Despite several decades of work in improving childhood nutrition in Ethiopia, stunting remains a major public health concern with lack of evidence on its effect on school performance. Therefore, this study aimed at determining prevalence of stunting and the impact on academic performance among schoolchildren attending primary schools in Meskan District of Southern Ethiopia.During October 2016, we interviewed primary school children and their parents, collected anthropometric measurements of children, and conducted school record reviews among 408 randomly selected children attending ten primary schools. Univariate and multivariable linear regression were performed to assess stunting associated with academic performance among schoolchildren. A total of 378 primary schoolchildren were involved in the study giving a response rate of 93%. The prevalence of stunting was 16.9%. Grade repetition, place of residence, class rank of students and absenteeism from the class was negatively associated with the academic performance. In general, the present study children had relatively high prevalence of chronic undernutrition. We found that school underperformance was significantly high in the study area. The study underlines the need for nutrition interventions targeting rural areas to improve children’s academic performance. Poor academic achievement has been a major area of concern for educators, parents, and school children for more than three decades. There are several reasons for children to underperform at school such as age at enrollment, absenteeism, concentration in the classroom, psychosocial factors, illness and nutritional problems. Nutritional problems are among major factors that affect learning and school performance –3. Some Over the past decade, Ethiopia has shown encouraging progress in reducing undernutrition. However, as local studies conducted in Ethiopia revealed the baseline levels of chronic undernutrition (stunting) still remain high. According to the Ethiopia National Nutrition Program (NNP) baseline survey conducted in 2010, 23% of adolescent girls were stunted . Study cAlthough chronic undernutrition is a public health problem in Ethiopia, there is little documented information on the nutritional status of schoolchildren and the impact on their academic performance in Southern Ethiopia. Recently, the Ethiopian Ministry of Education recognized the impact of poor health and nutrition on children’s ability to learn, their school attendance, and concentration . The natA school-based randomized cross-sectional survey was employed from October to November, 2016 in primary schools of Meskan District in Southern Ethiopia, a food insecure district. Up to 150 undernutrition cases are reported monthly from all health facilities of the District . AccordiThe source population included all primary school children in Meskan District, with children from randomly selected primary schools constituting the study population. Primary school children, who did not intend to transfer schools during the intervention period, were included. Exclusion criteria included; schoolchildren with obvious congenital or physical growth measurements. We excluded schoolchildren whose parents did not provide written consent or were unwilling to participate.The sample size was calculated using StatCalc in Epi Info 7. The following parameters used to determine the sample size: 23% of adolescents were stunted , 95% conTen primary schools were selected from the forty primary schools using proportional to size (PS) allocation method. Number and list of students were obtained from school. From each selected schools study participants were selected by simple random sampling based on sampling frame existing in the schools (students’ roster).A structured questionnaire was developed by reviewing relevant literature, such as the nutrition baseline survey report for the National Nutrition Program of Ethiopia by Ethiopian Public Health Institute (EPHI) . The queHeight was measured in an erect position using a calibrated wooden height-measuring board  with a sliding head bar while children were barefoot. Their height was measured in duplicate and recorded to the nearest 0.1 cm or in triplicate whenever the deviation between the first two measurements was > 0.5 cm and the average of all measurements was taken. The age of children in completed years was obtained from student’s records and confirmed from their parents. In this study, stunting was defined as height-for-age z-score (HAZ) of equal to or less than minus two standard deviation (− 2 SD) below the mean of a reference standard .The overall subjects the students were given in the academic year 2015–2016 were considered to evaluate the academic performance of the students. Annual average grade score was evaluated by taking the result of two consecutive semesters of the year. To assess the relationship of chronic undernutrition (stunting) on academic performance, average marks of the overall subjects the students received were divided into two categories, poor scores and good scores, based on a cut-off mark of 50%. This cut off point was determined by considering the pass mark set by federal ministry of education, Ethiopian .The outcome variable in the study was academic performance, operationalized as students’ average marks of the overall subjects. Stunting in children was considered as an independent variable in this study. Durable household assets, home animals, housing conditions and vehicles were considered in the construction of household relative wealth index (RWI) using a principal component analysis after checking all the assumptions. RWI divided and ranked into quintile .The data were entered in double, cleaned and checked for missing values and outliers, and analyzed using IBM SPSS statistics for windows, version 23.0. The Z score value for height-for-age was calculated using the WHO AnthroPlus software .Descriptive analyses were performed using frequencies and percentages for categorical variables. Variables which were significant at p-value < 0.2 in the bivariate linear regression model analyses were candidate for entering into the multivariable linear regression model to identify the independent predictors for academic performance. A p < 0.05 was considered statistically significant. We present the results of the linear regression as parameter estimates (ß), p-values and 95% confidence intervals.We identified 408 primary school children in the study; however, 30 schoolchildren (17 boys and 13 girls) declined participation, leaving 378 (93%) schoolchildren paired with their parents from ten primary schools who were enrolled in our study. The study children (n = 378) had girl to boy ratio of 1.21:1. The mean age of the children was 12.8 years (SD ± 1.3) with a range of 10 to 15 years. Two hundred two (53.4%) of the schoolchildren were attending grade 6 and while a few students 30 (7.9%) were attending grade 8. Majority of the study participants 273 (72.2%) were from families who were currently lived in rural areas. Two hundred five (54.2%) of them had fathers who attended primary school. Two hundred twenty eight (60.3%) of enrolled students had fathers who were employed as farmers.One hundred five (27.8%) respondents were categorized in the fourth wealth quintile Table .Table 1SIn the academic year 2015–2016; 42 (11.1%) students repeated a grade due food shortages of which 28 (66.7%) were in their households. The overall prevalence of stunting was 16.9% (95% CI 13–20.6%). The prevalence of stunting was significantly higher in boys (18.7%) than girls (15.5%). The average score for among children was 64.52 (SD ± 8.45) and the majority  scored poorly (Table The majority of the schoolchildren 357 (94.4%) reported that they have good relationships with their peers, and 369 (97.6%) had good relationships with their families. Two hundred thirty nine 63.2%) schoolchildren reported being bullied by one of their families and 233 (61.6%) reported being punished at school. A majority of the parents 303 (80.2%) reported that they have to perform non-school work at their home (Table % schoolcThree hundred twenty five (86%) of the parents reported that they regularly attend the school meeting (parent–teacher meeting). The majority  of the parents said that they always discuss their children schooling with students while 34.9% of the parents support their children in doing their homework Table .In bivariate linear regression Table , grade rThe overall prevalence of stunting in this study was 16.9%, similar to a study conducted in West Ethiopia 17%) [% [7] andThe current study revealed underperformance when compared with a study conducted in Uganda and India of the primary school children was achieved better academic performance , 26. ThiThe finding of this study showed that grade repetition, class rank and absenteeism were associated with academic performance of students. Consistent with this study finding, a study done in Ethiopia, India and Malaysia, chronic undernutrition is associated with grade repetition , 26, 27.In general, the present study children had relatively high prevalence of chronic undernutrition. We found that school underperformance was significantly high in the study area. But, chronic undernutrition was not associated with poor academic performance of grade. The majority children were from subsistence farming households and consume foods grown in their local area. In study settings, the production of foods are mainly using for cash crops. However, there is ample evidence that children’s academic performance outcome can be affected by other factors that were not captured in this study, including micronutrient deficiencies, lack of food availability in their households, medical problems, below average intelligence, specific learning disabilities, attention deficit hyperactivity disorder, emotional problems, a poor sociocultural home environment, psychiatric disorders, or environmental factors, and parasitic infections. The study underlines the need for nutrition interventions targeting rural areas to improve children’s academic performance of grade.This study used only anthropomorphic measurements and did not assess the micronutrient status of study participants.There may have been differences in the evaluation system for schoolchildren academic performance among the study schools.Furthermore, physical performance capacity and motor skills were not measured in this study.


