7.142)]  and NA [OR: 2.789 (1.222–6.361)]  were risk factors for ADHD with comorbidity; TA (vs. no-TA) and NA  were risk factors for ADHD among male groups , while low birth weight (LBW) [OR: 2.054 (1.063–3.967)]  was a risk factor for ADHD among females groups .The etiopathogenesis of ADHD is not clear; however, both genetic  and enviFirst, we found a strong correlation between TA and ADHD in the total study population, which is not consistent with the results of previous studies . This inCatechol-O-methyltransferase (COMT) gene expression; COMT gene was shown to be associated with ADHD or ADHD traits (Second, in the present study, NA was found to increase the risk of ADHD in the total population, which is consistent with previous findings. For example, D traits . All theMTHFR gene is associated with infant birth weight (Finally, in order to test the stability of results, we performed subgroup analysis after disaggregating the ADHD population by presence or absence of comorbidity and gender. In the subgroup analysis of ADHD population with/without comorbidity, we found that TA and NA were associated with ADHD without other comorbid mental disorders and ADHD with other comorbid mental disorders, respectively, this finding is consistent with the findings observed in the total study population. In addition, we also found that ADHD with other comorbid mental disorders was associated with more events during pregnancy and perinatal period. Instrumental delivery also increased the risk of ADHD with other comorbid mental disorders, which is a novel finding of our study. Prior studies have shown that instrumental delivery increases the risk of fetal injury, included head injuries ; howeverh weight  as well h weight . Lastly,h weight , which ih weight . HoweverSeveral limitations of this study should be acknowledged. First, despite the large sample size in this study, the cross-sectional design of our study does not permit causal inferences. Therefore, a large cohort study is required to further examine the relationship of events occurring during pregnancy and ADHD. Second, the possibility of recall bias for parental rearing attitude and behavior, and events related to pregnancy and the perinatal period cannot be ruled out. Third, parental smoking during the perinatal period has been shown to be associated with a higher risk of ADHD in the progeny ; howeverIn this study, TA, NA, instrumental delivery, and LBW were found to be associated with an increased risk of ADHD in the total study population or in subgroups of subjects with ADHD disaggregated by comorbidity and gender. Among these, TA was found to increase the risk of ADHD without other comorbid mental disorders. Instrumental delivery and NA were found to increase the risk of ADHD with other comorbid mental disorders. TA and NA were found to be risk factors for ADHD among males, but not among females. LBW was a risk factor for ADHD among females.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Ethics Committee at the Second Xiangya Hospital. All parents or guardians and students in this study gave written informed consent.JBL was responsible for data processing and writing of the manuscript. YQH participated in the investigation and writing of the manuscript. YMS participated in the design, investigation, and evaluation of the study. YYZ participated in data collection and analysis. TTM participated in the investigation and evaluation of the study. BX and XLC participated in the investigation and evaluation of the study. YMF participated in data processing. XRL participated in the design, investigation, and evaluation of the study and contributed to critical revision. YTX contributed to critical revision. JPL contributed to critical revision and evaluation of the study. All authors have read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Due to the loss of agro-biodiversity, there is a strong effort to find apparent and efficient mechanisms for the conservation and sustainable use of genetic diversity. A joint monitoring of the diversity and collections structure of the Montenegrin maize landraces conserved in the Serbian (MRIZPGB) and Montenegrin (MGB) gene banks has been conducted in order to improve the composition of the collections and to identify and eliminate possible redundancy. Based on a separate analysis of white- and yellow-orange maize landraces, it can be concluded that the diversity and evolution of distinct maize landraces grown and collected in Montenegro have been simultaneously shaped by both environmental  and socially driven factors . Although it has been determined that the authenticity and variability of the Montenegrin maize landraces gene pool have largely been preserved in the MRIZPGB collection, a significant amount of redundancy was observed. The obtained results will contribute to the cost-efficient conservation of the maize gene pool in the Montenegrin and Serbian gene banks. The recognized and well-preserved original variability of the MRIZPGB and MGB Montenegrin gene pool represents a valuable source for pre-breeding activities on broadening the white and flint maize breeding programmes under temperate conditions. Numerous studies underline the importance of accessions conserved in gene banks for their potential use as donors of favourable traits, especially those for overcoming different abiotic and biotic stresses ,3,4,5,6.Over 1700 gene banks worldwide conserve over 7 million accessions, out of which only a small portion is fully characterized, with a very limited number being used for crop improvement . A largeNumerous problems that make the management and the use of genetic resources more difficult arise from the collection process itself. Many gene banks’ collections started as working collections for taxonomic studies and breeding activities. Afterwards, the collections expanded through collection activities and exchanges with other gene banks. In many cases, this resulted in collections of a considerable size, with an unbalanced composition and an unknown value . FrequenThe high operational costs of collection maintenance  impose tThe Montenegrin plant gene bank (MGB) in Montenegro, and the Maize Research Institute Zemun Polje Gene Bank (MRIZPGB) in Serbia, have initiated a cooperation with the main goal of studying the diversity and the structure of maize landrace collections, gathered in the territory of Montenegro and conserved in these two institutions.The aim of the present study was the following: (i) to conduct a detailed analysis of passport data for maize landraces collected in Montenegro and conserved by the MRIZPGB and the MGB; (ii) to classify the MRIZPGB accessions into homogenous groups based on the morphological analysis , and later to assign MGB accessions into defined groups ; (iii) to identify possible redundancies based on passport data and the morphological analysis.According to the analysis of passport data from the MRIZP gene bank, 64, 225, and 31 accessions were collected in Montenegro during the 1960s, 1970s, and the 1980s, respectively. In relation to the collecting/sponsoring institutions, the Institute of Agriculture Titograd (IAT), the MRIZP, and the Yugoslav Association of Researchers (YAR) together with the United States Department of Agriculture (USDA) collected 136, 124, and 60 accessions, respectively. A pronounced imbalance in terms of the number of collected samples per municipality was observed .A great number of accessions collected in relatively small areas could result in the existence of duplicates, particularly in the municipalities where two different institutions collected a great number of accessions in the same period . Furthermore, there is an issue of similarities/dissimilarities of accessions collected in the area of the same municipality, in different periods, and by various institutions. For instance, out of 42 accessions collected in the area of Ivangrad, 12 were collected during the 1960s by the YAR and the USDA, 13 during the 1970s, including four accessions collected by the IAT and nine by the MRIZP, whereas the IAT collected another 17 accessions in the same municipality during the 1980s .Sixty-eight maize landraces were collected during seven collecting missions at the MGB through the regional project “South East European Development Network on Plant Genetic Resources (SEEDNet)”, (2004–2010). Two accessions were collected in 2016 . AccordiThe MRIZPGB collection of white maize landraces was classified into five clusters (CLs I–V) by hierarchical cluster analysis based on 26 standardized phenotypic traits. CL I encompassed early maturing flint landraces with a short plant and small ears. CLs II and V consisted of dent maize landraces with more robust plants, whereas CLs III and IV represent transitional forms .To emphasize the complexity of interrelations among the studied maize landraces, a correspondence analysis was applied according to morphological similarities. The first and the second dimensions of the correspondence analysis encompass 96%, and 4% of inertia (“variability”), respectively. The CL I landraces, with negative values in the first dimension (D1), exhibit the longest vectors and the greatest scattering, especially landraces 6, 2, 22, 13, 14, and 9, collected by the YAR and USDA in 1963. Landraces 57, 170, 29, 77, 169, 162, and 165, collected by the IAT and the MRIZP in territories of various municipalities during the 1970s and the 1980s, are closely positioned to landrace 1, collected by the YAR and the USDA in 1963 . AccordiThe CL II landraces demonstrate positive values for the D1 axis and negative for the D2. The YAR and the USDA collected only two accessions (landraces 3 and 8) in Podgorica and Danilovgrad in 1963 . HoweverThe differentiation between accessions within CL V, which show positive D1 values, is more pronounced; however, a close positioning of certain accessions can be noticed. These accessions were collected by different institutions in territories of the same or various municipalities in the mid-1970s as follows: accessions 84 (MRIZP) and 157 (IAT), 97 (MRIZP) and 64 (IAT), as well as 82 and 90 (MRIZP), and 148 and 161 (IAT).The greatest number of accessions belongs to CL III (49) and IV (49), with only seven, i.e., ten accessions, respectively, collected by the YAR and the USDA in 1963. The majority of accessions were collected by the MRIZP and IAT in the 1970s. The accessions of these clusters are placed between CL I , and CLs II and V  . FurtherMaize is a cross-pollinated species, and it is very difficult to determine reliably whether two accessions of open-pollinated varieties are duplicates or not. Regardless of the type of information, which is important for decision making , it is very difficult to establish the threshold value that defines the duplicate. Based on the obtained results, it was concluded that collecting activities performed by the YAR and USDA at the beginning of the 1960s were accomplished correctly. Since hybrids were not grown at that time, the 26 accessions of white maize landraces were thought to undoubtedly represent original Montenegrin maize landraces. Based on the distance matrix (Squared Euclidean Distances < 200) of these 26 accessions, a great morphological similarity was established between landraces 6 and 22, and 9 and 14 (CL I), as well as between landraces 15 and 24 (CL III). However, according to the passport data  and the referent gene bank seed collection, these accessions cannot be considered duplicates.Regarding the accessions collected subsequently, according to results obtained, the existence of numerous redundant/duplicate accessions can be expected. Based on the performed statistical analyses, distance matrices (Squared Euclidean Distances < 200), passport data, and the