Flo2 gene is a member of a conserved gene family in plants. This gene has been found to be related to thousand grain weight (TGW) in rice. Its orthologs in hexaploid wheat were cloned, and the haplotype variation in TaFlo2-A1 was tested for association with TGW.The TaFlo2-A1, TaFlo2-B1 and TaFlo2-D1 contained 23, 23 and 24 exons, respectively. The deduced proteins of TaFlo2-A1 (1734 aa), TaFlo2-B1 (1698 aa) and TaFlo2-D1 (1682 aa) were highly similar (>94%) and exhibited >77% similarity with the rice FLO2 protein. Like the rice FLO2 protein, four tetratricopeptide repeat (TPR) motifs were observed in the deduced TaFLO2 protein. An 8-bp InDel (−10 to −17 bp) in the promoter region and five SNPs in first intron of TaFlo2-A1 together formed two haplotypes, TaFlo2-A1a and TaFlo2-A1b, in bread wheat. TaFlo2 was located on homeologous group 2 chromosomes. TaFlo2-A1 was inferred to be located on deletion bin ‘2AL1–0.85-1.00’. The TaFlo2-A1 haplotypes were characterized in the Chinese Micro Core Collection (MCC) and Pakistani wheat collection using the molecular marker TaFlo2-Indel8. TaFlo2-A1 was found to be associated with TGW but not with grain number per spike (GpS) in both the MCC and Pakistani wheat collections. The frequency of TaFlo2-A1b (positive haplotype) was low in commercial wheat cultivars; thus this haplotype can be selected to improve grain weight without negatively affecting GpS. The expression level of TaFlo2-A1 in developing grains at 5 DAF (days after flowering) was positively correlated with TGW in cultivars carrying the positive haplotype.The cloned sequences of Flo2 gene in hexaploid wheat. Furthermore, the newly developed molecular marker ‘TaFlo2-InDel8’ could be incorporated into the kit of wheat breeders for use in marker-assisted selection.This study will likely lead to additional investigations to understand the regulatory mechanism of the The online version of this article (10.1186/s12870-017-1114-3) contains supplementary material, which is available to authorized users. Enhancing the grain yield potential of wheat is a key focus of wheat breeders. Grain yield is the product of various yield components. Wheat grain yield per unit area is the product of grain yield per spike (GYS) and the number of spikes per unit area. The latter depends on sowing density and is highly affected by environmental factors. The GYS is determined by grain number per spike (GpS) and thousand grain weight (TGW), which are variably correlated in different wheat collections/populations. A significant negative correlation between these two traits has been reported in bi-parental populations –3, but n−1 in 1992 to ~5.4 T ha−1 today is mainly due to the genetic increase in TGW from ~20 g to ~45 g, respectively [−1 can be obtained by just a 1-g increase in TGW [TGW in wheat has been one of the target traits for selection during domestication and breeding , 8. For e in TGW . Howevere in TGW . For exae in TGW  discovere in TGW .TaTGW6 [TaCwi-A1 [TaSus2-2B [TaSus2-2A, TaSus1-7A [TaGW2-6A, 6B [TaCKX6-D1 [TaSAP1-A1 [TaGS1a [TaGS-D1 [TaGASR-A1 [Nevertheless, from this plethora of QTLs, few loci/genes have been cloned by map-based cloning approaches mainly because of the large and complex hexaploid genome ~17 Gb) that consists of three homeologous genomes  and an abundance of repeat sequences (80%) . StudiesTaTGW6 , 17, TaC Gb that TaCwi-A1 , TaSus2-aSus2-2B , TaSus2-aSus1-7A , TaGW2-62-6A, 6B , 12, 21,aCKX6-D1 , TaSAP1-aSAP1-A1 , TaGS1a  [TaGS1a , TaGS-D1[TaGS-D1 , and TaGaGASR-A1  were disFLOURY ENDOSPERM2 (Flo2) gene is a member of a conserved gene family in plants. In rice, this gene has been shown to have a tetratricopeptide repeat (TPR) motif consisting of 3–16 tandem repeats of 34 aa residues that mediate protein–protein interactions in the nucleus [OsFlo2 gene was cloned in the indica cultivar ‘Kasalath’; this gene was found to have 23 exons and 22 introns and coded for a protein consisting of 1720 amino acid residues that had three TPR motifs in the middle [Flo2 was constitutive in both vegetative tissues and developing seeds, and the expression was relatively high level in developing seeds. The flo2 mutants exhibit a significant reduction in amylose content and grain weight and exhibit altered expression of various starch synthesis-related genes, indicating its key role in regulating rice grain weight and starch quality [Flo2 orthologs in hexaploid wheat, the association of TaFlo2-A1 sequence polymorphisms with TGW and the comparison of temporal expression profiles of TaFlo2-A1 haplotypes in flag leaves and developing caryopses.The  nucleus , 28. Thee middle . The exp quality , 28. In TaFlo2 gene in hexaploid wheat, Chinese Spring (CS) and two sets of cultivars with lower and higher TGW were used; the set of cultivars with higher TGW included Dixiuzao (49.5 g), Enmai4 (49.2 g), Liying 5(49.4 g) and Laizhou 953 (52.2 g), and the set with lower TGW included Jinyang 60 (23.5 g), Baihuamai (24.1 g), Sanyuehuang (25.2 g) and Zipi (25.5 g). The Chinese Spring nulli-tetrasomic lines were used to assign TaFlo2 genes to wheat homeologous chromosomes. The Chinese Micro Core Collection  and Pakistani wheat collection (130 accessions) were used to confirm the association between TaFlo2-A1 haplotypes and TGW. To avoid the effect of population structure, normalized MCC subpopulations were used for association analysis [For cloning the analysis , 29. Theanalysis  considerOsFlo2 gene (NC_008397) was used as a query