Staphylococcus lugdunensis is an emergent virulent coagulase-negative Staphylococcus that is increasingly responsible for severe infections. In an attempt to generate informative sequence data for subtyping S. lugdunensis, we selected and sequenced seven polymorphic variable number of tandem repeats (VNTRs) to develop two new methods: a classic length-based multiple-locus VNTR analysis (MLVA) method and a tandem repeat sequence typing (TRST) method. We assessed their performances compared to two existing methods, multilocus sequence typing (MLST) and multivirulence-locus sequence typing (MVLST) for 128 isolates from diverse clinical settings and geographical origins. The clustering achieved by the four methods was highly congruent, with MLVA discriminating within clonal complexes as defined by MLST. Indeed, MLVA was highly discriminant compared to MLST and MVLST in terms of number of genotypes as well as diversity indexes. Sequencing of the seven VNTRs showed that they were stable, and analysis of sequence polymorphisms provided superior discriminatory power. The typeability, reproducibility, and epidemiological concordance of these new methods were excellent. Of note, no link between clustering and clinical settings was identified. This study demonstrates that MLVA and TRST provide valuable information for molecular epidemiological study of S. lugdunensis, and represent promising tools to distinguish between strains of homogenous lineages in this clonal species. Staphylococcus lugdunensis is a coagulase-negative Staphylococcus (CoNS) belonging to the normal human skin microbiota2 that is increasingly recognized as a virulent pathogen in both community-acquired and healthcare associated infections4. This commensal bacterium has mainly been associated with serious infections, such as skin and soft tissue infections6, bone and joint infections8, but also bacteremia and endocarditis10. Thus, the pathogenicity of S. lugdunensis is closer to that of S. aureus than that of other CoNS; it may be related to virulence factors such as a fibrinogen binding protein11, a von Willebrand factor-binding protein12, synergistic hemolysins13 or a metalloprotease14.S. lugdunensis strains such as macrorestriction analysis using pulsed-field gel electrophoresis15 and multilocus sequence typing (MLST)16. The latter is considered as the gold standard method for epidemiological typing and population genetic study of S. lugdunensis. Recently, we developed a multivirulence-locus sequence typing (MVLST) method, based on the sequence data of seven known or putative virulence-associated loci17. MVLST was significantly more discriminant than MLST, even allowing a trilocus sequence typing method for microepidemiological purposes. Phylogenetic analyses by MLST and MVLST revealed a clonal population structure, a mutational evolution of this pathogen, and a lack of hypervirulent lineages.Several molecular typing methods are currently available for 18. Molecular typing based on the analysis of repeat copy number at multiple VNTR loci, known as multiple-locus VNTR analysis (MLVA), is a genotyping method that is being used for strain comparison, and can also provide insights into population structure19. This method has already been successfully applied to many other bacterial species and showed a high level of discrimination23. Especially, measuring repeat polymorphisms in clonal microorganisms can provide a solid basis for genetic type assignment24. In addition, to increase the discriminatory power of MLVA, sequencing repeated sequences that display internal variability can be performed25.An interesting source of genetic polymorphism is provided by tandemly repeated sequences, known as variable number of tandem repeats (VNTRs), whose number of repetitions varies at different rates depending on the different loci and even allelesS. lugdunensis, and (ii) to evaluate and compare the added value of these new methods with the existing methods MLST and MVLST in terms of S. lugdunensis strain discrimination within clonal groups.The aims of this study were to identify VNTR loci and to sequence them in order (i) to develop the first length-based MLVA method and a sequence-based MLVA (tandem repeat sequence typing) method for S. lugdunensis clinical isolates by MLST and MVLST. Data for 20 isolates were derived from our previous studies17, while we performed MLST and MVLST analysis of 108 additional clinical isolates for the purposes of the present study.To determine their diversity and whether they belong to major clonal complexes, we analyzed 128 http://bigsdb.web.pasteur.fr/staphlugdunensis). ST3 was the most common ST (n = 29), followed by ST2 (n = 15) and ST24 (n = 15). The 25 STs were distributed over 7 clonal complexes (CC1 to 7) and 2 singletons, as shown in the minimum spanning tree . Two new STs, designated ST31 and ST32, were incorporated in the online database (atlLR3) to 11 (for isdJ). Twenty-one trilocus virulence types (VTTs) were assigned; 3 major VTTs  were shared by 81 isolates. Five new VTT were identified (VTT34 to 38). The clustering obtained by MVLST was similar to that observed by MLST. For instance, VTT15 and VTT16 corresponded to ST2 and ST3 respectively, VTT1 clustered into 4 STs  from CC1 and 3 linked VTTs  belonged to ST24.MVLST performed on the 128 isolates revealed that the number of individual alleles for each of the three genes ranged from 10 (for SLUG_16930 and T16). Pathogenic and carriage isolates overlapped in their genotype distribution, with 4 of 7 CCs containing isolates from both sources. However, although carriage isolates did not cluster in a unique lineage, 59% (10/17) belonged to CC3 and 53% (9/17) belonged to VTT16.No correlation was found between genotype and clinical origin; for example, CC1 was shared by isolates from 8 different clinical contexts. Isolates recovered from hematogenic infections (blood or osteoarticular isolates) or from skin and soft tissue infections did not cluster in separate lineages. Nevertheless, an interesting trend was observed: 4 of 5 device-associated infection isolates belonged to CC3  were located in non-coding regions. The tandem repeat (TR) unit size of the 7 VNTRs was between 24 and 58 bp  tree Fig. . For theThe distribution of the isolates did not allow us to identify any correlation between MCs and clinical settings. However, 12 of 17 (70%) carriage isolates and 4 of the 5 device-associated infection isolates clustered into MC2 Fig. , as obseS. lugdunensis isolates as 55 MTs were clustered into 6 MCs as opposed to 25 STs into 7 CCs. Interestingly, MLVA efficiently subdivided some CCs such as CC1 and CC5  in the TRs of all loci, and showed that no insertion or deletion was present in flanking sequences. For each VNTR, alleles defined for the overall panel of 128 isolates ranged from 4 (SLU7) to 27 (SLU3) . A complete overview of these allelic profiles is presented in Table Analysis of the 7 VNTR sequences revealed a total of 21 TRST types (TRTs) among the 30 panel test isolates; epidemiologically-related isolates belonged to the same TRT, which is in favor of an versus 5 STs) and for CC6 (2 STs versus 10 TRTs).Figure As for MLVA, no correlation was observed between TRTs and clinical settings, but the majority of carriage (n = 11) and device-associated infection (n = 4) isolates remained clustered (TRST cluster I). Of note, 23 isolates collected from Strasbourg University Hospital between November 2013 and March 2016 could not be distinguished by both MLST (ST3) and MLVA/TRST (MT1/TRT1). As a fact, they could be considered as a clone. Interestingly, 14 isolates from 7 different clinical settings and 9 of the 16 carriage isolates belonged to this clone.26. Simpson’s DI calculated on the basis of the 123 remaining unrelated isolates showed that MLVA (DIMLVA = 0.933) was more discriminatory than MLST (DIMLST = 0.898) and MVLST (DIMVLST = 0.845) (Table TRST = 0.943) was higher than the analysis of the size of the VNTRs.To assess the respective discriminatory power of the MLST, MVLST, MLVA and TRST methods, we excluded 5 of the 10 isolates that were isolated from the same patient and that were shown to be identical by all typing methodsTable S. lugdunensis clustering was highly congruent with MLVA typing, and the probability that a pair of isolates with the same MT also shared identical TRT was 84%  of MLVA, compared with that of MLST and MVLST, showed that MLVA had high congruence with MLST (AW = 0.826) and MVLST (AW = 0.815) Table . This inth, 20th and 30th subcultures, indicating that the 7 VNTRs were stable.Examination of VNTR loci stability revealed that, for each isolate, identical MLVA profiles (in terms of TR number and sequences) were generated for the original isolates and their 10IAS). The linkage disequilibrium was shown to be significant in the LIAN 3.7 analysis, indicating a clonal population structure for the S. lugdunensis isolates. IAS was calculated at 0.1819 for MLVA and 0.4639 for TRST at a significance of P < 1.00 × 10−5.A quantitative analysis of the linkage between alleles from the 7 VNTR loci was performed by calculating the standardized index of association , and compared them with MLST as a reference method and with the virulence-associated trilocus method (MVLST). The relevance of these new molecular tools was investigated against a genetically diverse set of 128 S. lugdunensis isolates collected from various clinical settings and geographical origins.Because S. lugdunensis genomes available in GenBank database were initially identified in silico. Finally, seven VNTRs were selected because they met the preconditions described in the methods section and for their discriminatory power. PCR amplification and sequencing of the seven VNTRs were achieved for all the 128 isolates tested representing a typeability of 100%. MLVA proposes here a set of markers with different DIs (0.090 to 0.679). Markers with a moderate DI (<0.5), presumably reflecting their slow rate of evolution, define clusters. Conversely, VNTRs displaying more diversity and more rapid evolution reflect variability within clusters. Our MLVA method is similar to those proposed for S. aureus20 or S. epidermidis21, in terms of number of VNTRs and diversity indexes. Of note, 5 of the 7 VNTRs presented similarities (80 to 90% Blast identity) with other staphylococci, including S. aureus and S. haemolyticus, but the 7 primers are specific to the species S. lugdunensis excluding the possibility of false positive amplifications.One hundred TR sequences contained in the sls genes, as described by Heilbronner et al.28. However, these VNTRs could be attractive because the number of repeats is possibly more subjected to selective pressure. Indeed, previous MLVA studies of Staphylococcus29 and Streptococcus agalactiae22 found high variability in genes encoding Sdr adhesins and FbsA surface protein, respectively. Thus, these targets would be interesting to include in an MLVA method based on a whole genome sequencing approach.In the midst of all VNTRs identified with Tandem Repeats Finder software, some have been eliminated because fragments were too long to be amplified and sequenced in standard PCR conditions. For example, some were located in genes encoding putative surface-exposed proteins, like T group. Thus, this comparison confirms the coevolution of virulence-associated loci studied with housekeeping genes as shown previously17, and validates the use of the trilocus method as a good epidemiological tool while significantly reducing costs.We compared MLVA with the widely used MLST and the alternative trilocus MVLST. This latter method allowed us to define nearly the same number of profiles as MLST and was consistent with MLST, as unique