referent seed collection of white MRIZPGB maize landraces, a list of possible duplicate accessions for each cluster was made . AccordiThe discriminant analysis showed that 88.3% of originally classified (MRIZPGB) accessions had been correctly classified, i.e., 20 accessions were incorrectly classified by cluster analysis. Obtained probabilities point out that the highest number of incorrectly classified accessions  were placed between CL III and CL IV, indicating a weak differentiation of these two clusters (probability data are not presented).Since the first discriminant function encompasses 85.5% of variance , and theTwenty-four MGB white maize accessions were assigned to five existing clusters. The majority of MGB white landraces  were assigned to CL I, which encompassed white, early maturing flints, whereas four landraces were distributed to CLs II, III, IV, and V .Based on the distance matrix (the squared Euclidean distance < 200), passport data, and ear photos of the MGB white landrace gene pool, it was determined that landraces 13 M and 30 M, and 18 M and 37 M—all belonging to CL I—represent possible duplicates.The location of collecting sites for both gene bank accessions is provided in The grouping of yellow-orange maize landraces was also performed with the creation of five clusters . Based on cluster mean values, flint landraces prevail in each cluster .The correspondence analysis showed that the first (D1) and the second (D2) axes encompass 98.0% and 1.7% of inertia (“variability”), respectively. The highest positive values of the D1 dimension in the correspondence analyses , were recorded for the CL V accessions . These lLess pronounced positive values of the D1 dimension  were detected in the CL II accessions. In comparison with CL V landraces, these landraces are also flints, but of medium-early maturity (FAO 300), with somewhat smaller kernels and a not-so-robust plant. Only five accessions  were collected by the YAR and the USDA in 1963. The rest (23 accessions) were collected by the MRIZP and the IAT in the mid-1970s, mostly in the Nikšić municipality, where numerous landraces express a great morphological similarity. Hence, the existence of possible duplicates among these landraces is a consequence of the poor coordination of collecting activities among different institutions, as well as the poor implementation of a standard collecting protocol.The CL IV accessions had extremely negative values for the D1 dimension. CL IV encompasses the smallest number of landraces, with the hardest and the smallest orange kernels, with the shortest plants and 14 kernel rows per ear . Only onThe CL I accessions have negative values for D1, while the CL III accessions have negative values for the D2 dimension . The higTwenty-two yellow-orange landraces  collected by the YAR and the USDA in the 1960s can be considered as original Montenegrin maize landraces. These landraces were collected first and there is a lower probability of duplicate occurrence. Although closely positioned in In comparison to early collecting missions, subsequent ones produced a certain number of duplicate accessions that were collected by the MRIZP and IAT in addition to the original accessions. Based on the distance matrix, passport data, and the referent seed collection, the accessions that could be considered duplicates within each cluster were identified . The aveThe discriminant analysis showed that 94.5% of yellow-orange MRIZPGB accessions were correctly classified by the cluster analysis, while the weakest discrimination was between the CL I and CL III clusters. Seven accessions were misclassified. Eigenvalues  show thaOut of the total number of MGB accessions, 46 are yellow-orange landraces. The majority of the MGB accessions were designated to CL IV (32) and CL III (11), while only one accession for CLs I and II was assigned. Not a single accession was designated to CL V.The distance matrix, passport data, and photos of ears determined the absence of duplicate accessions within the CL III gene pool of the MGB yellow-orange maize landraces. Three pairs of possible duplicates were determined within the CL IV accessions . Even though almost half of these accessions were collected in one municipality, only one pair of possible duplicates (26 M and 57 M) was identified.The locations of collecting sites of the MRIZPGB and the MGB yellow-orange maize accessions are presented on the map of Montenegro . The larSince its foundation 1945), the MRIZP breeding programme has included the collecting of local maize populations as well as the introduction of maize germplasm from abroad. In time, the gene bank for maize has established its own comprehensive research programme, using appropriate scientific methods and adequate facilities, and thus affirming itself as the national Yugoslav gene bank for maize. Nowadays, the MRIZP gene bank conserves 2217 accessions of maize landraces from the former Yugoslavia, including 320 landraces collected in Montenegro ,19.45, the MThe initial collecting missions by autochthonous populations of agricultural plants in Montenegro were performed at the end of the 1940s, exclusively for scientific purposes. The implementation of the SEEDNet Project allowed for the establishment of the Montenegrin plant gene bank—MGB in 2004. Currently, the MBG conserves 70 maize landraces and very little has been done so far to study the maize gene pool in Montenegro ,22. PrioThe hilly/mountainous regions of Montenegro, where agricultural production is performed in small and isolated fields, is favourable for great diversity of maize populations. A close study of the passport data of the MRIZPGB accessions shows that the organised collecting missions began at the right time , but also that the large number of accessions were collected in a relatively small area, which could generate the existence of numerous redundant accessions. These findings are in line with the fact that the various institutions collected a very unbalanced number of accessions in the territories of the same municipality (even in the same period), as a consequence of poor planning and coordination of collecting activities . On the The large number of accessions and the presumed high level of duplication, both within and between collections, burdened the efficient use of existing variability. Given the similar issues, many gene banks have become more concerned with the assessment of diversity within their collections and with the improvement of the efficiency of genetic resource management, than with the acquisition of new material ,27. On tOn the map of white landraces collecting sites , sites iMost of the CLs V and II landraces (dents) grown at 450 m, i.e., 250 m above sea level, was collected in the central part of Montenegro. CL II landraces are characterised by the tallest plants, while CL V landraces have the largest kernels . InteresObtained results indicated that a large number of similar accessions within the MRIZPGB white maize gene pool were found in CLs III, IV, and II. Suggested potential redundancies according to passport and morphological data should be validated with molecular markers . When grThe poorest discrimination was between the CLs III and IV landraces. The vicinage of collecting sites for accessions within these clusters indicates a possible common origin and a significant exchange of genetic material via pollen. Similar to reports of Salami et al. , the disThe assignment of white MGB accessions into CL I—early flints, indicated far less variability of these landraces compared to the Montenegrin landraces conserved in the MRIZPGB collection. Although significant efforts have been made within the SEEDNet Project on collecting and conserving maize landraces, this study supports the assertion that the preservation of the maize gene pool in Montenegro was approached with a considerable delay . The rurIn the collection of yellow-orange MRIZPGB maize landraces, accessions with the flint type predominate. According to Pavićević , the higA clear differentiation of CLs IV and V accessions in the correspondence analysis plot indicates the authentic morphology of these landraces, which is opposite to numerous overlapping of landraces belonging to the remaining clusters . AccordiA great concordance is observed on the map comparing collecting sites  along wiIn relation to CLs V and II, an opposite spatial distribution was found for the CL IV and I accessions ; this isThe central position of CL III accessions  indicateWithin the MRIZPGB collection, a large number of Montenegrin indigenous maize landraces are early maturing flinty landraces, and as such, they are carriers of traits important for overcoming different types of biotic and abiotic stresses. Studies performed by Babić et al. ,45 and PThe assignment of the MGB accessions into existing groups indicated that the majority of accessions were classified into CLs III and IV. The landraces similar to CL V accessions no longer exist, while landraces similar to CLs I and II landraces are on the edge of the extinction. It is most probable that the numerous ecotypes ceased to be cultivated due to rural depopulation, and due to the transition to the cultivation of hybrid varieties . In addiThe morphological characterisation of 320 MRIZPGB accessions was conducted according to the CIMMYT/IBPGR Descriptors for Maize  in 2015 The following 26 phenotypic traits were evaluated: plant height, ear height, number of leaves above the ear, total number of leaves, leaf length, leaf width, tassel length, tassel peduncle length, length of the branching part of tassel, number of primary tassel branches, number of secondary tassel branches, kernel row number, number of kernels per row, ear length, ear width at the top of ear, ear width in the middle of ear, ear width at the base of ear, cob width, rachis width, kernel length, kernel width, kernel thickness, kernel hardness, kernel dentiness, and 1000-kernel weight.Maize landraces differing in kernel colour express distinct properties, genetic background, and utilisation value . TherefoAfter the definition of homogenous groups of the MRIZPGB accessions by cluster analysis, 70 MGB accessions were assigned to the appropriate cluster using a discriminant analysis , which aThe authenticity and variability of the Montenegrin maize landrace gene pool have largely been preserved due to collecting activities initiated prior to the introduction of hybrid varieties into commercial production. A higher genetic variability of the Montenegrin maize landrace gene pool was observed in the MRIZPGB collection. Diversity and the evolution of landraces have been simultaneously shaped by both environmental  and socially driven factors . Identified possible duplicates within the MRIZPGB and MGB collections will contribute to the rationalisation of conservation activities for maize genetic resources in Serbia and Montenegro; these will also promote the performance of more efficiently planned studies on the genetic variability and structure of the Montenegrin maize landrace gene pool by molecular markers. The direct incorporation of genetic resources into breeding programmes is inefficient due to the lack of the possibility to collect all the desirable characteristics from a small number of generations while maintaining all the positive characteristics of the elite material. The ambition of the cooperation between MRIZPGB and MGB is to start and speed-up the mobilization of currently scattered, unrecognized, and underused maize genetic resources maintained in two collections, as well as facilitate their use in pre-breeding activities in order to make them suitable for the broadening of the base of white and flint commercial maize breeding programmes under temperate conditions.