for BLAST searches against the wheat sequences database in the URGI (https://urgi.versailles.inra.fr/). All wheat scaffold sequences with high similarity to the rice OsFlo2 sequence were assembled to construct a putative TaFlo2 gene using DNAMAN (http://www.lynnon.com). Based on the scaffold sequences, six conserved primer pairs were used to specifically amplify TaFlo2 coding and promoter sequences from the three wheat sub-genomes: A, B and D (Table TaFlo2 mRNA of 4902 bp was cloned in Chinese Spring using three primer pairs designed from the predicted mRNA sequence (Table http://www.lynnon.com).The genomic sequence of the rice  D Table . The TaFce Table . GenomicTaFlo2-A1a or TaFlo2-A1b) for the TaFlo2-A1 gene. For MCC, the average values of TGW of the two haplotype groups were calculated using replicated data collected from 3 years  of plants in Beijing [The 262 MCC and 130 Pakistani varieties were genotyped with the primer pair TaFlo2-InDel8, and PCR product was run on 8% polyacrylamide gels. Based on TaFlo2-InDel8 scoring, the MCC and Pakistani accessions sorted into two groups according to their haplotypes  among different samples were made by ANOVA using SPSS 13.0.The Yangmai 19, Chinese Spring, Pubing3228, Shannong23 and Zhengmai9405 varieties were sown at the experimental station of the Institute of Genetics & Developmental Biology, CAS in Beijing, China in October 2014; three rows of each variety were planted. The length of each row was 2 m, and the row-to-row distance was 20 cm. The plants were managed in accordance with standard agronomic practices; irrigation and fertilizer were supplied for optimal growth. Twelve- day-old flag leaves of five plants from each variety were harvested and stored at −80 °C. Unfertilized grains were collected from each variety 1–2 days before flowering (DBF). Fertilized grains were collected from each variety at 5, 10, 15, 20 and 25 days after flowering (DAF). The flag leaf and developing grain samples were processed for the preparation of total RNA as described previously . Three beviously . Statisthttp://www.lynnon.com). Amino acid sequence alignment was accomplished using ClustalW2 in EMBLEBI . Potential signal peptide sequences in the deduced proteins of TaFLO2 and its homologs were predicted using Softberry software (http://www.softberry.com/berry.phtml). The predicted TaFLO2 protein was BLASTed both in the NCBI smart blast system (http://blast.st-va.ncbi.nlm.nih.gov/smartblast) to search for homologous proteins and in the NCBI CD system (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) to search for conserved domains.Nucleotide and protein identities among the compared sequences were calculated using DNAMAN  was used as a query against the wheat genome sequences database in the URGI (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Three bread wheat scaffolds  with high similarity  were identified as potential orthologs to the rice Flo2 gene. The sequences of these scaffolds were downloaded and assembled with DNASTAR (http://www.dnastar.com/) to construct a putative TaFlo2 sequence.To select potential candidate TaFlo2 homologs and predict their deduced protein sequence and structure, Softberry (http://www.softberry.com/berry.phtml) and NCBI (https://www.ncbi.nlm.nih.gov/) tools were used. The deduced proteins of TaFlo2-A1 (1734 aa), TaFlo2-B1 (1698 aa) and TaFlo2-D1 (1682 aa) were highly similar (>94% identity among themselves) and exhibited >77% similarity with the rice FLO2 protein. Like in the rice FLO2 protein [Aegilops tauschii, Brachypodium distachyon and long-grain rice proteins  at position 1417–1419 bp . Further analysis revealed that the TaFlo2-A1 gene was located on deletion bin ‘2AL1–0.85-1.00’.To assign chromosomal locations to  2D Fig. . The cloTaFlo2-A1 with TGW, two natural populations, the Chinese Micro Core Collection (MCC) and the Pakistani collection, were used. In the MCC, the homozygous TaFlo2-A1a haplotype was found in 219 (83.5%) accessions, whereas the TaFlo2-A1b haplotype was found in 43 (16.5%) accessions. In the Pakistani wheat collection, the number of accessions carrying TaFlo2-A1a and TaFlo2-A1b were 85 (64.6%) and 45 (35.4%), respectively. Both in the MCC and Pakistani collections, the positive haplotype TaFlo2A-A1b had a lower frequency, which showed the scope of improving grain weight.To associate TaFlo2-A1a and TaFlo2-A1b was statistically significant in both populations  across the 3 years of data. The mean differences in TGW between the two haplotypes in 2002, 2005 and 2006 were 7.00 ± 1.12 g, 7.80 ± 1.11 g and 8.40 ± 0.94 g, respectively. Consistent with the results of the MCC, the mean difference in TGW between groups of accessions having TaFlo2-A1a and TaFlo2-A1b was also significant (P ≤ 0.05) across both years of data in the Pakistani wheat collection. The values of the mean difference between the two haplotypes (TaFlo2-A1a and TaFlo2-A1b) in the Pakistani wheat population were 4.50 ± 0.71 g and 5.20 ± 0.72 g for 2009 and 2010, respectively. The phenotypic variance for TGW explained by TaFlo2-A1 haplotypes was 6.19% in 2002, 7.76% in 2005 and 8.37% in 2006 in the MCC. In the Pakistani collection, the phenotypic variance for TGW explained by TaFlo2-A1 haplotypes was 4.42% in 2009 and 5.11% in 2010 , an association analysis was performed for GpS in both populations. However, the differences in GpS between the haplotypes TaFlo2-A1a and TaFlo2-A1b were not significant in either population  for