STs or their single locus variants (SLVs) and double locus variants (DLVs) were found in each VTS. lugdunensis isolates16. Thus, our findings suggest that the loci targeted by MLVA provide a robust measure of genetic relationships. The greater discriminatory index of MLVA made it possible to distinguish between isolates within CCs defined by MLST, but this appeared highly CC-dependent. Indeed, some clones, especially CC1, showed a significant diversity with many genotypes identified by MLVA and TRST. On the contrary, CC2 was less discriminated by these methods, suggesting that CC2 has emerged more recently than CC1. The higher discriminatory power of MLVA in some lineages defined as clonal by MLST has previously been described for several other species such as Listeria monocytogenes23 and Streptococcus agalactiae22.Analysis of the seven VNTRs on the overall panel of isolates revealed that MLVA was highly discriminant compared to MLST in terms of number of profiles as well as discriminatory index. This could be explained by the fact that VNTRs evolve more rapidly than housekeeping genes, limiting the MLST resolution. However, although these two methods measure different evolutional mechanisms at multiple genomic loci, similar clustering was obtained, probably due to the rarity of recombination among i.e. size homoplasy)31. Thus, sequencing was selected not only as a method for repeat number verification, but also to detect single nucleotide polymorphisms in addition to allele size, SNPs being less prone to homoplasy32. It allowed us to propose a TRST method, which proved more discriminating than MLVA. Indeed, 55 MTs were identified by analyzing repeat array sizes, whereas 69 TRTs were identified by sequencing, indicating that a quarter of these alleles were misclassified by conventional MLVA. Considering that the growing use of whole genome sequencing, with its continuously decreasing costs and increased rapidity33, the present TRST method offers an additional tool for a worldwide traceable picture of S. lugdunensis clones. New alleles and TRST types will be certainly identified by in silico-TRST analysis as new genome sequences become available, contributing to a global evaluation of S. lugdunensis epidemiology.On the other hand, MLVA is generally considered as an unreliable phylogenetic method because of the high frequency of the independent evolution of the same repeat array sizes in different isolates  resulted in a highly similar clustering of isolates (44 MTs) while slightly decreasing the discriminatory capacity (DI = 0.920). This simpler version of the MLVA method could allow potential future use of multiplex PCR assays with fluorescent primers and determination of allele size by capillary electrophoresis.MLVA and TRST have strong potential for inter-laboratory comparisons, providing accurate and reproducible data that can easily be exchanged. We have proved that the VNTRs studied were stable during laboratory subcultures, in terms of number of repeats as well as nucleotide sequences, and found complete reproducibility. Furthermore, the epidemiological concordance of the related strains has been validated. Thus, our new MLVA and TRST methods fulfill a number of criteria recognized as important for the successful implementation of a typing method and the interpretation of its resultsS. aureus, for which MLST data do not provide any information regarding the pathogenicity of isolates, because changes in virulence correspond primarily to changes in the accessory, rather than core genome34. Like S. aureus, S. lugdunensis evolution is predominantly clonal17, therefore the study of the accessory genome could contribute to a better understanding of the genetic basis of these different clinical behaviors. On the other hand, the majority of carriage isolates and those collected from device-associated infections of this work were distributed in one complex whatever the typing method. Thus, it would be interesting to analyze a larger number of worldwide invasive and carriage S. lugdunensis isolates to measure the ratio of pathogenic to carrier strains for the various lineages. This ratio, combined with the study of the accessory genome, would help to investigate the existence of successful commensal clones.In this study, no strong correlation was observed between genotypes and clinical settings of the 128 isolates. This could suggest that host susceptibility plays an important role in the propensity of isolates to cause disease, and/or that the virulence potential of isolates is not reflected by MLST or MLVA data. This has been described for 16, CC1 (n = 31) and CC3 (n = 26) were the most common clonal complexes identified by MLST for the 98 isolates collected at Strasbourg University Hospital. Within these CCs, the most prevalent STs were the primary founders of these complexes, ST6 (n = 14) and ST3 (n = 26) respectively. Interestingly, a particularly prevalent clone (n = 23), including virulent and carriage isolates, was identified by both MLST (ST3) and MLVA/TRST analysis (MT1/TRT1), even if the discriminatory power of these latter typing methods is high. Thus, whole genome (wg) sequencing, and particularly wgMLST, wgTRST and/or virulome determination, would be useful to identify markers to distinguish between such isolates. On the other hand, the fact that these isolates have been regularly identified from different patients and different hospital wards over a 24-month period suggests that this could be a clone capable of long-term persistence. While ST3 has already been identified16, this present study is the first to describe it as a persistent clone. This has already been described by Cheng et al.35, who identified a major endemic ST6 clone of oxacillin-resistant S. lugdunensis in a tertiary medical center in Taiwan. However, unlike Cheng et al., ST3 isolates are not multiresistant to antibiotics, so their persistence cannot be directly related to antibiotic pressure; it could be linked to a higher fitness under certain environmental or clinical conditions. In the light of these results and that of Kassem et al.36, who showed that clinical surfaces are frequently contaminated by S. lugdunensis, further investigations are needed to explore the mode of transmission and environmental reservoirs of this species.As previously reported in other studiesS. lugdunensis typing. MLVA and TRST are the most discriminant typing methods available to date. Furthermore, the analysis of the polymorphism of seven VNTRs in terms of variations in copy number of repeats, as well as internal sequence, allowed us to predict CCs with high accuracy while distinguishing strains belonging to some lineages. Sequence-based MLVA may prove particularly useful because it yields unambiguous data that can easily be exchanged and used for inter-laboratory comparisons. Resolving phylogenetic diversity to a high level in this clonal species, MLVA and TRST represent promising tools which could help to elucidate the global epidemiology and evolution of S. lugdunensis as well as to identify outbreaks.In conclusion, this work provides a description of the first two VNTR-based methods for S. lugdunensis human isolates were used in this study , collected during the VISLISI trial (Virulence of Staphylococcus lugdunensis in Severe Infections)14, and 16 carriage isolates  were included. These 98 isolates were collected from November 2013 to March 2016 at Strasbourg University Hospital, France. All the 128 isolates were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer.A total of 128 Isolates (stored at −80 °C before use) were grown overnight at 37 °C on tryptic soy agar with 5% horse blood agar plate. DNA was extracted using the InstaGene Matrix kit  according to the manufacturer’s recommendations. PCRs were performed on a Veriti Thermal Cycler  in a final volume of 25 µl containing 12.5 µl GoTaq G2 Green Master Mix , 0.50 µM each primer and 5 µl of extracted DNA.aroE, dat, ddl, gmk, ldh, recA and yqiL) as previously described16, except that the PCR products were purified and sequenced by GATC Biotech SARL . Allelic profiles and corresponding sequence types (STs) were assigned with BioNumerics software . The sequences of all new alleles and the new STs identified were entered into the international MLST database (http://bigsdb.web.pasteur.fr/staphlugdunensis/). STs were clustered into clonal complexes (CCs) when they shared at least five identical alleles (single [SLV] or double locus variants [DLV]) with another member of the group, by using the eBURST V3 program (http://eburst.mlst.net).MLST genotyping was performed by sequencing seven gene loci . Sequences of the three loci were analyzed using BioNumerics software  to determine allelic profiles and trilocus virulence type (VTT).MVLST genotyping was based on the sequence determination of the intragenic regions of three virulence-associated loci, S. lugdunensis isolates N92014328 and HKU09-0137 were screened in silico for the presence of tandem repeats (TRs) by using Tandem Repeats Finder software, version 4.07b38. Primers flanking 28 potential VNTR loci were designed using the OLIGO Primer Design software ; their specificity was evaluated by Blastn. Primers were tested on the panel of 30 genetically diverse S. lugdunensis isolates to assess amplification ability, repeatability and the level of polymorphism of these loci. A VNTR was validated as a good candidate for MLVA typing if the locus (i) was present in all isolates, (ii) exhibited a minimum size of 18 bp for an individual TR, and (iii) required a single primer pair for amplification and sequencing of the internal fragment. Of the 28 VNTRs tested, seven fulfilled the above mentioned criteria, and were named SLU (for S. lugdunensis) followed by a number.The genome sequences of The primers used for VNTR amplification and sequencing are presented in Table et al.39. The numbers of TRs at each locus were combined and listed in the order SLU1-SLU2-SLU3-SLU4-SLU5-SLU6-SLU7 e.g. 1-2-10-4-3-2-2. Each unique combination was converted into a distinct MLVA type (MT).The number of TRs of each VNTR was determined with BioNumerics software  by using the “polymorphic VNTR typing” plugin. To check that the entire repeat array had been sequenced, the flanking sequences (start and stop pattern) were spotted but were not included as typing information. For incomplete repeats, the TR copy number was rounded down to the nearest complete copy number, in accordance with the guidelines published by Nadon Sequences of TR were determined for each VNTR, and each unique repeat sequence was identified by the name of the VNTR in lower case, followed by a number Table ; for exaAll typing data were uploaded into the BioNumerics software  and clustered using the appropriate settings. Two different techniques were used to represent the relationships between isolates. A dendrogram was generated based on the categorical coefficient provided by the BioNumerics software and the algorithm of unweighted pair group method with arithmetic mean (UPGMA). Cutoff values of 67% and 91% similarity were applied to define respectively MLVA and TRST clusters. Minimum spanning trees were constructed using the categorical coefficient and the priority rule to first link types that have the highest number of single locus variants. MTs were grouped into MLVA complexes (MCs) if they differed by no more than a single VNTR. To generate a minimum spanning tree based on TRST data, a composite dataset was constructed using BioNumerics software, based on a multiple alignment of each VNTR allele.40 and adjusted Wallace coefficients (AW)41  as described by Grundmann et al.42) were calculated by resorting to the online tool at http://www.comparingpartitions.info/?link=Tool.The discriminatory ability as well as the congruence of the results obtained by using the different typing methods and/or their combination were evaluated. To this aim, both the Simpson’s diversity index (DI)IAS) between alleles was calculated to test for linkage disequilibrium between alleles of MLVA and TRST using the LIAN Linkage Analysis 3.7 online tool (http://guanine.evolbio.mpg.de) as described by Haubold and Hudson43. The null hypothesis of linkage equilibrium, IAS = 0, was tested with 100000 Monte Carlo simulations.The standardized index of association (Since VNTR regions are considered to be one of the fastest evolving sequences of a genome, it was important to test the stability of the selected VNTR regions. To determine this stability, two isolates were subcultured on tryptic soy agar for 30 consecutive days by streaking a single colony from each strain on agar plates. The original culture, subculture numbers 10, 20 and 30 were used for DNA extraction and the total DNA was subjected to the MLVA and TRST assays.Supplementary information