There is evidence showing that maternal demographic and pregnancy-related characteristics are associated with LBW. Little attention is given to paternal characteristics, which may be associated with a higher risk of LBW. This study aimed to assess the effect of paternal characteristics on LBW among singleton deliveries at Kilimanjaro Christian Medical Centre (KCMC) zonal referral hospital in Kilimanjaro region, northern Tanzania.this was a secondary analysis of a hospital-based cohort study from maternally-linked medical birth registry data at KCMC between 2000 and 2018. A total of 47,035 singleton deliveries were included in this study. Data analysis was performed using statistical package for social sciences (SPSS), version 20 . Relative risk and corresponding 95% confidence intervals (CI) were used to determine association between LBW and paternal characteristics using log-binomial regression models, with robust standard errors to account for clustering of deliveries within mothers.the proportion of LBW during the study period was 9.6%. After adjusting for maternal characteristics, higher risk of LBW was among fathers with low education level , aged ≤24 years old , and those unemployed . Lower risk of LBW was among fathers aged ≥40 years , but this association was not statistically significant.the study confirmed paternal young age (≤24 years old), paternal low education level and unemployment as predictors for LBW. Current evidence on the effect of paternal characteristics on LBW might suggest that programs and policies should target their engagement as a key strategy for improving birth outcomes during the perinatal period. Future studies should assess how paternal factors are associated with the risk of adverse birth outcomes. Low birth weight (LBW) remains to be devastating adverse pregnancy outcomes in low- and middle-income countries (LMICs) . AccordiPaternal and maternal unemployment has been associated with LBW in Nigeria  while hiStudy design, setting, and population: we conducted a retrospective cohort study utilizing data from the birth registry at KCMC referral hospital, situated in the Moshi Municipality of Kilimanjaro region, Northern Tanzania. KCMC is one of the four zonal referral hospitals in Tanzania, and serves over 15 million people, including women from the local communities (who may come to deliver on their own accord) and referred cases from neighboring regions. An average of 4000 births occur at KCMC each year [ach year . The stuach year ,24. We tData source and collection methods: the data collection methods for the KCMC medical birth registry are well described elsewhere [lsewhere ,25. In blsewhere ,18,26.Study variables: the dependent variable in this study was LBW. LBW was defined as a birth weight of less than 2500g regardless of gestational age, [nal age,  and was nal age, ,11. OtheStatistical analysis: we performed data cleaning and analysis using the Statistical Package for Social Sciences (SPSS version 20) [sion 20) . NumericSocial demographics of the participants: the mean paternal age among 47,035 deliveries in this study was 33 (SD=7.0) years. Majority (54%) were aged between 25-34 years. More than half (60%) had secondary or higher education level, two-third (71%) were unemployed. The majority (55%) of the mothers were aged between 25-34 years, with their mean age of 28 (SD=6.0) years. In all, 86.3% were married and 50.1% had secondary/higher education, 76% were unemployed, and 34% were residing in rural areas with only 55% attended more than 4 ANC visits  among fathers aged less than 24 years, compared to fathers aged 25-34 (9.1%). LBW was also higher among fathers with no or primary education (16.3%), and those unemployed (10.4%) ( (10.4%) .Paternal characteristics associated with: there was a significant association between LBW and paternal age, education level, and occupation in the unadjusted/crude analysis. The risk of LBW was high among fathers aged ≤24 years old , and ≥40 years  compared to those aged 25-34, with low education , the unemployed   . However0, 1.50) .The study aimed to determine the association between paternal background characteristics and LBW among singleton deliveries at KCMC referral hospital, northern Tanzania. The proportion of LBW in all recorded deliveries from 2000-2018 was 9.6%. Independent of maternal characteristics, low paternal education, unemployed, and low paternal age ≤24 years) increased the risk of LBW. The proportion of LBW in this study is higher than the national estimate (7%)  years in with preUnemployment significantly increased the risk of LBW, which could be associated with poor socio-economic status.,13 Poor Strengths and limitations: the large hospital-based birth registry data recorded from 2000-2018 in the KCMC zonal referral hospital enabled us to analyze and establish the association between paternal characteristics and LBW controlling for potential maternal confounders. This study is probably the first, to the best of our knowledge, to report the association between paternal characteristics and LBW in Tanzania and sub-Saharan Africa. As with other epidemiological studies, some key variables, for instance lifestyle factors of the fathers, such as smoking and alcohol use, were not measured and included in the birth registry, which has been associated with LBW in other studies [The study confirmed paternal age, education level and occupation as predictors for LBW. Current evidence on the effect of paternal characteristics on LBW might suggest that programs and policies should target their engagement as a key strategy for improving birth outcomes during the perinatal period. Future studies should assess how paternal factors are associated with the risk of adverse birth outcomes.Maternal characteristics are known factors that increase the risk of LBW;Preconception health counseling which has been shown to improve maternal health status and, in some cases, have been demonstrated to be effective in limiting the incidence of adverse pregnancy outcomes such as LBW has mainly targeted women.This study confirmed paternal young age (≤24 years old), paternal low education level and unemployment as predictors for LBW;Programs and policies should target paternal engagement as a key strategy for improving birth outcomes during the perinatal period.