high TGW instead of loci for low number of kernels per spike and/or low grain yield.Collectively, our data demonstrated that TaFlo2-A1a and TaFlo2-A1b on TGW at the gene expression level in flag leaves and developing grains, two polymorphic accessions were used. The expression level of TaFLO2 was positively correlated with TGW, which is consistent with previous results in rice [TaFlo2-A1a, which exhibits low expression levels, represented the group of accessions that have low average TGW, and the haplotype TaFlo2-A1b, which exhibits high expression levels, represented the group of accessions that have high average TGW in both Chinese and Pakistani wheat populations. Quantitative RT-PCR assays showed that for both types of haplotypes, the expression level was maximum in 12-day-old flag leaves followed by expression in developing grains sampled at 5 DAF. However, the expression of both types of haplotypes decreased rapidly in the fertilized caryopses collected at 10, 15, 20 and 25 DAF. The expression level of TaFlo2-A1b was higher than that of TaFlo2-A1a at all tested stages but significantly differed only in flag leaves and developing grains at 5 DAF  to 8.37% (in the MCC) of phenotypic variation. The TGW-related QTL identified on 2AL includes ‘Xgwm339-Xbarc311’ in 139 RILs between two hard red spring wheat lines [QTgw.ipk-2A (Xgwm372) in 111 BC2F3 lines derived from the cross ‘Flair × XX86’ [QGwt.crc-2A (Xgwm558-Xgwm294) in a double-haploid population generated from the cross ‘RL4452 × AC Domain’ [QTkw.sdau-2A (Xwmc181a-Xubc840c) in 131 RILs derived from ‘Chuan 35050’ × ‘Shannong 483’ [QSZ.uaf-2A.1 (Xwmc455) in natural populations of 108 CIMMYT and Pakistani spring wheat accessions [QTkw.hwwgr-2AL (Xgwm312–IWA6090) in 127 RILs derived from ‘Ning7840’ × ‘Clark’ [In our study, at lines ; QTgw.ip × XX86’ ; QGwt.cr Domain’ ; QTkw.sdcessions ; and QTkXgwm71.2/Xgwm558 and Xgwm294, with an interval of 22 cM according to the consensus map of Somers [http://wheat.pw.usda.gov/ggpages/SSRclub/GeneticPhysical/). From this TGW-QTL-rich region, only one gene, TaCwi-A1, has been isolated thus far between the Xgwm 71.2 and Xbarc15 deletion bin ‘C-2AL1–0.85’, which is adjacent to the centromere [http://wheat.pw.usda.gov/ggpages/SSRclub/GeneticPhysical/) and ‘Yu 8679 × Jing 411’ SSR + SNP [TaFlo2-A1 was inferred on deletion bin ‘2AL1–0.85-1.00’. Hence, the TaFlo2-A1 is a TGW-related gene located on the distal deletion bin of chromosome 2AL, and the molecular marker ‘TaFlo2-InDel8’ is an addition to the kit of wheat breeders for marker-assisted selection.These QTL on 2AL are located between f Somers  or 16.1 ntromere . By inteSR + SNP  maps, thThe relationship between the number of grains per spike (GpS) and the TGW was traditionally found as being negatively correlated –3. HowevTaFlo2-A1b) identified in this study would help to increase TGW without reducing the average GpS in wheat.In many genome-wide association studies (GWAS) for TGW and GpS, many loci were found to be associated with only one of the traits , 29, 50.TaFlo2-A1, which is represented by two haplotypes in our study, was found to be significantly associated with TGW. Polymorphisms of an 8-bp InDel in the promoter and of five SNPs in the first intron were observed in TaFlo2-A1. The orthologs TaFlo2-B1 and TaFlo2-D1 lacked sequence variations associated with TGW  was higher than that of the negative haplotype (TaFlo2-A1a). The phenomenon of higher expression of the positive TaFlo2 haplotype is consistent with the results for rice OsFlo2, in which the overexpression of the positive haplotype significantly increases grain size [flo2 mutation in the promoter and in the open reading frame hinders the expression of genes involved in the synthesis of starch and protein [flo2 mutations negatively affect grain quality attributes such as amylose content, grain appearance and physiochemical properties despite maintaining or increasing grain size [OsFlo2 and TaFlo2-A1 at the sequence, structure and expression levels, the 8-bp InDel mutation in the TaFlo2-A1 promoter likely regulates grain size by affecting the expression of genes involved in the synthesis of starch and protein in wheat grains. Therefore, the increased expression of TaFlo2-A1 has a positive effect on grain yield but may have a negative effect on some grain quality attributes in wheat, which shall need further investigations.Flo2 is considered to be a member of a conserved gene family in plants . TaFlo2-ain size . In rice protein , 28. In ain size , 28. BasFlo2 gene in wheat.The newly developed molecular marker ‘TaFlo2-InDel8’ is an addition to the kit of wheat breeder for marker-assisted selection. This study likely lead to additional investigations to unveil the exact regulatory mechanism of the Flo2 orthologs in hexaploid wheat were cloned, and TaFlo2-A1 was found to be associated with TGW but not with grain number per spike (GpS) in both the MCC and Pakistani wheat collections. The frequency of TaFlo2-A1b (positive haplotype) was low in commercial wheat cultivars; thus this haplotype can be selected to improve grain weight. This study likely lead to additional investigations to understand the regulatory mechanism of the Flo2 gene in hexaploid wheat. The newly developed molecular marker ‘TaFlo2-InDel8’ could be incorporated into the kit of wheat breeders for use in marker-assisted selection.The