Simulated atmospheric N addition was applied in a factorial experiment with N addition level  and N type  treatments. The experiment was conducted over the 2010 growing season (May-September) at the Kailaqi farm of Genhe Forestry Bureau, located in the northern Great Xin’an mountain range, northern China. Monthly N addition treatments were applied in three replicate plots per treatment (n = 36), and measurements of dissolved organic carbon (DOC) and dissolved inorganic carbon (DIC) were derived from monthly sampling of the organic and mineral soil horizons. There was a significant effect of N type, with the combined N source (NH4NO3) producing significantly higher DOC than the control (ambient addition) or the NH4Cl treatment in both the organic and mineral layers. The N addition treatment increased DIC in the organic layer at the low levels only, while N type did not have a significant effect. There was a significant interaction of the month and the N level treatment, as low level N addition tended to increase the content of soil DOC while high level N tended to inhibit soil DOC content, with these trends being most pronounced in the middle of the growing season. These results elucidate the importance of the type and timing of N additions to the dynamics of soil carbon pools.Understanding the effects of nitrogen (N) addition on dissolved carbon in boreal forest soils is essential for accurate evaluation of regional carbon balances. The objective of this study was to determine the effects of different levels and types of N addition on soil dissolved carbon concentration in a cold-temperate coniferous forest through an  The annual addition rate of reactive N in the atmosphere has increased from 15 Tg yr−1 in 1860 to 187 Tg yr−1 in 20051, and it is expected that the rate will double in next 25 years3. China currently ranks as the region with the third highest N addition in the world following Europe and United States, with a mean total atmospheric dry and wet N deposition across the country of 12.9 kg ha−1yr−1, and values reaching up to 63.5 kg ha−1 yr−1 in some areas5. Increases in atmospheric N level affect processes such as carbon uptake, photosynthetic allocation, plant growth, litter decomposition, soil organic matter turnover and soil respiration6. Hence, increased N levels greatly interfere with forest ecosystem processes and functions7.The amount of atmospheric nitrogen (N) has increased considerably in recent decades as a result of higher anthropogenic emissions, resulting in significant changes in carbon and N cycles in terrestrial ecosystems8, and the organic carbon source for microorganisms present in the soil. The DOC can be derived directly from litter, humus decomposition, precipitation input and fine root secretions9. In terrestrial ecosystems, DOC accounts for only a small fraction of C output10, but it plays an important role in the interactions between the C and N cycle. Specifically, C decomposition is coupled with N availability, dissolved organic N (DON) decomposition resembles that of DOC, and DOC decomposition is fueled by N mineralization9. Despite these known connections, the effects of increasing N deposition on soil DOC concentrations remain unclear. Reports have indicated that increased N levels either increase11, decrease, or do not affect12 the soil DOC concentration. Complicating the interpretation of these conflicting reports, the mechanism underlying the effect of N addition on soil DOC is still unknown.The dissolved organic carbon (DOC) is a component of the soil’s active organic carbon2, H2CO3, HCO3− and CO32−. These are compounds derived mainly from CO2 which reacts with water present in the soil to form H2CO3 and HCO3−. Since soil DIC content is much lower than soil DOC content, DIC is often overlooked in soil carbon balance studies. In addition, information on DIC dynamic responses to increasing N concentration are still needed13.Soil dissolved inorganic carbon (DIC) includes CO15. These studies mainly focused on the influence of different management  and land use practices on soil dissolved carbon content, distribution, leaching, migration, conversion and accumulation. Reports on the effects of N addition on soil dissolved carbon are rare. Moreover, the impact of N deposition on dissolved carbon in boreal coniferous forest ecosystems is unknown. The atmospheric N deposition in Great Xin’an mountain region is substantial, ranging from 9.87 to 14.25 kg N ha−1 yr−1 6. Long-term inputs of low-level N will increase N availability in boreal forest ecosystems, affecting regional soil CO2 emissions and the soil carbon balance16. Although the mechanisms controlling the interaction of C and N in soils have not yet been fully understood, a possible response of DOC and DIC to changed N conditions can be expected based on several previous findings. For example, N addition increases microbial activity and metabolism, which in turn may change the DOC status in the soil17. Mineral N sometimes interacts with lignin, increasing the stability of soil organic matter, so mineral N additions could decrease DOC. Furthermore, a site-specific C: N ratio may exist for the organic layer or other soil layers in a given ecosystem, which may influence the DOC/DIC responses to N changes19. Therefore, more experimental studies are needed to help clarify the effects and interactions of different N addition scenarios. The objective of this study was to determine the effects of different levels and types of N addition on soil dissolved carbon concentration in a cold-temperate coniferous forest through an in-situ fertilization experiment. We hypothesized that the type and level of nitrogen addition would significantly affect the soil dissolved organic carbon. We anticipate that our results will provide new insights into forest soil carbon dynamics in the context of N addition in northern forest ecosystems.In recent years, studies on soil dissolved carbon and its dynamics have been conducted in forest, grassland, wetland and agricultural ecosystems−1, 390.08% greater than that of the mineral soil layer ; but the level of N addition has no significant effect (p > 0.05). Specifically, N addition in the form of nitrate generally affected soil DOC content in a stronger way than ammonium N addition.The balance of soil carbon pools is mainly affected by the input from plants and the loss from leaching of active carbon components, such as DOC, DIC or the release of CO21. However, N addition can increase above-ground biomass and subsequent litter return22. At same time, N form is a restrictive condition, and the plants and soil microbes have a preference for NO3− 24. During the growing season, plants and microbes typically use more NO3− than other forms of available N26. Therefore, our results can be interpreted as further evidence to suggest that plants and microbes absorbed higher amounts of N in the form of NO3− from the soil during the experiment. For humus-rich topsoil, increased microbial metabolic activity will promote soil DOC release. The fact that the soil DOC content increased even under the increased microbial activity in presence of nitrate N is notable23, given that the treatments appeared to increase substrate utilization and alter the soil biota activity and composition.In coniferous forest systems, the DOC content in the soil is mainly affected by litter degradation and rhizosphere deposition, although degradation of humus may also increase the percentage of soil DOC27 may explain this pattern. Hagedorn28 suggests that experimental N addition changes the physical and chemical adsorption capacity of mineral soil organic matter due to increasing ionic strength, soil N availability and decreasing soil pH. In our study, since application of high level nitrate led to soil acidification , potentially increasing soil CO2 emissions29. Sodium nitrate addition consistently increases DOC, whereas ammonium salts additions usually decreases DOC30. We also reported that three levels of N input slightly increased DOC content in the organic and mineral soil layer compared to the control condition; however, the differences were not statistically significant.It is also possible that alternative microbial mechanisms31. Previous studies have shown that in the boreal forest, where soil pH is acidic, soil inorganic carbon changes very little32. However, in our study, it was found that in the initial progress of N addition, soil acidification did not have a significant effect on soil DOC and DIC content , white birch (Betula platyphylla), with understory species including safflower Pyrola (Pyrola incarnata), Ledum  and red beans cranberry (Vaccinium Vitisidaea). A complete description of the site is given in Table The study was conducted at the Kailaqi farm of Genhe Forestry Bureau , located in the Great Xin’an mountain range, northern China. The study site is in the southern edge of the boreal temperate permafrost zone. This area is characterized as a cold-temperate semi-humid climate, with a mean average annual temperature of −5.4 °C. About 60% of its annual precipitation (550 mm) occurs in July and Augustays Fig. . Soil is4+, NO3− as well as their combined effects, three N addition type treatments were used in this study, NH4Cl, KNO3, and NH4NO3. Based on the measured atmospheric N deposition flux at the study area (8.5 kg N ha−1yr−1), we designed four N addition level treatments representing different fertilizer input: (Control), which meant no N in addition to ambient background levels, low level N  which was a small increase to the in-situ addition level, medium level N , and high level N (High-N 40 kg N ha−1yr−1). The last two fertilization treatments simulated realistic future atmospheric N deposition scenarios50. The amount of N input in last three treatments increased approximately 1.25, 2.0 and 4.0 times compared to the control treatment, respectively. Three replicate plots were established for each combination of N addition type (3) and N addition level (4) for a total of 36 plots , and calculated according to the following equations:Repeated measures analysis of variance (ANOVA) models were applied to examine mean differences in soil dissolved organic carbon, dissolved inorganic carbon and pH, with N addition level and N addition type as between subjects effects, and month as a within subjects effect. The ANOVAs were followed by a Tukey-HSD post-hoc tests to identify significant mean differences for each response variable. All statistical analyses were conducted using SPSS version 24.0  and SigmaPlot version 12.5 . The level of statistical significance was set at p < 0.05.