Hall Effect study confirmed that prepared CH3NH3PbI3−xClx samples are p-type semiconductor having carrier concentration of the order of ~ 1016 cm−3. The charge carrier mobility, mean free path and mean free life time were found to decrease with increasing temperature due to polaronic effect. The excitonic effective mass is estimated to (0.090–0.196)me and excitonic binding energy (15–33) meV, well consistent with Wannier-Mott hydrogenic model and the nature of exciton is likely to be Mott-Wannier type. From electrical measurement, it was observed that charge carrier conduction in CH3NH3PbI3−xClx is governed by migration of 3N In this paper we explain the temperature dependence of excitonic effective mass and charge carrier conduction mechanism occurs in CH When X site is replaced by another halogen element such as chlorine (Cl) into AMX3, it converts to mixed halide perovskites CH3NH3PbI3−xClx. This mixed halide perovskites exhibit semiconducting nature with some exciting properties for photovoltaic and optoelectronic device applications. The single-junction solar cell architecture, based on CH3NH3PbI3−xClx perovskite, shows power conversion efficiency of 25.5%1. Besides, CH3NH3PbI3−xClx thin film shows multi-colored emission5, second harmonic generation or frequency doubling5, bimolecular recombination6, trap-assist recombination7, strong tunable interband transitions8, and ambipolar charge transport9. However, mixed halide perovskite suffers a compositional instability due to halide  segregation10. But a very small addition of Cl into (I/Br) lattice restrains the halide phase segregation, improves the charge carrier mobility, carrier lifetime and stability of solar cells without changing the crystallographic domain size11. These properties are directly correlated with the temperature dependent charge transport behavior in CH3NH3PbI3−xClx and expected to improve device configuration compared to (I/Br) composition.Now-a-days, methylammonium lead halide perovskites (MLHP) are regarded worldwide as an emerging material class in the field of photovoltaic and optoelectronics. The typically chemical formula of MLHP is AMX12, combination of time-resolved terahertz with optical transient reflection spectroscopy (TRTS-TR)13, photoluminescence quenching (PLQ)14, time resolved electro-absorption spectroscopy15, time-of-flight (TOF) photoconductivity16 and Hall effect measurement17 have been carried out to explore the transport properties of lead halide perovskites. These pioneer studies reveal the presence of non-Langevin charge carriers6, strong back-scattering on free carrier dynamics12, existence of higher hole mobility compared to electron mobility13, large electron–hole diffusion lengths (˃1 μm)14, electric field assisted charge carrier seperation15, grain size dependent mobility16, and influence of self-doping in carrier concentration17. Despite the advancement in deep-seated perceptive in transport behavior, a lot of issues such as charge-carrier type, the mechanisms for restraining carrier mobility, origin of electron–hole recombination rates, donor or acceptor ionization energy, position of Fermi level, effective mass of charge-carriers, grain boundary barrier height, carrier mean free path-life time in CH3NH3PbI3−xClx still perplexes the researchers. It is well known that the mixed halide perovskite undergoes a phase transition from orthorhombic-tetragonal-cubic at 160 K and in between (315–330) K, respectively19. However, the study of charge carrier dynamics is limited to tetragonal phase while it is also important to explore such properties for cubic phase above room temperature (RT).Up to now, multiple approaches such as transient terahertz (THz) spectroscopy3N 25. The conception of polaron is used to explain intrinsic electrical properties because it describes the interaction between charge carriers and phonons. The interaction of charge carriers with the organic part has significant influence on the effective mass, temperature and magnetic field dependent carrier transport and electrical conductivity of the mixed halide perovskite material.Recently, it is reported that charge carries generate self-induced rotation of the organic part 2, spray pyrolysis (Spray)5 and repeated dipping-withdrawing (Dipping)3 techniques in an ambient atmosphere. Our study confirmed the presence of tetragonal and mixed cubic-tetragonal phase in CH3NH3PbI3−xClx thin film. The film morphology was composed with spherical, rod, cuboid and polyhedral like crystal grains of sizes 100 nm to 2 μm5. However, this article concentrates on the charge carrier transport of CH3NH3PbI3−xClx thin films using Hall Effect and dc-electrical conductivity measurement as a function of temperature, ranging from RT to 378 K. The carrier concentration, mobility, excitonic effective mass, excitonic binding energy, Fermi level, grain boundary barrier height and mean free life time of charge carriers are calculated from Hall Effect study. Furthermore, activation energies in CH3NH3PbI3−xClx thin films are exposed from electrical study. The effect of polarons on charge carrier dynamics and possible carrier conduction mechanism in CH3NH3PbI3−xClx thin films has been discussed. Therefore, in-depth study of charge carrier transport and conduction mechanism above RT for CH3NH3PbI3−xClx may provide a broader impact in the arena of perovskite research.In our previous work we report the characterization of nano-crystalline CHH) and carrier concentration (nc) have also been calculated.Temperature dependent Hall voltage measurement is done at a constant magnetic field of 9.815 KG and the variation of Hall voltage is shown in Fig. 3NH3PbI3−xClx is a p-type material. It is reported that a lot of Pb, CH3NH3, I and Cl vacancies present in the films27 as because precursor solution was prepared by dissolving CH3NH3I and PbCl2 at a molar ratio 3:1. However, Pb and CH3NH3 vacancies play the vital role for p-type conductivity of prepared CH3NH3PbI3−xClx thin films because of the lower formation energy of Pb and CH3NH3 compared to halogen vacancies.From Fig. 3NH3PbI3−xClx thin films are shown in Fig. 32. Table 3NH3PbI3−xClx obtained in different studies. It is reported that the breaking of inversion symmetry generates Rashba effect33 which enhance longitudinal optical phonon scattering by displacing the organic part or Pb-halogen bending or stretching and limits the increase of charge-carrier mobility at room temperature.The temperature dependent carrier concentration  and mobility  for (a) CDC, (b) spray and (c) dipping deposite CH3NH3PbI3−xClx samples is found to increase exponentially  and excitonic effective mass μ* = 0.1me for calculating excitonic binding energy of perovskite. However, in this study ε = 9 has been used for calculating 35. The values of e, 0.42me, 0.38me (me is the rest mass of electron) in the TR-I and 2.16me, 2.23me, 3.04me in the TR-II for CDC, spray and dipping deposited samples, respectively. The estimated 37. Taking the effective mass of electron e from theoretical calculations38, the excitonic effective mass μ* is estimated to (0.090–0.196)me using equation, 1/μ* = 1/e35, found for tetragonal phase (≥ 145 K). However, μ* in TR-II is found high because of non-parabolic nature of valance bands in CH3NH3PbI3−xClx. Theoretical study39 suggested that lead vacancies diminish anti-bonding atomic orbital overlap resulting in flatten of valance bands. Moreover, polaronic effect40 and strong hydrogen bonding due to van-der-Waals interactions39 may also increase μ*.The acceptor ionization energy, Fermi level and effective mass of charge carriers have been calculated from the temperature dependent Hall voltage measurement. The temperature dependent carrier concentration in the presence of a constant magnetic field can be represented by following equations,entclass1pt{minimab of all perovskite samples has been calculated using the equation,The excitonic binding energy, Eb in TR-I and TR-II is due to temperature dependent polaronic effect of CH3NH3PbI3−xClx. The extending radius of the lowest bound state r* is calculated to ascertain the nature of excitons in CH3NH3PbI3−xClx using the equation, r* = ε (me/μ*) rb, where, rb is the Bohr radius. The r* is estimated to 5.29 nm, 3.40 nm, 3.52 nm in TR-I and 2.49 nm, 2.48 nm, 2.43 nm in TR-II for CDC, spray and dipping deposited samples. The value of r* is larger than the lattice constants  of CH3NH3PbI3−xClx indicating that the exciton is weak and likely to be Mott–Wannier type.The excitonic binding energy is found (15–24) meV for TR-I and (32–33) meV for TR-II, which is well consistent according to Wannier–Mott hydrogenic model. This variation of EF can be determined by knowing the value of Ea, Na and The position of Fermi level Ea, Na and EF for both higher and lower temperature regions are tabulated in Table c and Na are not equal but vary with temperature and film processing methods as well, indicates CH3NH3PbI3−xClx is very sensitive to environment, chemical composition, growth parameters etc. The Fermi energy is found negative means EF lies below the acceptor level.The values of E41, the trapping states create a depletion region in the grain and a potential barrier at the interface. In a semiconductor sample the relation between charge carrier mobility and grain boundary barrier height can be expressed asb is the grain boundary barrier height and hT1/2) vs 1/T graph  and tabulated in Table m) and mean free time (τm) in CH3NH3PbI3−xClx thin films can be calculated from the following equationsAccording to conventional Drude-Sommerfeld model, the carrier mean free path  K.In the mixed halide perovskite, the transition from tetragonal to cubic phase occurs at temperature ~ 330 K3NH3PbI3−xClx is rather complex. It is strongly dependent on temperature, vacancies or interstitial defects and activation energies of the materials. In general, CH3NH3PbI3−xClx includes Pb, CH3NH3 and halogen vacancies as suggested from theoretical and experimental studies46. The low energy vacancies create a rotational re-organization of organic CH3NH3 dipoles resulting in free dipolar polarons in mixed halide perovskite20. Eames et al.45 proposed that these vacancies migrate from one site to the neighboring site due to low activation energy. They found activation energies involved 0.58 eV and 0.84 eV for migrating 3N 47 also reported that the predicted activation energies for migration of 3N 48. In Fig. B is the Boltzmann constant, T is the absolute temperature, I, ΔEII and ΔEIII are activation energies for region I, II and III, respectively. From the slope of lnσ vs 1000/T plot  decreases with increasing temperature in region I (294–317) K. The initial decrease is caused by the strong interactions in between organic–inorganic part and rotational disorder of methylammonium cation within perovskite structure. In addition, longitudinal optical phonon scattering may also decrease the conductivity with temperature in region I. The activation energy ΔEI  K, σdc increases rapidly and ΔEIII ˃ ΔEII. The activation energies ΔEIII are found close to 0.84 eV for CDC and spray deposited and ΔEIII = 0.57 eV for dipping deposited samples. The rapid increase of σdc in region III occurs because of diffusion or migration of CH3N 6 octahedra or neighbor central vacant site47. Therefore, the conduction processes are attributed to the migration of 3N 3NH3PbI3−xClx. Moreover, the concentrations of charge carriers (majority holes) which are activated with acceptor ionization energy (Ea) favorable for the transport across grain boundaries of CH3NH3PbI3−xClx samples.From Fig. on I 294–17 K. The3NH3PbI3−xClx thin films have been evaluated as a function of temperature ranging from room temperature to 378 K. Hall measurement study confirms that the prepared CH3NH3PbI3−xClx films are p-type semiconductor with carrier concentration of the order of ~ 1016 cm−3. The carrier mobility found to decrease with increasing temperature due to polaronic effect. At RT, the mobilities for CDC, spray and dipping deposited samples are 46, 49 and 20 cm2V−1 s−1, respectively. The excitonic effective mass (μ*) is estimated to be (0.090–0.196)me and excitonic binding energy (Eb) varies from (15–33) meV, suggesting Mott-Wannier type exciton. Additionally, the grain boundary barrier height, mean free path and mean free life time of charge carriers have also been evaluated. Electrical study reveals that multi-conduction processes are involved in CH3NH3PbI3−xClx, which is governed by the migration of vacancies, vacancy-assisted diffusion of mobile ions and accumulation of holes across grain boundaries.In summary, the charge carrier conduction mechanism of CH3NH3PbI3−xClx thin films was prepared using the following procedure. Methylamine (CH3NH2) solution was mixed with hydroiodic acid and ethanol at room temperature. The solution was perturbed continuously by stirring with a magnetic stirrer at a constant speed in an ice bath for 2 h. The color of CH3NH3I solution transforms from red to transparent and heated at 100 °C for 4 h in a furnace to achieve white-colored CH3NH3I powder. Finally, CH3NH3I and lead(II) chloride powders were dissolved in anhydrous N,Ndimethylformamide at a molar ratio 3:1 to achieve a mixed halide perovskite precursor solution.Lead halide perovskite solution for fabricating CH3NH3PbI3−xClx thin films have been fabricated at ambient atmosphere by three different methods. These methods are (a) Chemical dip-coating (CDC) (b) Spray pyrolysis (spray) and (c) Repeated dipping-withdrawing (dipping). The details of preparing CH3NH3PbI3−xClx solution and different steps which have been made in these methods to deposit CH3NH3PbI3−xClx thin films have been reported elsewhere5.CHTemperature dependent Hall voltage was measured at constant magnetic field of 9.815 KG using the conventional van-der-Pauw method. The magnetic field used for the study of Hall Effect was provided by electromagnets designed and produced by Newport instruments Ltd. England. The circuit arrangement for temperature dependent Hall Effect measurement is shown in Fig. The resistivity of all samples was measured by van-der-Pauw method. Experimental setup of the van-dar-Pauw’s specimen to measure the resistivity with varying temperature is shown in Fig. Supplementary Information.