Supplemental Digital Content is available in the text Accumulating studies have explored the effect of thymidylate synthase enhancer region (TSER) variation on risk of pediatric acute lymphoblastic leukemia (ALL) with controversial results. Therefore, this quantitative meta-analysis was performed to assess synthetically the association of TSER variation with susceptibility to develop pediatric ALL.The PubMed, ScienceDirect, Google Scholar, Wanfang Database, and China National Knowledge Infrastructure were systematically retrieved to obtain the published case-control studies about the relationship between TSER variation and pediatric ALL risk. The quality assessment of the included studies was preformed and relevant information was collected. Odds ratios (ORs) and 95% confidence intervals (CIs) were applied to evaluate the strength of association.P = 0.41; 2R/2R vs 3R/3R: OR = 0.99, 95% CI = 0.84–1.16, P = 0.90; 2R2R vs 3R/3R+2R/3R: OR = 1.05, 95% CI = 0.92–1.21, P = 0.45; 2R/3R+2R/2R vs 3R/3R: OR = 0.97, 95% CI = 0.87–1.09, P = 0.63; 2R vs 3R: OR = 1.03, 95% CI = 0.92–1.15, P = 0.61). Similarly, there was no significant association existed in the stratification analyses according to ethnicity, control source, and quality score.This meta-analysis finally included 2681 children with ALL and 3854 matched controls from 11 investigations. The quantitative synthesis results found no significant association between TSER variation and susceptibility to pediatric ALL in overall comparisons under 5 genetic models (2R/3R vs 3R/3R: OR = 0.95, 95% CI = 0.84–1.07, This meta-analysis shows that TSER variation is not related to the development risk of pediatric ALL. Folate metabolism not only supplies the methyl group for proper DNA biosynthesis, it also provides the universal methyl donor for DNA methylation . Plenty of studies have clarified that low folate intake causes uracil misincorporation in the process of DNA replication reactions, resulting in DNA double-strand breakage, chromosomal deletion, and catastrophic DNA repair.,6 What is more, hypomethylation of DNA may also cause the activation of proto-oncogenes.,8 Emerging evidence has shown that variations in genes encoding folate-metabolizing enzymes disturb the balance of folate metabolism and have been associated with an altered susceptibility to cancer.–11Pediatric acute lymphoblastic leukemia (ALL) accounts for 30% of all malignancy diagnosed in children and 80% of pediatric leukemia.,13 Therefore, TYMS functions as an essential regulator in the process of DNA biosynthesis, repair, and methylation. The TYMS gene with 7 exons locates at 18p11.32. There are several functionally important variants in the TYMS untranslated regions, of which thymidylate synthase enhancer region (TSER) variation has been most widely investigated.–16 TSER, a tandem-repeat polymorphism, which includes double (2R) or triple (3R) repeats of a 28 bp sequence in the TYMS 5′-untranslated enhanced region, may be associated with an alteration in TYMS mRNA expression.,18 Considering the pivotal role of folate in the development of cancer and the potential influence of TSER polymorphism in the TYMS gene on DNA biosynthesis and methylation, it is reasonable that TSER variation might be related to susceptibility to develop malignancies. Increasing studies have found that TSER polymorphism has been linked to human various cancer risks, such as non-Hodgkin lymphoma, breast cancer, and colorectal cancer.–21 Recently, numberous investigations have explored the effect of TSER variation on development risk of pediatric ALL, yet the reported results remain controversial. The discrepancies among these studies may be ascribed to the genetic backgrounds difference and relatively small sample size in individual investigation. Therefore, a quantitative meta-analysis was performed to evaluate synthetically the association of TSER variation with pediatric ALL risk.Thymidylate synthase (TYMS) catalyzes the methylation of deoxyuridine monophosphate (dUMP) to deoxythymidine monophospate (dTMP), and maintains the balance of deoxynucleotide pool, which is needed for normal DNA replication and damage repair.22.1The PubMed, ScienceDirect, Google Scholar, Wanfang Databases, and China National Knowledge Infrastructure were systematically searched to screen reports about the association of TSER variation and risk of pediatric ALL utilizing the following keywords: “childhood” or “pediatric” or “children,” “leukemia” or “acute lymphoblastic leukemia” or “ALL,” “thymidylate synthase” or “TS” or “TYMS,” “polymorphism” or “mutation” or “variation” or “variant.” The latest literature search was performed on January 20, 2016 and there was no language restriction. In addition, the reference lists in the retrieved articles were screened to identify relevant investigations. Ethical approval was not necessary because this study was a meta-analysis.2.2The following inclusion criteria were applied for literature selection: case-control designed study; confirmed diagnosis for the case group of pediatric ALL; available genotypes distribution data for cases and controls. The letters, case reports, commentary, and review articles were excluded. If the same or overlapping data was reported by multiple articles, we chose the one with larger sample size.2.3 The NOS method, with a maximum score of nine points, includes 3 quality categories: selection, comparability, and exposure evaluation. Studies with more than 6 scores were identified as high quality. Any disagreement was resolved by reevaluation of the originally included