Autism has been linked with the changes in brain connectivity that disrupt neural communication, especially involving frontal networks. Pathological changes in white matter are evident in adults with autism, particularly affecting axons below the anterior cingulate cortices (ACC). It is still unknown whether axon pathology appears early or late in development and whether it changes or not from childhood through adulthood. To address these questions, we examined typical and pathological development of about 1 million axons in post-mortem brains of children, adolescents, and adults with and without autism (ages 3–67 years). We used high-resolution microscopy to systematically sample and study quantitatively the fine structure of myelinated axons in the white matter below ACC. We provide novel evidence of changes in the density, size and trajectories of ACC axons in typical postnatal development from childhood through adulthood. Against the normal profile of axon development, our data revealed lower density of myelinated axons that connect ACC with neighboring cortices in children with autism. In the course of development the proportion of thin axons, which form short-range pathways, increased significantly in individuals with autism, but remained flat in controls. In contrast, the relative proportion of thick axons, which form long-range pathways, increased from childhood to adulthood in the control group, but decreased in autism. Our findings provide a timeline for profound changes in axon density and thickness below ACC that affect axon physiology in a direction suggesting bias in short over distant neural communication in autism. Importantly, measures of axon density, myelination, and orientation provide white matter anisotropy/diffusivity estimates at the level of single axons. The structural template established can be used to compare with measures obtained from imaging in living subjects, and guide analysis of functional and structural imaging data from humans for comparison with pathological states.The online version of this article (10.1007/s00401-018-1904-1) contains supplementary material, which is available to authorized users. Autism spectrum disorders (ASD) have been linked with the changes in brain connectivity that disrupt neural communication , 23, 79.Moreover, both typical and atypical development of cortical pathways remain grossly understudied at the single axon level. In view of the appearance of ASD symptomatology at an early age, we reasoned that the disorganization of axons below the ACC may start early. Comparison with adults may thus provide insight about the developmental trajectory for both typical and disrupted neural communication.To begin to address these issues, we used post-mortem brain tissue from children (ages 3–10 years), teenagers (ages 14–15 years) and adults (ages 30–67 years) with and without ASD, and systematically sampled and imaged individual myelinated axons in the white matter below ACC at high resolution in the light (LM) and electron microscope (EM). Our goal was to investigate the typical postnatal development of vulnerable ACC pathways  and compWe provide evidence for systematic changes in the density, size and trajectories of axons of ACC pathways in typical postnatal development from childhood through late adulthood. Against the normal profile of development, the ASD groups showed lower density of myelinated axons in childhood, followed by an increase in adulthood in pathways that travel over short distances and a decrease in pathways that course over long distances, in patterns that differed significantly from the control groups. These findings reveal fine structural underpinnings of persistent pathology that may be key anatomical correlates of disrupted neural communication in ASD.3 beneath cortical areas 32 and anterior 24, collectively called ACC here. At levels of the cingulate that surrounded the genu of the corpus callosum, we included both dorsal  and ventral  ACC regions, as described in detailed cytoarchitectonic studies and atlases mapping the human brain , 8628, 8We stained other series of sections using the Gallyas silver technique to label intracortical and white matter myelin , 48, 86.We processed adjacent series of 50-μm-thick coronal sections for EM, using a high-contrast method. We confirmed the presence of ACC cortical gray matter in adjacent sections that were stained with Nissl (see above). We rinsed sections in 0.1 M PB and postfixed them in a variable wattage microwave oven  with 6% glutaraldehyde at 100–150 W. Under a dissecting microscope, we cut small regions of sections containing the superficial or deep parts of the white matter below ACC. White matter regions of interest were rinsed in 0.1 M cacodylate buffer, and then postfixed with intermediate water rinses first in 0.1% tannic acid, then in 1% osmium tetroxide with 1.5% potassium ferrocyanide, then in TCH aqueous solution (0.1 g of thiocarbohydrazide), and finally in 2% osmium tetroxide. Sections were washed in water, stained overnight with 1% uranyl acetate followed by lead aspartate and dehydrated in an ascending series of alcohols. Tissue sections were then cleared in propylene oxide and embedded in araldite or LX112 resin at 60 °C and then in Aclar film for storage. Pieces of the resin-embedded sections were cut and re-embedded in resin blocks. We cut serial semithin (1 μm) or ultrathin (50-100 nm) coronal sections with a diamond knife , using an ultramicrotome . Lipids in the myelin sheath and in membranes were rendered electron-dense and appeared dark after EM processing .For the Nissl staining of semithin sections, we prepared 1% aqueous solution with toluidine blue powder  in distilled water. The solution was filtered and mixed with sodium borate , filtered again and diluted 1:1 with 70% ethanol. Semithin sections floating in water were mounted on gelatin-coated slides and placed on a heater plate until the water evaporated. Sections were then covered with the final toluidine blue solution for 1 min, rinsed with water, differentiated with 70% ethanol and rinsed with water, before coverslipping as described above.We collected ultrathin sections on single slot pioloform-coated grids to view with a transmission electron microscope  or a scanning electron microscope (Zeiss Gemini 300 with STEM detector and Atlas 5 software modules), at magnifications between 2000 and 50,000×, as described , 80, 84.We estimated the overall density of neurons, astrocytes, and oligodendrocytes in ACC gray matter regions of interest (ROIs) overlying the white matter regions examined, based on previous maps of the human brain , 73. ForWe counted Nissl-stained neurons, astrocytes, and oligodendrocytes in the outlined ROI volume at 1000×, using systematic random sampling. We identified neurons and glia based on their characteristic features, following a detailed cytology algorithm we have developed . Briefly3).We set the counting frame (disector) size for cell counts at 50–60 μm with a height of 5 μm and grid spacing of 100–300 μm. To ensure unbiased estimate of the number of cells we first measured the thickness of each section, and set a guard zone at the bottom and top of each section to correct for objects plucked during sectioning (minimum 2 μm in 10–15 μm sections after tissue shrinkage); the disector thickness was thus smaller than the thickness of the section , 75. TheFollowing a similar approach, we used uniform random sampling to image axons systematically at the optical microscope. To estimate axon density, we captured images of semithin toluidine blue-stained white matter sections at 1000× with the same light exposure to minimize background variability. We then converted to grayscale, inverted, and thresholded images in ImageJ so that myelinated axons were highlighted, and we estimated the ratio of the surface area of myelinated axons within each sample and averaged for each subject.We estimated the relative density of axons and the thickness of axons and myelin in the white matter below ACC at the EM, using a systematic random sampling fraction of 1:1,000 that yielded more than 6000 axons, per case, per ROI. We divided the white matter into a superficial part  and a deep part (DWM), as described , and orientation angle. We estimated myelinated axon orientation variability within each white matter region by measuring axon profile angle trajectories in each image and then calculating the standard deviation (SD) in each image Fig. b, c. We We gathered data blind to condition and white matter region and conducted independent analyses by at least two investigators. Random codes for human subjects and images were broken after completion of each part of the study. To estimate the sample size, we took into account the number of cases and volume fraction of ACC sampled so that the number of individual cells and axons examined produced estimates with a small coefficient of error (< 10%), as described , 82, 86.It should be noted that, to the best of our knowledge, this study included the largest sample size and cohort of brain tissue from post-mortem human cases examined to date at such high resolution. We imaged and analyzed an estimated 1,000,000 individual myelinated axons from post-mortem brain tissue from 32 individuals. Most axons were imaged at 1000× magnification with the optical microscope, where we could examine large white matter regions of interest and reliably resolve myelinated axons down to 0.2 μm in diameter. A quarter of the axons were independently imaged at high resolution in the EM , where we could reliably identify all myelinated axons, including about 20% with diameters below 0.2 μm thickness. For the EM analysis of axon densities and sizes, we obtained samples from widely spaced ultrathin sections (one every ten) and fields of view through systematic random sampling to minimize the likelihood of sampling axons from the same parent branch. This sampling scheme and the fact that most axons branch very close to or after they enter the gray matter minimized the likelihood of counting segments of the same axon more than once.We quantitatively estimated 16 morphometric structural features of myelinated axons in the white matter (feature dimensions), including: relative axon density , relative axon density (only elongated profiles with AR ≥ 3), outer diameter, inner diameter, major diameter of outlined axon profile, axon orientation, myelin thickness, g-ratio, relative proportion of thin, medium, thick and extra-large axons, myelin thickness of thin axons, myelin thickness of medium axons, myelin thickness of thick axons, and myelin thickness of extra-large axons.t tests. We used ANCOVA, with age as a covariate, to test for differences in the developmental trajectories of axon features. For all analyses, p values < 0.05 were taken as statistically significant.We performed statistical analyses using Statistica , SPSS (IBM), or Matlab (MathWorks). We evaluated data through scatter and frequency distribution plots and K-means cluster analysis, with parameters set to maximize initial between-cluster distances, to segregate axons into four groups by thickness, based on their outer diameters, which included the myelin sheath. Data distributions for continuous variables were not significantly different from normal as determined by the Kolmogorov–Smirnov test, and thus allowed the use of parametric statistics. We initially used Chi square and Kolmogorov–Smirnov tests to examine axon size distributions and multiple linear