Bartonella spp. are zoonotic bacteria with small mammals as main reservoirs. Bartonella spp. prevalence in small mammals from Myanmar and Sri Lanka are yet unknown. (2) Methods: Small mammals were snap trapped in Sri Lanka and Myanmar in urban surroundings. Spleens-derived DNA was screened for Bartonella spp. using conventional PCR based on three target genes. Positive samples were sequenced. (3) Results: 994 small mammals were collected comprising 6 species: Bandicota bengalensis, Bandicota indica, Rattus exulans, Rattus rattus, Mus booduga, and Suncus murinus. In Myanmar, the Bartonella prevalence in Bandicoot rats (68.47%) was higher than in Rattus rattus (41.67%), Rattus exulans (21.33%), and Suncus murinus (3.64%). Furthermore the prevalence in Myanmar  was twice as high as in Sri Lanka . In Sri Lanka, Bartonella spp. occurred almost exclusively in R. rattus. In Myanmar, Bartonella kosoyi was mainly detected (56%), followed by Bartonella sp. KM2529 (15%), Bartonella sp. SE-Bart D (12%) and Bartonella henselae (1%). In Sri Lanka, B. phoceensis (60%) and Bartonella sp. KM2581 (33%) were predominant. (4) Conclusions: Bartonella spp. were detected in all investigated small mammal species from Myanmar and Sri Lanka for the first time. Bartonella kosoyi and B. henselae are zoonotic. As these small mammals originated from urban settlements, human bartonellosis seems likely to occur.(1) Background:  Bartonella spp. are vector-borne and commonly transmitted via blood-sucking arthropods. Rodents are known to be the main reservoir of different Bartonella spp. with fleas the main vector within and between rodent populations [Bartonella elizabethae, Bartonella grahamii, B. henselae, Bartonella tribocorum, and Bartonella vinsonii subsp. arupensis are described in association with rodent reservoirs [The genus Bartonella includes over 40 species and subspecies  of gram-ulations ,3. Amongservoirs ,4,5.Bartonella spp. infections are associated with various human diseases such as cat-scratch disease (Bartonella henselae), trench fever (Bartonella quintana), and Oroya fever (Bartonella bacilliformis). Cases of bartonellosis associated with rodents are described from all over the world with clinical symptoms such as fever, muscle and joint pain, neuroretinitis, and endocarditis [B. henselae in human patients from Laos and Taiwan [Bartonella tamiae and other Bartonella spp. were found in febrile patients from Thailand [carditis ,7. Regard Taiwan ,9. FurthThailand . Until n2 and population size is 54 million people [2 [Nonetheless, rodent-associated zoonotic Bartonellae can pose a risk in countries such as Myanmar and Sri Lanka where close contact between humans and rodents is common ,11. Myann people  of whichn people ,14. Aboueople [2 . As of 2eople [2 .The genera Bandicota, Rattus and Mus include synanthropic pest rodent species that live in close contact with humans in those countries . In otheBartonella spp. prevalence in fleas associated with cats and rodents in an area along the Thai-Myanmar border. Six out of 90 fleas were tested positive for Bartonella DNA and a newly described Bartonella species was detected from one flea collected from a Rattus surifer [Bartonella spp. infections in dogs with positive individuals showing even multiple infections [In Myanmar, a previous study  detected surifer . In Sri fections .Bartonella spp. to assess the risk for human health and companion animals.To date, no study of Bartonella prevalence in small mammals as possible reservoir hosts has been conducted in Myanmar and Sri Lanka. Because of the close contact between rodents and humans in various parts of these countries, it is important to study rodent-associated Thus, the main aim of this study was to detect small mammal species living close to human settlements in Myanmar and Sri Lanka and to evaluate the Bartonella prevalence in those small mammal species for the first time. Another purpose was to identify the Bartonella species in these samples by molecular techniques to evaluate their zoonotic potential.Small mammals were trapped at five sites in Myanmar and Sri Lanka in the years 2018 and 2019. Traps were set in villages at structures used for rice storage and rice processing in Myanmar and Sri Lanka .In Myanmar, study sites were located in granaries or mills in the rural delta and coastal zone, where the main agricultural production is located. Dayēbo  and Pike Kye We  are rural areas mainly characterized by rice production . At the Small mammal trapping in Sri Lanka was conducted in Pasyala (Gampaha District) i , Pasyala (Gampaha District) ii , Kahapathwala  i  and Kahapathwala  ii  that are characterized by an increase in housing units indicating population growth in these areas. Sinhapura (Polonnaruwa District) i  is characterized by rural agriculture with 39.1% employment in this sector ,33. TherSmall mammals were obtained and killed by snap trapping as part of regular pest rodent control operations. In the countries where the work was conducted, snap trapping is not regarded as animal experimentation. Hence, no animal ethics permits were required. In Myanmar, small mammals were trapped from June to December in 2018 and from May to December in 2019. In each site, 10 snap traps  and 5 local-made bamboo traps were baited with unhulled rice and set opportunistically in and around 10 small-holder rice storage facilities for 450 trap nights per site and year . In Sri Lanka, small mammals were captured in two trapping sessions in June–December 2018 and two trapping sessions in March–August 2019 (for details see ). In eaccytochrome b gene [Bartonella spp.Trapped small mammals were identified to species level based on morphology  supplemee b gene . Standar® 24 tissue homogenizer  was used for mechanical disruption with 5500 rpm for 2 × 15 s with a 10 s break between both runs.Before processing the spleen samples for DNA extraction, the ethanol was washed out with phosphate-buffered-saline  by transferring in PBS and incubating for four hours and drying afterwards. Then, all samples were homogenized with 0.6 g sterile ceramic beads  four times the sample’s weight with PBS (pH 7.2). The PrecellysAfterwards the DNA was extracted using the QIAamp DNA Mini Kit  according to the manufacturer’s instructions. The DNA samples were measured qualitatively and quantitatively with the NanoDrop ND-1000 (PeqLab Biotechnologie GmbH). Samples with a DNA concentration >40 ng/μL were diluted with the kit’s elution buffer to receive a DNA concentration of 20–40 ng/μL for each sample.Bartonella spp. was detected in DNA samples by conventional polymerase chain reaction (PCR) targeting the NADH dehydrogenase subunit (nuoG) with an amplicon size of 346 base pairs (bp). Additionally, all samples were further analyzed in two PCRs targeting the gltA gene (378 bp) and a fragment of the 16S-23S rRNA ITS region (453–780 bp) [The presence of –780 bp) ,37,38,39nuoG was detected with previously published PCR protocols and the genus specific primers nuoGF (5′-GGCGTGATTGTTCTCGTTA-3′) and nuoGR (5′-CACGACCACGGCTATCAAT-3′) [Bartonella spp. DNA based on the gltA gene the genus specific primers BhCS.781p (5′-GGGGACCAGCTCATGGTGG-3′) and BhCS.1137n (5′-AATGCAAAAAGAACAGTAAACA-3′) were used. The amplification consisted of 45 cycles of denaturation at 95 °C for 30 s, annealing at 53 °C for 30 s and elongation at 72 °C for 1 min [The amplicon of the CAAT-3′) . For theor 1 min ,38. The https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 25 February 2021)) [PCR products were prepared with DNA Gel Loading Dye (6×)  for gel electrophoresis in 2% agarose. The results were visualized by UV light using the UVP GelSolo streamlined gel documentation . Afterwards 125 positive samples were selected for sequencing to cover all small mammal species detected at each location in Myanmar and Sri Lanka in both seasons of each collection year. The samples were purified for sequencing using the NucleoSpin Gel and PCR clean-up kit  as recommended by the manufacturer. Further, samples were sequenced commercially with forward and reverse primers of the 16S-23S rRNA ITS region . The sequences were trimmed using Bionumerics v.7.6.1.  