studies.Two authors independently preformed the quality assessment of included studies according to the Newcastle–Ottawa Scale (NOS).2.4The information was collected from each eligible investigation independently by 2 authors: first author's name, publication year, country, ethnicity, sample size, control source, method used for genotyping, genotypes distribution data of the TSER variation in case and control group.2.5χ2 test was employed to check Hardy–Weinberg equilibrium (HWE) of genotypes distribution frequencies in control groups and P < 0.05 was considered as departure from equilibrium. The strength of association between TSER variation and pediatric ALL risk was measured by odds ratios (ORs) and 95% confidence intervals (CIs) under the homozygote model (2R/2R vs 3R/3R), heterozygote model (2R/3R vs 3R/3R), dominant model (2R/3R+2R/2R vs 3R/3R), recessive model (2R2R vs 3R/3R+2R/3R), and allele model (2R vs 3R), respectively. The χ2-test-based Q test was performed to estimate the heterogeneity between included studies. When P > 0.05, showing that no statistically significant heterogeneity existed, the fixed-effects model (Mantel–Haenszel) was employed to compute the pooled ORs; alternatively, the random-effects model (DerSimonian–Laird) was used. Stratification analyses were carried out based on ethnicity, control source, and NOS score. Sensitivity analysis was conducted by omission of studies deviated from HWE to assess the stability of combined results. Both qualitative funnel plot and quantitative Egger test were employed to assess publication bias. All the statistical tests were done with RevMan v5.3  and STATA v12.0 , and P < 0.05 was deemed to have statistical significance.The 33.1–33Table –29 4 studies on Asians,–33 and 2 investigations on mixed population.,24 Four investigations were hospital-based ,30,31,33 and 7 were population-based –29,32 designed when classified according to the control source. Four studies were divided into low quality with a NOS score of 4 or 5 points, and 7 with score 6 or greater were assigned as high quality. The alleles and genotypes distribution data of TSER variation in case group and control group are summarized in Table ,29Figure 3.2P = 0.41; 2R/2R vs 3R/3R: OR = 0.99, 95% CI = 0.84–1.16, P = 0.90; 2R2R vs 3R/3R+2R/3R: OR = 1.05, 95% CI = 0.92–1.21, P = 0.45; 2R/3R+2R/2R vs 3R/3R: OR = 0.97, 95% CI = 0.87–1.09, P = 0.63; 2R vs 3R: OR = 1.03, 95% CI = 0.92–1.15, P = 0.61). Similarly, no significant association was found in the stratification analyses according to ethnicity , NOS score , and control source   and 2R allelic carriers  in de Jonge et al study. To elucidate this inconsistency, a synthetical meta-analysis was conducted. In our study, no significant heterogeneity was observed among overall studies under all 5 genetic models. The combined data demonstrated that there was no significant association of TSER variation and risk of pediatric ALL in overall comparison under all genetic models. No significant association was found in the stratification analyses based on ethnicity, control source, and quality score. Our results were not in accordance with the conclusion published by Weng et al, which showed TSER variation might dedicate to significantly increased risk of childhood ALL . Since our study added several new investigations and included 2681 children with ALL and 3854 matched controls, which allowed for sufficient statistical power and more precise estimation, our conclusion is more reliable.To date, many epidemiological studies regarding the association of TSER variation with risk of pediatric ALL have been reported, but the published results remain controversial. Gast et alHowever, several limitations in our study need to be addressed in interpreting the results. First, due to data insufficiency, 2 relevant investigations were removed from the quantitative synthesis. Second, our analysis largely focused on single-factor estimates not adjusted for other confounders such as gender, lifestyles, and other potential factors, which may cause confounding bias and influence the combined results. The combined analyses of some subgroups may have no sufficient testing power to accurately assess the real association. In addition, the gene-environment interactions that may modify cancer susceptibility were not assessed in our study ascribed to the limited relevant information.5TYMS gene was not related to susceptibility to develop pediatric ALL. However, in the future, well-designed studies with more participants are demanded to verify this conclusion.In brief, this meta-analysis suggested that TSER polymorphism in


This study found a significant increase in PM H+-ATPase (PMA) activity in grape root exposed to NaCl. Furthermore, 7 Vitis vinifera PM H+-ATPase genes (VvPMAs) were identified within the grape genome and the expression response of these VvPMAs in grape root under salinity was analyzed. Two VvPMAs (VvPMA1 and VvPMA3) were expressed more strongly in roots than the other five VvPMAs. Moreover, roots exhibited diverse patterns of gene expression of VvPMA1 and VvPMA3 responses to salt stress. Interestingly, two transcripts of VvPMA1, which were created through alternative splicing (AS), were discovered and isolated from salt stressed root. Comparing the two VvPMA1 cDNA sequences (designated VvPMA1α and VvPMA1β) with the genomic sequence revealed that the second intron was retained in the VvPMA1β cDNA. This intron retention was predicted