regression analysis to examine correlations. To compare axon or cell densities across cases and conditions we employed one-way ANOVA or two-tailed Finally, we examined potential effects of sex, PMI, and other diagnoses  on all estimates for axon size as well as axon and cell density, using correlation analysis. In addition, we compared all estimated variables across cases using MANCOVA with sex, PMI, and other diagnoses as covariates. These analyses did not yield additional significant effects.Digital images that were used for analyses were not modified. We assembled figure panels by importing images into Adobe Illustrator software . Images were not retouched or edited other than applying minor adjustments of overall brightness and contrast using Adobe Photoshop.We first assessed the density of myelinated axons. This analysis showed that myelinated axons made up approximately 34% of the white matter below ACC in both ASD and CTR groups. The density of myelinated axons (% of space occupied by myelinated axons) increased progressively with increasing distance from cortical layer 6 and depth in the white matter [ and ]. The rest of the white matter was occupied mostly by unmyelinated axons and glia, mainly oligodendrocytes.We then measured axon diameters, which ranged between 0.1 and 9.7 μm, consistent with findings in human cortex , 80, 84.t test, p = 0.00001; Fig. We next measured the orientation of myelinated axons. Results showed significantly more axon orientation variability in the SWM than in the DWM in each group (ASD and CTR) and for each individual brain  had relatively more thick myelinated axons than children 4–10 years), who had a higher proportion of thin axons  on all estimates for axon density, size, as well as cell densities at the 95% confidence level.N = 3). Further studies of the brain in adolescence will be required to reliably describe the rate of changes in the white matter from childhood to adulthood.We provide direct evidence for typical and pathological changes in ACC white matter pathways with age. These findings were based on the largest cohort of human brains (32) and number of axons investigated (~ 1 million) at high resolution to date. Our findings revealed significant heterogeneity in ACC pathways in the course of postnatal development in ASD, in stark contrast to modest, possibly incremental, but biologically important, changes seen in typical postnatal development. Axon myelination in ACC pathways followed a heterogeneous trajectory, consistent with cortical changes seen with age in early childhood, adolescence and late adulthood e.g., , 58, 77), 7758, 7In typical postnatal development, the ratio of thick to thin axons and the density of myelinated axons increased from childhood to adulthood, consistent with the developmental processes of axon growth and myelination , 51, 77.The detailed analyses of normal development of axons provided a foundation to compare with a pathologic state, which revealed significant differences in the density and thickness of myelinated axons below ACC in ASD. A key difference was the lower density of myelinated axons in the SWM below ACC in children with ASD, coinciding with the period when the white matter is enlarged , 32, 58.The opposite developmental trajectories of ACC pathways in CTR and ASD groups are also supported by the significant decrease with age in the thickness of myelinated axons in the ASD group, which remains relatively flat in typical development. The primary driver for the pathology in ASD is likely the thinning of the axolemma, but not the surrounding myelin, as evidenced by a steeper slope for the inner, compared to outer axon diameter in ASD. This change led to small but significant decrease in the g-ratio with age, a relationship that affects conduction velocity and neural synchrony between connected areas, in processes that are disrupted in ASD , 59.Our study also provides an estimate of white matter axon trajectory anisotropy at the level of single axons, based on high-resolution microscopic imaging, and can be used to compare with measures obtained from diffusion tensor imaging (DTI) in living subjects, albeit at lower resolution. Axon parameters of density, thickness, and trajectory correlate well with imaging studies, and provide specific structural underpinnings of typical and pathological changes in the directional preference of diffusion [fractional anisotropy (FA)], and other estimated diffusion rates , which are associated with white matter integrity and myelination , 44, 65.In conclusion, we identified distinct changes from childhood to adulthood in the relative density, size composition, and trajectory of myelinated axons in the white matter below ACC. These novel findings revealed the developmental course in CTR brains and provided the basis to disentangle typical developmental processes from continued pathology in axons in ASD. Our findings revealed differences in the development of axons in ASD within a long lifespan, exhibiting a course that deviated from the CTR groups, biasing communication within the ACC and neighboring prefrontal cortices, and compromising communication over long distances.Supplementary material 1 (PDF 20 kb)Below is the link to the electronic supplementary material.


Background and Objectives: Limited research has evaluated the effects of acute exercise on cognition under different conditions of inspired oxygenation. Thus, the purpose of this study was to examine the effects of high-intensity interval exercise (HIE) under normoxia (inspired fraction of oxygen (FIO2): 0.209) and moderate hypoxia (FIO2: 0.154) on cognitive function. Design: A single-blinded cross-over design was used to observe the main effects of exercise and oxygen level, and interaction effects on cognitive task performance. Methods: Twenty inactive adults  performed a cognitive task  before and immediately after an acute bout of HIE under normoxic and hypoxic conditions. The HIE comprised 10 repetitions of 6 s high-intensity cycling against 7.5% body weight interspersed with 30 s passive recovery. Heart rate, peripheral oxygen saturation (SpO2) and rating of perceived exertion were monitored. Results: The acute bout of HIE did not affect the reaction time  but the accuracy rate decreased significantly after HIE under both normoxic and hypoxic conditions . Moreover, moderate hypoxia had no influence either on reaction time  or response accuracy . Conclusions: These results indicate that an acute session of HIE may impair response accuracy immediately post-HIE, without sacrificing reaction time. Meanwhile moderate hypoxia was found to have no adverse effect on cognitive function in inactive young adults, at least in the present study. Moderate-intensity level exercise or physical activity is generally accepted to have positive effects on cognitive function , among b2max in untrained individuals) can attenuate cognitive performance [Much evidence has emerged in the past decade illustrating that high intensity interval exercise (HIE), an exercise modality comprising repeated bursts of vigorous exercise with less intense or passive recovery periods , is an eformance . Furtherformance . Thus, m2) level of 0.154, an integrated understanding of the effect of moderate hypoxia at FIO2 of 0.154  and exercise on cognition might be more practical than other hypoxic levels.Combining hypoxia with exercise training may have synergistic effects on weight loss  and card2 = 0.154) on cognitive function. We hypothesized that cognitive function would be improved following HIE. However, we anticipated that moderate hypoxic exposure would cause an adverse effect on cognitive function in comparison to normoxia.Using a single-blinded cross-over design, the purpose of this study was to examine the effects of single bouts of HIE under normoxia or moderate hypoxia , a HIE trial under normoxia (NOR) and a HIE trial under simulated hypoxia (HYP) on four separate occasions. During the familiarization trial, subjects were familiarized with the experimental procedures, practiced the cognitive task , and undertook anthropometric assessments and a maximal graded exercise test. The three formal experimental trials were performed at the same time of day (from 19:00 to 20:00) under a well-controlled laboratory environment with stable temperature (22 ± 1 °C) and relative humidity (72 ± 5%). The order of the main trials was randomized and counterbalanced across subjects, who were unaware of the normoxia or hypoxia conditions. Seven-day periods were interspersed between trials to remove any potential residual effects.2: 150 mmHg, FIO2: 0.209) or hypoxic  conditions. The normoxic and hypoxic gas mixtures were generated using a modified gas mixing system  and supplied to subjects through tubes and a breathing mask. On the days of the main experiments, subjects arrived at the laboratory before 19:00 and put on a breathing mask at 19:00. After exposure to normoxia or hypoxia for 30 min, they performed a pre-exercise GNG task, a HIE session (or rest for the corresponding time) on an ergometer, and a post-exercise GNG task immediately after HIE under the respective experimental conditions.On the experimental days, subjects performed HIE (or rest) and cognitive tasks under either normoxic . The initial workload was 50 W and the power output increased by 25 W every 3 min until exhaustion. The highest VO2 value averaged over 30 consecutive seconds during the last stage was recorded as VO2peak.A maximal graded exercise test was performed by each subject on an ergometer  to measure the VO2) values before and after each bout of HIE were monitored using a pulse oximeter  placed on the left middle finger. Ratings of perceived exertion  were recorded after the completion of each cycling repetition.The 6-min HIE protocol consisted of 10 repetitions of 6 s of high-intensity cycling bouts interspersed with 30 s of passive recovery. Subjects pedaled as fast as possible against a resistance of 7.5% of body weight during the 6 s work durations, and underwent passive recovery during the 30 s rest periods on a cycle ergometer . Heart rate (HR) and peripheral oxygen saturation . If a red square appeared on screen followed by a number, or a blue square appeared on screen followed by a letter, the subjects needed to respond by pressing the “J” button with the right index finger. In contrast, if a letter showed following the red square or a number emerged following the blue square, the subjects needed to press the “F” button with the left index finger. Each stimulus remained on screen for a maximum of 2 s, or until a response was given. Following each trial, a signal of “+” would appear on screen as an inter-stimulus interval lasting for 2 s. The GNG task included 40 trials, and the number of correct responses assigned to “F” and “J” was the same. Reaction time (ms) and response accuracy (%) were recorded to measure cognitive performance. Functional imaging studies have demonstrated associations between GNG performance and activation of several brain structures, including in the prefrontal and anterior cingulate regions of the brain ; both stSubjects were asked to maintain their normal diet and daily routines and avoid extra exercise during the study period. Daily physical activity was monitored using a pedometer  on 3 days (2 days before and the day of the experiment) for each experimental trial. This pedometer has demonstrated evidence of validity .t-tests were conducted to evaluate demographic differences across sex (2: 0.209 