and compared to available data in GenBank with BLASTn (y 2021)) . ObtaineBartonella spp. in small mammals were determined by the Clopper and Pearson method [Confidence intervals (95% CI) for the prevalence of n method  with setP (probability) < 0.05. Only samples tested positive for all three target genes were considered positive for Bartonella spp.The independence of compared prevalence rates concerning small mammal species, age, sex, trapping year, season, and in Myanmar additionally regarding the habitat, was tested using Pearson’s Chi-squared test. Tests with two variables were considered to be significant if Bandicota bengalensis ; B. indica ; Rattus exulans ; R. rattus ; Suncus murinus   and 166 at granaries . In Sri Lanka, 499 small mammals were captured representing six species: B. bengalensis (n2019 = 1); B. indica ; Mus booduga (n2018 = 4); R. exulans ; R. rattus ; S. murinus   . Of thes19 = 10) . All smaBartonella spp. DNA by conventional PCR targeting all three target genes altogether .Overall 248 out of 994 samples  tested positive for together . Altoget2 = 41.273, df = 1, P < 0.0001). In contrast, Bartonella prevalence in Sri Lanka was similar between years (10–28%) and sites (10–28%)  .Bandicota than in Rattus spp. and S. murinus . The prevalence of Bartonella in the genus Rattus was significantly higher compared to S. murinus  in Myanmar. Likewise, in Sri Lanka the prevalence in R. rattus was higher than in all other small mammal species which were caught by lower numbers .In Myanmar, prevalence was higher in Bartonella spp. in Myanmar was detected between males and females , between the years 2018 and 2019  and the rainy and dry season . In Myanmar, the prevalence of Bartonella in small mammals caught in granaries was significantly higher than in small mammals caught in mills . However, due to the unequal distribution of small mammal species in granaries and mills, these results may be regarded as slightly distorting. The prevalence of adult small mammals was significantly higher compared to the lower prevalence of sub-adult small mammals . There was no significant difference in Sri Lanka for the Bartonella prevalence between adults and sub-adults , males and females , the years 2018 and 2019 , and the seasons  [No significant difference in the prevalence of  0.9443)   and R. rattus  and at the sites Dayēbo  and Kadoke Phayargyi . In Sri Lanka, 11 out of 80 positively tested samples  showed double infections. Here, co-infections were detected only in one rodent species, R. rattus, caught at the sites Pasyala and Kahapathwala.Several Bartonella co-infections were detected in small mammals from Myanmar and Sri Lanka. In Myanmar 30 out of 168 positively tested small mammals  with double infections were detected by PCR targeting the ITS region. Double infections in Myanmar were most common in Bartonella-positive samples based on the ITS region (53%) and 11 different Bartonella strains were detected . Bartonella sp. KM2529  and Bartonella sp. SE-Bart-D  were detected in small mammals from Myanmar as well. The most frequently identified Bartonella species in Sri Lanka was B. phoceensis  followed by Bartonella spp. KM2581 . The detected Bartonella spp. were evenly distributed and no effect of small mammal species, age, season and habitat was apparent. However, B. kosoyi. was exclusively detected in B. indica from Myanmar.Sequence analyses were performed for 125 detected . In MyanBartonella spp., (Accession Number: EF407566) and (Acc. No.: EF213776) were detected in one R. exulans caught in Kan Nyi Naung, Myanmar. Two R. norvegicus from China were earlier positive for Bartonella sp. RN25BJ, respectively, Bartonella sp. RN28BJ. Furthermore, 10 samples showed <97% identity with sequences in GenBank, but 98–100% within group-similarity and can be assembled in groups with >98% agreement  in Sri Lanka which are most similar to Bartonella sp. SE-Bart-D. Three samples from S. murinus caught in 2019 at two different sites in Myanmar form Group 3 and show high similarity to an uncultured Bartonella clone 5199  are in agreement with the previously mentioned studies [Especially in Southeast Asia rodent-associated infectious diseases are emerging . The Baral hosts . Severalal hosts ), Laos , China al hosts ,21, and al hosts ). The Ba studies .R. rattus was introduced via ship trading to this island [Xenopsylla cheopis) [Bartonella spp. prevalence in rodents at different islands. An explanation for this distribution was the varying ecological condition affecting the flea populations as essential vectors for the infection of rodents [Bartonella spp.; however, the interaction between host and vector has not been clarified, yet [The prevalence of Bartonella in small mammals from Sri Lanka was significantly lower than in Myanmar. In Sri Lanka, the cosmopolitan species s island . A possicheopis) . Moreove rodents . Variousied, yet . The misied, yet .Bartonella spp. prevalence among small mammal species in Myanmar. Bandicoot rats had the highest (63%) and S. murinus the lowest prevalence (4%). In a recent study in Nepal, a low prevalence of Bartonella spp. was detected in B. bengalensis (26.3%) and high prevalence in S. murinus (64.1%) while R. rattus (43.3%) was intermediate. In Bangladesh both B. bengalensis (63.2%) and S. murinus (42.9%) showed higher prevalence than R. rattus [S. murinus might be due to a lack of host-specific vectors in Sri Lanka in our study. However, information about the ectoparasite fauna of S. murinus is lacking. Additionally, low Bartonella prevalence was detected in other insectivores (Sorex araneus) in England [S. araneus showed that some blood-sucking arthropods harboring rare Bartonella spp. may feed and transmit these Bartonella spp. exclusively to shrews. Furthermore, S. araneus was found to be less affected by ectoparasites compared to endemic rodents [S. murinus may be related to an adapted ectoparasite fauna in Bangladesh but not in Myanmar or Sri Lanka. In Sri Lanka, the predominant rodent species R. rattus showed the highest prevalence (18%)—apart from the single B. bengalensis, which also tested positive. The prevalence for R. rattus and the high prevalence of B. bengalensis in Myanmar are in line with R. rattus in Asia .Bartonella spp. in small mammals from urban regions [Bartonella spp., which can infect several mammals [Bartonella spp. in Sri Lanka was detected concerning the different sites. All small mammals were caught in small-holder rice storage facilities in Sri Lanka and thus conditions were not as contrasting as at the sites in Myanmar.In Myanmar, the prevalence was higher in urban sites (Pike Kye We and Dayēbo) compared to the three rural sites. In line with our study, other authors reported higher prevalence of  regions ,59,60. ABartonella spp. These bacteria can persist in the reservoir host without harming them drastically [Sigmodon hispidus). The decrease of prevalence may be due to the development of immunity with age [Bartonella spp. was detected in B. bengalensis and B. indica and in Sri Lanka especially in R. rattus. Paziewska et al. [Myodes glareolus overcomes Bartonella infections within 1–2 months, however the length of time it takes to overcome the infection in Apodemus flavicollis is not definitely known. Furthermore, reinfections in M. glareolus were detected less frequently than in A. flavicollis [Bandicota spp. (more often collected in Myanmar) longer than in Rattus spp. (more often collected in Sri Lanka), which would explain the contrasting results in both countries.In Myanmar, the prevalence in adult small mammals showed significantly higher prevalence than in subadults, possibly due to the life cycle of stically . In contstically  reportedwith age . No diffa et al.  showed tvicollis . Thus, BIn the present study, there was no effect of the seasons. Morway et al. showed that Bartonella prevalence rates were influenced by climatic conditions . MoreoveBartonella spp. strains for a long time and that single strains replace each other. This infection strategy may have an impact on the probability of double infections in small mammals [Bartonella spp. infections with more than one species and related Bartonella strains [B. bengalensis, R. rattus, and R. exulans in Myanmar and exclusively in R. rattus in Sri Lanka at all sites. High co-infection rates (56%) with two or more genotypes, including recombinant variants, were detected in gerbils and fleas from Israel [Bartonella spp. than their hosts [Kosoy et al.  showed t mammals . However strains . This dim Israel . In Taiwir hosts . In Soutir hosts ,71,72.B. kosoyi, first described in 2019, was the predominant species for the samples from Myanmar. Analysis confirmed a phylogenetic association of B. kosoyi with the zoonotic B. grahamii, B. tribocorum, and B. elizabethae [Bartonella elizabethae and B. elizabethae-like species show host-specificity for rodents of the genus Rattus but can infect other small mammals like Bandicoot rats and S. murinus as well [B. kosoyi mainly in Bandicoot rats . Before the Tel Aviv strain was named B. kosoyi sp. nov., this strain had been detected in Bandicoot rats and R. rattus from Bangladesh [B. kosoyi.Our sequence analysis of Bartonella-positive samples identified 11 different Bartonella strains. Moreover,  as well . We detengladesh  and Nepangladesh . Furtherngladesh . TherefoBartonella sp. SE-Bart-D isolated from the Oriental rat flea (X. cheopis) in Egypt. Bartonella sp. SE-Bart-D shared 85–90% similarity with B. tribocorum, B. grahamii, and B. elizabethae, which are all known as zoonotic agents [Some of the sequencing results from Myanmar were highly similar (99.81–100%) to c agents .Bartonella phoceensis was the predominant result for all sequenced samples. Bartonella phoceensis was first detected along with Bartonella rattimassiliensis in France in 2004 [B. phoceensis was detected in small mammals from Asia. In Japan and Thailand B. phoceensis was detected in R. rattus [S. murinus and Rattus tanezumi [B. phoceensis was associated, with a similarity of 94%, with uncultured Bartonella species found in Rattus norvegicus from Taiwan [B. phoceensis is not recognized as a zoonotic agent and no case of human illness has been described.In Sri Lanka,  in 2004 . Since t. rattus ,77 and itanezumi . Moreovem Taiwan . Until nBartonella henselae was detected in one S. murinus from Myanmar in the current study. The organism with the highest similarity B. henselae Q5BJ-CW was first isolated from a dog in China. Bartonella henselae is known to be mostly transmitted by wild and domestic cats to humans [B. henselae, is associated with fever and regional lymphadenopathy [B. henselae in Italy and New Zealand. Therefore, rodents can be a source of infection due to close contact to humans [Bartonella spp. can be an underestimated threat to public health. Although the number of zoonotic Bartonella spp. has emerged, bartonellosis in humans is reported very rarely [o humans . Cat screnopathy . Though o humans ,80. Due y rarely .Bartonella spp. were detected for the first time in small mammals from Myanmar (34%) and Sri Lanka (16%) in this study. The high prevalence is in line with previously reported results from other countries in Southeast Asia. The reasons for the lower prevalence in Sri Lanka may be missing vectors or different ecological conditions. All examined small mammal species were Bartonella-positive in Myanmar and Sri Lanka. Eleven different Bartonella strains were detected of which three are considered potentially zoonotic. Thus, small mammals may serve as reservoir hosts for zoonotic Bartonella spp. in these countries. In Myanmar and Sri Lanka, contact between humans and rodents is frequent in rice storage and processing facilities. With an increasing degree of urbanization and thus more frequent contact between humans and rodents, these rodent-associated pathogens may pose a higher risk for human health.