to generate a novel VvPMA1 through N-terminal truncation because of a 5′- terminal frame shift. Yeast complementation assays of the two splice variants showed that VvPMA1β could enhance the ability to complement Saccharomyces cerevisiae deficient in PM H+-ATPase activity. In addition, the expression profiles of VvPMA1α and VvPMA1β differed under salinity. Our data suggests that through AS, the N-terminal length of VvPMA1 may be regulated to accurately modulate PM H+-ATPase activity of grape root in salt stress.It has been well-demonstrated that the control of plasma membrane H Grapevine is adapted to semiarid environments, where drought and salinity are common problems. Grapevine is considered moderately tolerant to salt stress, and this moderate tolerance has been mainly attributed to salt exclusion or to restriction of toxic ions in the root system  under a 16/8 h light/dark photocycle. Shoot tips with a minimum of one bud were used to subculture every month. After 30 days, uniform plants were selected as the test material and were transferred into Hoagland solution 2, 5 mM KNO3, 1 mM KH2PO4, and microelements contained 0.2 mM Fe-EDTA, 2 × 10−3 mM MnCl2, 2.5 × 10−4 mM H3BO3, 5 × 10−4 mM CuSO4, 2 × 10−3 mM ZnSO4 and 5 × 10−4 mM H2MoO4) for 7 days. The growth conditions were kept the same as above. The test seedlings were treated with 50 mM NaCl in Hoagland solution for 3, 6, 12, 24, 48, and 72 h, respectively. And the seedlings grown in Hoagland alone were used as the control (0 h). Samples were then harvested at the same time in the same day to minimize diurnal effects wherever possible, then frozen in liquid nitrogen and stored at −80°C.The table-grape cultivar Crimson seedless (C133–199 × Emperor) was used in this study. The sterilized seedlings were grown on 1/2 MS solid medium  PVP, 2 mM DTT, 0.2 mM PMSF, 5% (V/V) glycerol, and mannitol, which produced the same osmotic potential as within leaves. Two milliliters of the medium was used for each 2 g of fresh material. The homogenate was filtered through four layers of cheesecloth and centrifuged at 10,000 g for 20 min (Beckman JA-20). The supernatant was loaded on a 0%: 36%: 45% (W/W) sucrose gradient solution  and centrifuged at 100,000 g for 2 h (Beckman SW40Ti). The plasma membrane-enriched vesicles were located at the 36%: 45% (W/W) sucrose interface. These vesicles were carefully collected and diluted 3–4-fold with dilution buffer  and centrifuged at 100,000 g for 30 min (Beckman type 65). The pellets were suspended in a storage buffer (40% (V/V) glycerol, 2 mM DTT, 10 mM HEPES, adjusted to pH 7.0 with KOH).Plasma membrane-enriched vesicles were isolated by discontinuous sucrose gradient centrifugation, according to the method of Wang et al.  and MandProtein content was determined using bovine serum album as a protein standard according to the Bradford method 2MoO4, 1 mM NaN3, 1 mM MgSO4, 0.03% (V/V) TritionX-100, 50 mM KCl, and 3 mM ATPNa2. Inorganic phosphate was assayed by using the method of Lin and Morales . Then the PM H+-ATPase functional domains of these candidate proteins were predicted using Pfam software , the supernatant was then transferred to an equal volume of extraction cocktail [chloroform/isoamyl alcohol (24/1)] in order to extract the RNA. The mixture was centrifuged at 10, 000 g for 10 min, after which, the supernatant was mixed with LiCl (3 M) and stored at −20°C for 12 h. After centrifugation , the pellets were suspended in SSTE buffer . A chloroform/isoamyl alcohol (24/1) mixture was added, and then the samples were centrifuged at 10, 000 g for 10 min. The supernatant was transferred to a new RNase-free tube and mixed with 0.7 volume isopropanol. After centrifugation at 10,000 g for 20 min, the pellets were washed with 1 ml 75% alcohol, dried, and resuspended in RNase free water. The total RNA was quantified with a NanoDrop 2000C spectrophotometer  at wavelengths of 230, 260, and 280 nm and the 260/280 nm ratio was determined. RNA integrity was confirmed by running samples through 1% agarose gels. After that samples were stored at −80°C.Total grape RNA was extracted using the CTAB method as previously described  for the same gene  according to the manufacturer's instructions. Quantitative PCR was carried out on a real-time PCR system (Bio-Rad iQ5) using the SYBR Green Kit . Thermal cycling conditions were 95°C for 10 min followed by 95°C for 10 s, 56°C for 10 s for 40 cycles, and a melting cycle was then performed from 65° to 95°C. A house-keeping gene VvPMA1 was isolated through PCR using Pfu DNA polymerase  and the primers listed in Supplementary Table E. coli DH5α cells. Finally, the 9 recombinant plasmids were prepared and then sequenced, respectively.RNA was extracted from the root of a Crimson seedless grape plant and cDNA synthesis was carried out as described above. The full-length cDNA encoding PMA1 (yeast endogenous PM H+-ATPase) was kindly provided by Dr. Palmgren . The full-length coding regions of VvPMA1α and VvPMA1β were amplified from pEASY-Blunt-VvPMA1α and pEASY-Blunt-VvPMA1β using the primers VvPMA1α-P1, 5′CTCGAGATGGGAGGCGACAAATCC 3′, which includes an underlined XhoI site, and VvPMA1-P2, 5′ GGGCCCTCATACTGTGTAATGCTGTTGGATT 3′, with an underlined ApaI site; VvPMA1β-P1, 5′CTCGAGATGCGCATTTCATTGAATT 3′ and VvPMA1-P2, 5′ GGGCCCTCATACTGTGTAATGCTGTTGGATT 3′. The PCR products were subcloned into XhoI and ApaI sites of pMP625 to generate plasmid pMP625-VvPMA1α and pMP625-VvPMA1β. All PCR amplifications