and 0.154), and interaction effects (exercise × condition) on cognitive task performance and physiological parameters . Tukey’s post hoc test was used to compare the group differences when there was a significant interaction effect. The effect size of partial eta squared (η2) was calculated to determine the effect sizes of the main and interaction effects. The effect size was considered small if η2 < 0.06 and large if η2 > 0.14. All data were expressed as mean ± standard deviation (SD), and p < 0.05 was taken as the level of statistical significance.The PASW software  was used for statistical analyses. Before the main analyses, the Shapiro-Wilk test was conducted to confirm that the outcome variables were normally distributed. Independent samples ross sex . In orde2 = 0.209) and hypoxia (FIO2 = 0.154) showed similar peak power, average power and fatigue index  in daily activity among the three trials .An acute bout of HIE under normoxia  and RPE  in both the NOR and HYP trials significantly increased in the post-exercise measurements . Under the hypoxic condition, SpO2 was significantly lower at rest  and after exercise  when compared to the normoxic condition  or FIO2 level  on the changes in reaction time (Δ RT), and no interaction between exercise and FIO2 level . After the acute bout of HIE, Δ accuracy was significantly decreased in both the NOR and HYP trials (p < 0.01). In contrast, the reduction in FIO2 had no effect on Δ accuracy  in the present study did not affect cognitive function.The current study firstly examined the effects of HIE under normoxia and moderate hypoxia on cognitive function in healthy young adults. The main findings of this study were: (1) HIE impaired cognitive function under both normoxic and hypoxic conditions, as indicated by reduced accuracy rates in post-exercise measurements; (2) moderate hypoxia  on cognitive function [2 level of 0.154 in the present study  may be below the threshold at which hypoxia would impose distinct adverse effects on cognitive function [The brain tissue is largely dependent on a constant and sufficient oxygen supply to function effectively . In a hyncreases , and thencreases ,23. In tfunction ,28, but function ,39. It afunction , and cogfunction . Therefofunction . AnotherIt is undeniable that cerebral blood flood (CBF) and brain oxygenation may also be involved in the regulation of cognitive processes during exercise under normoxia and hypoxia through conflicting cerebral hemodynamic responses . In norm2, including brain oxygenation and CBF were not monitored, which limited the elucidation of potential mechanisms. Finally, further interpretation of the impact of hypoxic exposure alone on cognitive function is limited because of the lack of a non-exercise hypoxic control group.In the present study, a relatively simple cognitive test, the GNG task, was used to measure cognitive function in relation to executive function. More sophisticated and higher-level cognitive tasks involving information processing, attention, intelligence and working memory should be included in further studies. In addition, according to previous studies ,13, we c2 = 0.154) on cognitive function. After HIE, the accuracy rates of the GNG task decreased under both normoxic and hypoxic conditions, suggesting that HIE may impair cognitive function immediately post-HIE. However, moderate hypoxia  was demonstrated to have no adverse effect on cognitive performance, suggesting that moderate hypoxia could be a strategy to improve cardio-metabolic health outcomes without any impairment of inhibitory-based cognitive function in sedentary adults.In summary, the present study firstly addressed the influence of a single bout of HIE under normoxia and moderate hypoxia (i.e., FIO


Here we describe a novel mouse model of temporal lobe epilepsy (TLE) that moves the site of kainate injection from the rodent dorsal hippocampus (corresponding to the human posterior hippocampus) to the ventral hippocampus (corresponding to the human anterior hippocampus). We compare the phenotypes of this new model—with respect to seizures, cognitive impairment, affective deficits, and histopathology—to the standard dorsal intrahippocampal kainate model. Our results demonstrate that histopathological measures of granule cell dispersion and mossy fiber sprouting maximize near the site of kainate injection. Somewhat surprisingly, both the dorsal and ventral models exhibit similar spatial memory impairments in addition to similar electrographic and behavioral seizure burdens. In contrast, we find a more pronounced affective (anhedonic) phenotype specifically in the ventral model. These results demonstrate that the ventral intrahippocampal kainic acid model recapitulates critical pathologies of the dorsal model while providing a means to further study affective phenotypes such as depression in TLE. Temporal lobe epilepsy (TLE) is characterized by spontaneous recurring seizures. TLE additionally features cognitive and affective comorbidities that impair quality of life. Animal models studying TLE are critical to advance our understanding of the disorder. A current popular model targets the dorsal hippocampus with a focal injection of kainic acid to induce epilepsy. Evidence suggests that targeting the ventral hippocampus may produce a model of TLE with distinct benefits. We present data demonstrating that targeting the mouse ventral hippocampus with kainic acid creates a TLE model with seizure and cognitive phenotypes similar to the standard dorsal model, but with additional, pronounced, affective features, including anhedonia. These results describe a new tool for epilepsy researchers to better study comorbidities of TLE.Temporal lobe epilepsy (TLE) is the most common form of epilepsy in adults and features spontaneous recurring seizures in addition to cognitive and affective comorbidities, such as depression and anxiety. Despite decades of research, there is still a strong need to better understand various TLE pathologies, as evidenced by the persistence of high rates of comorbid depression . Animal IHKA was first formally proposed as a model of human TLE decades ago . HoweverAs the epilepsy field cemented the location of IHKA injections to the dorsal hippocampus, hippocampal researchers were bringing to light important distinctions between areas along the septotemporal axis of the hippocampus. For example, the dorsal third of the hippocampus was found to project to the retrosplenial cortex, a region involved in navigation, while the ventral two-thirds of the hippocampus has projections to various amygdalar areas that are involved in emotional response . While din vitro  and compare epilepsy-related phenotypes to the standard dorsal intrahippocampal KA (dKA) model. Specifically, we sought to address the following. (1) Does the vKA model recapitulate key anatomic pathologies seen in the dKA model? (2) Does the vKA model produce a distinct ictal phenotype from the dKA model, with regard to both electrographic and overt motor seizures? (3) Does the vKA model exhibit cognitive or affective phenotypes? If so, how do these compare to the dKA model?All animal procedures were performed in accordance with the regulations of the animal care committee of the University of Minnesota.n = 24) and female (n = 33) C57B/6J mice were stereotaxically injected with either 100 nl of saline or 100 nl of 20 mm KA in saline at postnatal day 45 or greater . Injection coordinates for the dorsal hippocampus group, in centimeters from bregma, were as follows: anteroposterior (AP), −0.2; mediolateral (ML), 0.125; dorsoventral (DV), −0.16. Coordinates for the ventral hippocampus were as follows: AP, −0.36; ML, 0.28; DV, −0.28. After injection of saline or KA, the syringe was immediately withdrawn and the animal was returned to its home cage to recover. We observed no acute mortality with this dose of KA in these animals. One week after saline or KA injection, animals were implanted with a twisted wire bipolar electrode (Plastics One) approximately equidistant between the dorsal and ventral injection sites . During all surgeries, animals were maintained on isoflurane anesthesia (∼2%) and a heating pad. Animals were group housed until the electrode implantation, at which point they were singly housed. No significant differences were observed between dorsally and ventrally injected saline mice; therefore, the saline subjects were collapsed into one group.Male , animals were chronically housed with simultaneous video and local field potential (vLFP) monitoring . A subse10.1523/ENEURO.0158-18.2018.ed1Extended Data 1Methods – Code Accessibility section. Download Extended Data 1, ZIP file.Automated ictal spike counter. We have provided the code (created in Matlab 2014b on a Windows 7 computer) to run our custom automated spike counter, used in this article to quantify electrographic seizures. The code runs as a Matlab script, which launches a customizable user interface. A detailed user guide accompanies the code. A copy of the code and user guide can also be found at the URL listed in the To ensure robust quantification of electrographic seizures, only a subset of animals with high signal-to-noise recordings underwent analysis . Electrographic seizures were quantified using an automated algorithm set to customized parameters for each subject. These parameters were based on the mean amplitude of the hippocampal recordings, ictal spike width, and time between spikes. For all animals, a seizure was defined as at least four spikes of greater than twice the baseline amplitude within each 2 s bin. Interictal periods were set to a minimum of 1 s. The minimum seizure duration was originally set at 3 s. In separate analyses, to specifically examine “hippocampal paroxysmal discharges,” event criteria were set to a minimum frequency of 5 Hz for a minimum duration of 5 s  were defined as >3 on the extended Racine scale of https://github.com/KM-Lab/Electrographic-Seizure-Analyzer. These items are also available as The automated electrographic seizure analysis software described in the article and accompanying user guide are freely available on-line at SCR_001622) transformations (with “data” being an array of data and “fs” being the sampling frequency):To flag events that were likely to include a behavioral manifestation , a semiautomated approach was used. Raw LFP data were processed using a fast Fourier transform, bandpass filtered between 20 and 200 Hz, reverse fast Fourier transformed, passed through a Butterworth filter, and then smoothed; if the resulting output was greater than a threshold value customized for each subject, that section of data was manually reviewed for the verification of LBS occurrence and scoring of the LBS. This approach correctly flagged all LBSs in a testing dataset that had been previously manually identified, and on at least one occasion, flagged an LBS that escaped manual detection. The data were processed using the following Matlab  and elevated plus maze (EPM) test . The OF apparatus was a 50 × 50 × 40 cm (length × width × height) plastic box. Animals were allowed to roam freely for 15 min. ANY-maze software (Stoelting) was used to record and quantify movement. A 10 cm border around the edge of the apparatus delineated the center of the open field and was used to calculate thigmotaxis. The total distance traveled was also analyzed. In the open field, one subject remained largely immobile throughout testing and was excluded from analysis.The elevated plus maze (Med