Depression is one of the most common psychiatric disorders. Recently, studies demonstrate that antidepressants generating BDNF not only maintain synaptic signal transmission but also repress neuroinflammatory cytokines such as IL-6 and IL-1β. Therefore, promoting BDNF expression provides a strategy for the treatment of depression. Our recent research has indicated that programmed cell death 4 (Pdcd4) is a new target for antidepressant treatment by facilitating BDNF. Herein, we modified Pdcd4 specific small interfering RNA (siPdcd4) with the rabies virus glycoprotein peptide (RVG/siPdcd4) which enables it cross the blood-brain barrier (BBB). We found that RVG/siPdcd4 complex was selectively delivered to neurons and microglia and silenced the expression of Pdcd4, thereby up-regulating the level of BDNF and down-regulating IL-6 and IL-1β expression. More importantly, RVG/siPdcd4 injection attenuated synaptic plasticity impairment and protected mice from CRS-induced depressive behavior. These findings suggest that RVG/siPdcd4 complex is a potential therapeutic medicine for depression. Major depressive disorder (MDD) is one of the most common psychiatric disorders and causes a leading disease burden . The limProgrammed cell death 4 (Pdcd4) as a tumor suppressor has been discovered for several years. Pdcd4 inhibits translation initiation of protein indirectly by competitively combining with Eukaryotic Initiation Factor 4A (eIF4A) . The co-Small interfering RNA (siRNA)-mediated gene knockdown offers a therapeutic strategy to overcome neurological disorder. For the benefit of small molecular weight, flexible application and less immune response, siRNA has been applied to neuropathy treatment , 17. In In this study, we explicitly addressed the gene-targeting strategy for depression treatment. Based on our previous research, we found hippocampal local injection of lentivirus-carried siPdcd4 could rescue CRS-induced depression on mice . To makeWild-type C57BL/6J male mice aged 4–6 weeks (weight 22–25 g) were used in the animal experiments. All mice were purchased from Vital River Laboratory Animal Technology Co. Ltd  and carefully reared in SPF laboratory of animal experimental center of Shandong University.Mice were put into ventilated polypropylene bound stress tube during 9:00–11:00 a.m. every day, and the mice in control group were grabbed at same time. The food and water were deprived during experiment.Depressive-like phenotypes in these models were validated by tail suspension test (TST), and the detail protocol is related to Depressive-like phenotypes in these models were validated by forced swimming test (FST), and the detail protocol is related to Depressive-like phenotypes in these models were validated by sucrose preference test, and the detail protocol is related to For the preparation of siPdcd4 with RVG-9dR peptide complex (RVG/siPdcd4), the mixture was generated following the protocol which is related to The detail steps are related to Applying the RVG/siRNA complex for animal, the detail steps of experiment was illustrated in SH-SY5Y,BV2, HT-22, Hela cells, and the primary hippocampal neurons were used in this report, and the detail cell culture protocol is related to The cell and brain tissue lysates were used for western blot assay, and the detail protocol is related to Relative target gene mRNA expression was normalized to β-actin mRNA and calculated using the The concentration of IL-6, IL-1β, and BDNF were quantified by specific Enzyme-Linked Immunosorbent Assay (ELISA) kits  according to the manufacturer’s instructions. The detail steps are related to The cells were fixed in paraformaldehyde solution for 15 min, and cells nuclear were stained with DAPI for 10 min at room temperature. All the images were captured with Laser confocal high content imaging analysis system .The changes of synaptic plasticity in mice were evaluated by golgi staining, and the detail steps are related to The statistical analysis was performed using PrismGraphPad 7 Software. The analysis method is related to Our recent research found that Pdcd4 is an endogenous suppressor of BDNF, meaning that silencing Pdcd4 will increase the expression of BDNF in nerve cells . At firsOur previous results indicate that silencing of Pdcd4 expression in hippocampus has antidepressant effect in depressive mice, suggesting siPdcd4 can be applied for depression therapy as an antidepressant. How to cross the BBB is an important obstacle of siRNA drug delivery for mental illness therapy. Previous studies have found that siRNA mixture with 9dR peptide-modified rabies virus-derived-RVG (RVG-9dR) can realize nerve cells targeting . To testIn order to prove the facilitation of RVG/siPdcd4 on BDNF expression. We detected the changes of BDNF in brain-derived cells  by western blot and ELISA, and the results showed that BDNF were significantly up-regulated in RVG/siPdcd4 group compared with RVG/siNC group Fig. . MoreoveNext, to prove the ability of RVG-9dR peptide on blood–brain barrier (BBB) traversing, Cy5 labeled-siPdcd4 was modified by RVG-9dR peptide (RVG/Cy5-siPdcd4) to visually track the movement of RVG/siRNA, and then the mixture was injected into mice through tail vein. Cy5 fluorescence signal was captured by imaging system to trace the distribution of Cy5-siPdcd4 in vivo. The results showed that only a small part of fluorescence was detected in brain at 6 h after intravenous administration Fig. . At 24 hChronic restraint stress (CRS) is an established model for mice depression. Our previous work has shown that CRS could up-regulated the expression of Pdcd4 that contribute to synaptic plasticity impairment and depression-like behavior by inhibiting the expression of BDNF . To addrIn order to evaluate the antidepressant effect of RVG/siPdcd4 treatment, behavioral tests were performed on depressive mice. The results showed that compared with CRS group the immobility time of RVG/siPdcd4 group in TST and FST were significantly reduced Fig. , and sucIn this study, we have demonstrated the potential of RVG/siRNA therapy to induce the knockdown of Pdcd4 in the CNS. More specifically, this therapeutic research has been proved to prevent synaptic lesion and BDNF expression abnormalities in CRS-induced depression model.siBACE1 or sisynuclein are developed to help these proteins degradation [RVG peptide-guided siRNA system is firstly applied to depression treatment. RVG peptide is a rabies virus-derived 29 amino-acid residue fragment, and it has drawn much attention on the drug-modification research owing to its BBB penetrability . Becauseradation , 24. HowN-methyl-d-aspartate receptor (NMDAR) antagonist, as a rapid-acting antidepressant, has been approved for depression treatment by the FDA in 2019 [siPdcd4, as a new type of antidepressant, could be applied to depression molecular therapy. Based on the monoamine hypothesis of depression, we have developed conventional antidepressants. However, with these drugs wildly used, the drawbacks are becoming more and more manifest. (1) the delayed onset is associated with an increased risk of suicidal behavior in the first month of antidepressant treatment. (2) approximately one-third depressed patients fail to respond to multiple antidepressant treatments and are considered treatment-resistant depression . Ketamin in 2019 . With di in 2019 . Based o in 2019 , 33. Our in 2019 . Therefo in 2019 , 35. In  in 2019 , 37. In In summary, we have shown that RVG-9dR provides a method for siPdcd4 delivery to brain, and it fully has antidepressant effect via promoting BDNF expression. These finding have solved the problem of neuro-targeted drug delivery in vivo, significantly accelerating the prospect of siRNA-based therapeutic for depression.Supplementary information