were carried out using Pfu DNA polymerase, which exhibits the lowest error rate of any thermostable DNA polymerase, and all constructs were sequenced to confirm their identity.Plasmid pMP625 containing the promoter and terminator of MATa ade1–100 his4–519 leu2–3, 112), the natural constitutive promoter of the PMA1 has been replaced by the galactose-dependent GAL1 promoter. As PMA1 is essential for yeast growth, RS72 is only able to grow on galactose-containing medium. Using this strain, plasmid-borne plants' PM H+-ATPases under the control of the constitutive PMA1 promoter can be tested for their ability to rescue pma1 mutants on glucose-containing medium.The yeast strain RS72  was cultured as described previously  or 20 ml liquid synthetic medium at OD600 0.01 containing either 2% galactose at pH 5.5 or 2% glucose at pH-values of 5.5, 4.5, and 3.5. The media were buffered with 50 mM succinic acid adjusted to various pH-values with Tris. Growth was recorded after incubation for 3 days at 30°C. Each experiment was replicated independently three times.Yeast was made competent for plasmid uptake by treatment with lithium acetate and polyethyleneglycol according to Gietz et al. . Positivt-test for independent samples. In all cases, the confidence coefficient was set at 0.05.Statistical analyses were performed using the software SigmaPlot 10.0 . Values were expressed as mean ± SD. All comparisons were done using Student's P < 0.01). The maximum increase in enzyme activity resulted in an activity about 2.7-fold greater than that of the control at 24 h. Although the enzyme activity decreased between 24 and 72 h, the activity level remained above the control activity level (P < 0.01)  for 0, 3, 12, 24, or 72 h. ATP hydrolytic activity in PM vesicles obtained from plant roots increased under the NaCl treatments. Moreover, the enzyme activity was up regulated within 3 h of treatment , but expression levels did not significantly differ between VvPMA1 and VvPMA3 (P > 0.05). Additionally, although expression of VvPMA2, VvPMA5, and VvPMA7 was found in other tissues . Interestingly, two different cDNA sequences of VvPMA1 were obtained. Comparison of the two VvPMA1 cDNA sequences (designated VvPMA1α and VvPMA1β) with the genomic sequence revealed that the second intron (93 bp) was retained in the VvPMA1β cDNA  grew well in galactose medium only , Ski-interacting protein (SKIP) etc., can control the AS of pre-mRNAs encoded by salt tolerance genes under salt stress in plants (Syed et al., VvPMA1β must be controlled by one or some specific splicing factors in grape root under salt conditions, moreover, VvPMA1β expression maintained low level in root under salt stress probably because the specific splicing factor has weak activity. Alternatively, PM H+-ATPase activity could be confined by an unknown feedback mechanism under different environment and overexpression of its gene might be disadvantageous to plant growth (Hashimoto-Sugimoto et al., +-ATPase activity. The specific splicing factor will be further screened from grape root and the function of VvPMA1β will be ascertained by transgenetic effectiveness in plant under salt stress.Recent genome-wide gene expression analyses have suggested that mRNA splicing is under the control of developmental and stress signals (Seo et al.,  Figures , the expVvPMA1 in response to salt stress occurred at 3 h of treatment as early as transcriptional regulation of VvPMA3 (Figures VvPMA1β and VvPMA3 reached peak before 24 h. Meanwhile VvPMA1α expression was unchanged markedly. After that, VvPMA1α expression obviously increased, while the other two diminished. Combining these findings with the dynamic change of the activity of PM H+-ATPase and VvPMA1β activity (Figure VvPMA1β would have a positive effect on PM H+-ATPase activity in salt stress, albeit with phenotypic lag. Therefore, subtle changes in the ratio of intact VvPMA1 and the alternatively spliced variant would likely have profound effects on PM H+-ATPase activity in grape root under saline conditions in spite of changes to the quantity of PM H+-ATPase present. Further work is required to determine changes in the quantity of each PM H+-ATPase isoform under salt stress.Additionally, posttranscriptional regulation of  Figures , 7. And y Figure , we pred+-ATPase in grape root. Increases to enzyme activity were accompanied by transcriptional increases. Furthermore, cloning and sequencing of the PCR-amplified fragments of PM H+-ATPase transcripts revealed the expression of an N-terminal truncated splice variant in response to NaCl treatment. This variant had a higher activity rate than the intact variant. These findings suggest that alternative splicing, in coordination with gene transcriptional control, may help to maintain the appropriate level of the PM H+-ATPase activity in grape roots under saline condition. Furthermore, it is also possible that differential regulation of the PM H+-ATPase isoforms within the same cell could be an important mechanism for responding to a diverse range of environmental fluctuations.Taken together, the results of this study revealed that NaCl has a stimulatory effect on PM HNH, XZ, and HZ participated in the study conception and design. XJ contributed to RNA extraction and qPCR. NH, YD, and XH contributed to data analysis. NH wrote the manuscript. YD and XH critically revised the manuscript. All authors approved the final version of 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.