Associates) arm dimensions were 34 × 7 cm, at a height of 90 cm. Two of the four arms of the maze were enclosed with tall walls, while the other two arms were open. Animals were placed in the center of the maze and allowed to explore for 5 min. Movement was tracked and analyzed using Ethovision software (Noldus). The time in closed arms and open arms was calculated, in addition to the total distance traveled. Three subjects that fell from the open arms of the elevated plus maze were excluded from analysis. Additionally, one subject was excluded due to improper apparatus setup and another for remaining largely immobile throughout testing.For both open field and elevated plus maze, testing was performed in dim conditions, ∼50 lux at the center of the maze. On both tests, the arena was cleaned with 70% ethanol between subjects. Subjects were tested by experimenters blinded to experimental condition.Object location memory (OLM) and object recognition memory (ORM) testing occurred following OF and EPM testing . The order of the two tests was alternated between subjects, and there was 1 week between the end of one testing session and the beginning of habituation for the next memory test. The design of the tests was based on previously published protocols . Mice weSCT testing occurred on average 69 d postinjection . Following a published protocol , animalsm, pH 4, citrate buffer; 30% 0.5 m hydroquinone; and <1% of a 19% silver nitrate solution. Slices were mounted on a slide and dried for 3 h at room temperature, then submerged in the Timm stain for 45 min at room temperature. Slides were then rinsed with water and submerged in 5% sodium thiosulfate for 5 min. Slides were then dehydrated in a series of ethanol baths and mounted with DPX Mountant (Sigma-Aldrich). Light microscopy was used to capture images of the dentate gyrus. A region of interest was drawn around the granule and molecular layers, which were thresholded to capture blackened (Timm-stained) tissue and were expressed as a fraction of the total area of the molecular and granule cell layers. Observations were taken from two hippocampal sections per animal .To assess histopathology, hippocampi ipsilateral to kainate or saline injection were analyzed in coronal sections, comparing MFS and GCD near the dorsal site (∼1.8 mm posterior to bregma) and near the ventral site (∼3.4 mm posterior to bregma). For examination of MFS, Timm staining was performed using modifications of a published protocol . A subseSCR_002285). All tissue processing and analysis was performed blinded to experimental condition.Another series of brain slices was collected from a subset of animals and mounted on slides with DAPI  mounting media . As with MFS, observations for GCD were taken from two hippocampal sections per animal . The width of the widest point of the central two-thirds of the enclosed blade of the dentate gyrus ipsilateral to injection was measured to assess GCD. The boundaries of the granule cell layer were defined to end when there was a >20 µm gap between nuclei. Image quantifications were performed using ImageJ . For the vKA group, the reverse was true: MFS was greater at the ventral site compared with the dorsal site . Results from examining GCD followed a similar pattern . Likewise, for the vKA group, the GCD was greater in the ventral compared with dorsal hippocampus . These data indicate that vKA and dKA models both produce anatomic pathologies but that the models exhibit different pathologic focal points corresponding to their respective site of kainate injection. These histopathological results support the distinction of the two sites of KA administration.Epilepsy can present with histologic, electrophysiological, cognitive, and affective pathologies. We therefore sought to characterize the vKA model, in which kainic acid is injected into the ventral hippocampus, in addition to the standard dKA model, in which kainic acid is injected into the dorsal hippocampus, along these various phenotypes. In both human patients and animal models of temporal lobe epilepsy, including the dKA model, anatomic signs of epileptic pathology in the tissue of the temporal lobe are often present. In addition to hippocampal sclerosis , aberran pattern d. In theGiven the differing sites of maximal anatomic pathology, we investigated whether the dKA and vKA insults generate different seizure phenotypes. At least 2 weeks after KA or saline injection, animals were placed under chronic video-synchronized LFP monitoring. Hippocampal activity was recorded via a twisted wire electrode placed approximately equidistant between the two sites of KA injection. This setup continuously recorded video and hippocampal LFP data for 2 weeks. The data were analyzed off-line by a combination of blinded manual and semiautomated analyses. In total, 7943 h of data were analyzed for electrographic seizures.p = 0.78; dKA: n = 12 animals; mean, 20 ± 7 events/h; vKA: n = 11 animals; mean, 21 ± 5 events/h). To ensure that the lack of difference was not due to insufficient power, a power analysis was performed, which indicated that >300 subjects (150 dKA and 150 vKA) would be needed to demonstrate a statistically significant result.Examination of electrographic seizures revealed similar frequencies in both the dKA and vKA groups when a seizure was defined to have a minimum 3 s duration .Further analysis of the data to specifically examine the occurrence of hippocampal paroxysmal discharge-like events .An additional analysis was performed examining seizures not at the subject level but at the seizure level. Examination of electrographic seizure durations described only a small (0.6 s) difference between dKA and vKA seizures . Examination of electrographic seizure durations at this later time point again suggested only a small (0.6 s) difference between dKA and vKA seizures , duration , or median seizure severity  occurred much less frequently. Analysis indicated similar LBS burdens between dKA and vKA animals, with no significant differences in frequency , seizure severity remained similar . Therefore, for both electrographic and large behavioral seizures, no substantial differences in seizure burden were found between dKA and vKA models.An additional analysis was performed examining seizure duration and severity not at the subject level but at the seizure level. While there was a small (0.2 s) difference in LBS duration , as well as tests of hyperactivity, anxiety , and anhedonia (SCT).p = 0.74; saline: n = 13 animals; mean DI, 19.3 ± 1.2; dKA: n = 12 animals; mean DI, 18 ± 1.5; vKA: n = 11 animals; mean DI, 19.7 ± 2; The OLM test is known to be sensitive to hippocampal dysfunction, while the ORM test shows less sensitivity to hippocampal damage . On the p = 0.011; saline: n = 13 animals; mean DI, 22.5 ± 1.2; dKA: n = 8 animals; mean DI, 2.9 ± 1.6; vKA: n = 10 animals; mean DI, 3.6 ± 1.5; Mann–Whitney test: dKA vs saline, p = 0.012; vKA vs saline, p = 0.014; dKA vs vKA, p = 0.96; p = 0.79; ORM: Kruskal–Wallis ANOVA, p = 0.48). These data therefore reveal that dKA and vKA mice possess similar memory deficits that are sensitive to spatial memory function without impairments of more generalized, object identity memory.Previous reports have observed deficits for dKA animals on the OLM test . We invep = 0.041; saline: n = 15 animals; mean distance, 66.4 ± 0.7 m; dKA: n = 13 animals; mean distance, 80.8 ± 1.2 m; vKA: n = 14 animals; mean distance, 69.7 ± 0.9 m; Mann–Whitney ANOVA: dKA vs saline, p = 0.017; Mann–Whitney test: vKA vs saline, p = 0.42; In addition to spatial memory deficits, we asked whether either the dKA or vKA groups exhibit affective phenotypes. Two tests were used to examine hyperactivity and anxiety: the OF and EPM. In the open field, the dKA group traveled a greater distance than the saline group. In contrast, however, the vKA group did not exhibit this phenotype  and individual comparisons exposed a significant difference between the vKA and saline groups  but not between the dKA and saline groups , there was a strong trend in overall difference between groups . However, data from the elevated plus maze did not show any between-group difference in time spent in open arms of the maze—another measure of anxiety . Together, these data from the open field and elevated plus maze experiments indicate a hyperactive phenotype present in the dKA animals while suggesting a possible anxiety phenotype in the vKA animals.Repeated testing of these phenotypes in a different experimental paradigm, the elevated plus maze, suggested a similar increase in dKA distance traveled (supporting a hyperactive phenotype in the dKA model), while vKA animals trended toward this  in which vKA animals drank less sucrose water than saline animals . The dKA group appeared to show a more intermediate phenotype; analysis of consumed sucrose water trended toward reduced consumption , some studies report increased anxiety-like behavior , while oTissue from human patients with TLE often displays both GCD and MFS . In the in vitro work observed that the rodent ventral hippocampus generates more frequent spontaneous seizure-like bursting than the dorsal hippocampus  was found to be more ictogenic than the posterior hippocampus (corresponding to the rodent dorsal hippocampus; pocampus . Furtherpocampus . In a sipocampus . More repocampus . TogetheThe finding that the dKA and vKA models exhibit similar rates of both electrographic and large behavioral seizures in chronic epilepsy raises several interesting prospects. For one, differences in ictal frequency in acute studies may not generalize to chronic models of spontaneous seizures. Consider, for example, the increased current injections required for afterdischarges in kindling targeting the ventral hippocampus , which sWith both dKA and vKA groups exhibiting similar ictal phenotypes, the potential confound of secondary effects of ictal burdens is removed. This is important when evaluating the dKA and vKA models on multiple pathologic dimensions, as seizure burden can influence cognitive and affective outcomes. For example, seizure frequency and severity are known to correlate with progressive cognitive deterioration in people with TLE . AdditioBoth the dKA and vKA groups had a similar impairment on a test of hippocampal-dependent spatial memory, without showing impairments on a test of nonspatial memory. Though the dorsal hippocampus is generally associated with spatial navigation and memory, there are several possibilities that could account for the vKA group also showing spatial impairment. For one, following ventral KA injection, broad network changes may impair the ability of the dorsal hippocampus to mediate spatial memory. Alternatively, the ventral hippocampus is known to be involved with some aspects of spatial memory, and damage to these functions could cause the observed deficit. For example, in addition to the dorsal hippocampus, the ventral hippocampus also has place cells, although they generally govern a much larger place field than their dorsal counterparts . AdditioThe dKA model benefits from extensive prior characterization, such as the response to antiepileptic drugs  and optoOverall, the vKA model of TLE exhibits similar histopathological and ictal phenotypes compared with the standard dKA model, but displays additional affective phenotypes. This model provides a tool for epilepsy researchers to further examine important comorbidities of TLE, such as depression, while maintaining the benefits of a focal KA model.