Attention deficit/hyperactivity disorder (ADHD) is one of the most frequent neurodevelopmental disorders in childhood and adolescence. Choosing the right treatment is critical to controlling and improving symptoms. An innovative ADHD treatment is neurofeedback (NF) that trains participants to self-regulate brain activity. The aim of the study was to analyze the effects of NF interventions in children with ADHD. A systematic review was carried out in the CINAHL, Medline (PubMed), Proquest, and Scopus databases, following the PRISMA recommendations. Nine articles were found. The NF improved behavior, allowed greater control of impulsivity, and increased sustained attention. In addition, it improved motor control, bimanual coordination and was associated with a reduction in theta waves. NF combined with other interventions such as medication, physical activity, behavioral therapy training, or attention training with brain–computer interaction, reduced primary ADHD symptoms. Furthermore, more randomized controlled trials would be necessary to determine the significant effects. Attention deficit/hyperactivity disorder (ADHD) or hyperkinetic disorder is one of the most common mental disorders in childhood and adolescence . It is aThere are different genetic and environmental factors among the causes of attention deficit/hyperactivity disorder. ADHD tends to run in families, with a risk of 5 to 10 times higher among first-degree relatives [Throughout childhood, ADHD is related to inattention, poor planning ability, and impulsivity, causing further deterioration as external demands increase . This geTherefore, early interventions in children are essential, in order to reduce the repercussions in adolescence and adulthood. Among the effective treatments available for ADHD, the main differences are related to the type of intervention , the age of the patient, the cost, the available patient and caregiver time, the expected effectiveness in the reduction of symptoms, the adverse effects, safety, and tolerability ,11.The main treatment for ADHD continues to be pharmacological, with psychostimulant drugs (methylphenidate or amphetamines) being the most widely used . HoweverAmong the different non-pharmacological treatment strategies, neurofeedback (NF) has been considered an innovative ADHD treatment . NF is aThe NF focuses mainly on improving self-control over patterns of brain activity, decreasing theta waves , and/or increasing beta waves  ,18. ThisSome systematic reviews have analyzed the positive effects of non-pharmacological interventions ,21, and A systematic review was performed following the guidelines of the PRISMA statement  .The search was carried out through the following databases: CINAHL, Medline (via PubMed), Proquest, and Scopus. The search equation using the Mesh terms was “Child AND attention deficit hyperactivity disorder AND neurofeedback”. The search was conducted in June 2021.The inclusion criteria were: (1) randomized controlled trials, (2) children sample (up to 13 years), (3) NF as a treatment for ADHD, (4) comparison of NF with a controlled group or other interventions, (5) published in English or Spanish, (6) published in the last 5 years.The exclusion criteria were: (1) no randomization, (2) mixed samples (adults and children) or adult population sample, (3) interventions without NF, (4) only NF interventions without a control group or other interventions.The selection of articles was carried out in three steps. The title and abstract were read first, followed by reading the full text. Finally, a critical reading of the studies was carried out to assess the methodological quality and risk of bias.The quality of the included studies was assessed following the levels of evidence and grades of recommendation by the Oxford Center for Evidence-Based Medicine (OCEBM) . Risk ofFirst, two authors independently reviewed the title and abstract of the articles found. Second full text was read. A third author was consulted in case of disagreement.To extract the data from each study, a data collection table was created. The variables obtained in the selected articles were: (1) author, year of publication, country; (2) design; (3) sample size; (4) aim; (5) type of intervention, duration, and follow-up; (6) main results.n = 9 studies were included. The study selection process is shown in The initial search provided 332 articles. After removing duplicates and those that did not meet the inclusion criteria, a total of n = 3). The main characteristics of all the included studies are listed in All included studies were randomized controlled trials ,32,33,34Comparing the clinical efficacy of the NF-SCP and the computerized cognitive training (CogT), an improvement was found in the symptomatology perceived by parents and teachers after training. Furthermore, the effect of NF-SMR combined with game-based CogT in children with ADHD showed positive therapeutic effects on brain waves, except for sensory motor activity  as well as an improvement in symptomatology .p < 0.01 for the left hemisphere and t (17) = 3.97, p < 0.01 for right hemisphere) [On the other hand, it is found that NF-SMR training improved motor control and more specifically bimanual coordination among children with ADHD . In addiisphere) , althougisphere) .p = 0.01), being more effective compared to single medication treatment [Comparing NF with methylphenidate (MPH) treatment, teachers reported significantly lower ADHD symptoms in the group MPH, but there were no differences between groups in parental report . In addireatment . Some aureatment . Althougreatment , and othreatment .Some papers compared the effects of NF with a physical activity intervention ,33. CombAnalyzing NF and the behavioral therapy intervention, some authors found that both treatments achieved improvement on response control and attention .p = 0.0177) [Finally, comparing the NF with attention training based on the brain–computer interface, an improvement was found in inattentive symptoms in ADHD children, being an option for treating milder cases or as an adjunctive treatment  .ADHD is one of the most common neurological disorders in childhood. Although there are different therapeutic interventions, the goal of this research was to evaluate the efficacy of NF interventions in children.NF improved the symptomatology in children with ADHD, because of learning through video games, attending to their mistakes, and training functions nonconscious control—such as attention, achievement of objectives, self-control, and self-regulation of attention levels, and concentration—in addition to inhibiting distracting stimuli . Also, iIn addition, another of the benefits found was the improvement in motor control and bimanual coordination. ADHD in children is often associated with poor motor control, coordination problems, and difficulty controlling strength , which aIt seems that the NF is related to a reduction in theta waves, although the results were not clear. Literature also shows divergences, some authors found significant individual learning curves for both theta and beta over the course of the intervention, although individual learning curves were not significantly correlated with behavioral changes ,40. OtheRegarding drug treatment, NF is considered a viable alternative therapy for specific groups of children with ADHD who do not respond to medication or have severe side effects ,22. The Alternative therapies combining NF plus physical activity improved attention and short/long-term memory. Other studies indicate great benefits in the improvement of cognitive, behavioral and physical symptoms after mixed physical exercise programs, particularly moderate to intense aerobic exercise ,43. IndeBehavioral therapy with NF improved response and attention control, as pointed out by other authors . Even otNF combinate with the training of attention based on the brain–computer interface shows benefits in inattentive symptoms, even some authors indicate that this type of training program is beneficial in cases of ADHD that present more severe symptoms .One promising aspect of NF is its relationship to procedural learning, so its benefits can be more durable over time after treatment. Technology development provides different treatment options, combining traditional drug treatment, along with NF, physical activity, or cognitive training, all strategies provide positive therapeutic effects on brainwaves and ADHD symptomatology. Although NF can be used as complementary in patients who have significant side effects to stimulant medication or in patients whose family refuses to try the medication, further research is needed to corroborate its effects in each of the behavioural areas of the children with ADHD.This study had a number of limitations to be acknowledged. First, although all studies use NF as an intervention, the great variability in the duration of the intervention may influence the heterogeneity of the results. Also, the sample sizes of the controlled randomized trial were small. Future research would be necessary to analyze the NF effects in each of the behavior areas of children with ADHD. Furthermore, more randomized controlled trials would be necessary to determine the significant effects and the duration of the effects over time.NF showed a significant improvement of the symptoms in children with ADHD. This treatment improved behavior, allowed greater control of impulsivity, and increased sustained attention. In addition, it improved motor control, bimanual coordination, and was associated with a reduction in theta waves. NF combined with other interventions such as drugs, physical activity, behavioral therapy training, or attention training with brain–computer reduced primary ADHD symptoms. However, more scientific evidence is needed to support its efficacy and greater implantation in clinical practice.