Allergic asthma is an inflammatory lung disease that is partly sustained by the chemokine eotaxin-3 (CCL26), which extends eosinophil migration into tissues long after allergen exposure. Modulation of CCL26 could represent a means to mitigate airway inflammation. Here we evaluated procyanidin A2 as a means of modulating CCL26 production and investigated interactions with the known inflammation modulator, Interferon γ (IFNγ). We used the human lung epithelial cell line A549 and optimized the conditions for inducing CCL26. Cells were exposed to a range of procyanidin A2 or IFNγ concentrations for varied lengths of time prior to an inflammatory insult of interleukin-4 (IL-4) for 24 h. An enzyme-linked immunosorbent assay was used to measure CCL26 production. Exposing cells to 5 μM procyanidin A2 (prior to IL-4) reduced CCL26 production by 35% compared with control. Greatest inhibition by procyanidin A2 was seen with a 2 h exposure prior to IL-4, whereas IFNγ inhibition was greatest at 24 h. Concomitant incubation of procyanidin A2 and IFNγ did not extend the inhibitory efficacy of procyanidin A2. These data provide evidence that procyanidin A2 can modulate IL-4-induced CCL26 production by A549 lung epithelial cells and that it does so in a manner that is different from IFNγ. Procyanidins are polyphenolic secondary plant metabolites that are recognized as biologically active in the context of human health. They are the product of polymerization of monomeric flavan-3-ol units by a currently undefined mechanism in plants . The monInflammatory diseases, such as allergic asthma, are less prevalent in populations that consume procyanidin-rich diets ,7. The lProcyanidin A2 is one structurally defined A-type procyanidin  that is p < 0.001) and above. The time course for CCL26 production following 5 ng/mL IL-4 ranging from 1 to 72 h is shown in p < 0.01) and continued to increase in a linear manner until the final measured time point of 72 h . From these data we determined that 5 ng/mL IL-4 for 24 h provided a sufficiently robust induction of CCL26, which enabled us to progress into utilising this model for the evaluation of procyanidin A2.The concentration response characteristics for CCL26 production following IL-4 exposure (24 h) are shown in 2O2 was used, and demonstrated a measurable release of LDH (p < 0.001 compared with baseline control). Exposure to procyanidin A2 at 1, 5, 10 and 20 μM did not mediate any significant change in LDH release from the baseline control  from procyanidin A2-exposed epithelial cells as an indicator of the breakdown of the cellular plasma membrane. A positive control of 100 mM H control . Procyanp < 0.01) on IL-4–induced CCL26 production when incubated for 6 h prior to an inflammatory insult of 5 ng/mL IL-4 for 24 h  and 5.8 nM (high). The time courses for procyanidin A2, low IFNγ, and high IFNγ inhibition of CCL26 are shown in p < 0.05). Procyanidin A2 had a similar inhibition of CCL26 production at 6 h when compared with high concentration IFNγ  to a later (6 h) time point or improve inhibitory efficacy beyond that of procyanidin A2 alone . InvestiThis study sought to evaluate the potential modulation of cytokine-induced CCL26 by a commercially sourced, structurally-defined, pure dimeric procyanidin, procyanidin A2. We confirmed IL-4 exposure induces CCL26 production in A549 cells, thereby modelling an aspect of airway inflammation of relevance to allergic asthma. Exposure time and IL-4 concentration were optimized for CCL26 production. These experiments demonstrated that 5 ng/mL IL-4 for 24 h allowed for a robust induction of CCL26 production in A549 cells. Using these parameters we demonstrated that both 5 and 10 μM procyanidin A2 when incubated 6 h prior to an inflammatory insult had an inhibitory effect on CCL26 production. Experiments determining cytotoxicity by measuring LDH, along with previous work in our group using the WST-1 assay , demonstCCL26 gene [There is a growing pool of evidence that supports the ability of polyphenolic compounds, specifically procyanidins, to modulate key inflammatory events (see reviews) ,4. AberrL26 gene , and incL26 gene . The temTo date the demonstrated biological effects of A-type linked procyanidins include activity as an antioxidant, which is attributed to the catechol group in the B-ring of many flavonoids that can trap free radicals ,26,27, aPrevious work from our group in a similar cell culture model suggested proanthocyanidins as the bioactive components responsible for the ability of blackcurrant extracts to modulate CCL26 production in vitro . The stuIn order to progress our understanding of the role procyanidins may have in mitigating airway inflammation, future work could investigate procyanidin A2 by utilizing an air-liquid interface epithelial cell culture system. This would mimic and evaluate an application via inhalation as opposed to oral ingestion. Studies investigating the efficacy of IFNγ applied through inhalation have suggested its use in eosinophilic asthma, with effects limited to the respiratory tract . This ap® CCL-185™, ). Cell culture media, phosphate buffered saline (PBS), penicillin-streptomycin-neomycin antibiotic mixture, 100× l-glutamine, and 2.5% trypsin were purchased from Life Technologies . Fetal bovine serum (FBS) was purchased from Moregate Biotech . Procyanidin A2 -epicatechin) was purchased from Extrasynthese . The CCL26 DuoSet ELISA kit, human recombinant IL-4 and IFNγ were purchased from R&D Systems . Low-endotoxin bovine serum albumin was purchased from MP Biomedicals . Ecoteric T20 (Tween 20) and 30% hydrogen peroxide were purchased from ThermoFisher Scientific . Sodium acetate tri-hydrate was from BDH Laboratory Supplies . β-nicotinamide adenine dinucleotide reduced dipotassium salt (NADH), dimethyl sulfoxide (DMSO) and all other chemicals not specifically listed were purchased from Sigma-Aldrich .The human alveolar epithelial (A549) cell-line was purchased from the American Type Culture Collection (ATCC2 in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) containing 2 mM l-glutamine, 50 μg/mL penicillin, 50 μg/mL streptomycin, 100 μg/mL neomycin, and 10% FBS. The cell cultures were grown to form a monolayer (80% confluence) and then growth arrested for 24 h in the absence of FBS before conducting experiments. Experiments used A549 cells of passage 89–107.Cells were grown under standard tissue culture conditions as previously described . Briefly5 per well in 12-well plates with culturing and growth arrest as described above. Cells were then exposed to serum-free media containing IL-4. Extracellular media were collected and frozen at −80 °C until analysis for CCL26 using a commercially available sandwich ELISA kit following the manufacturer’s instructions.The production of CCL26 can be induced by IL-4 in A549 airway epithelial cells in vitro to model the inappropriate inflammation that occurs during allergic airway inflammation. In order to optimize a bioassay for CCL26 production, we characterized both the concentration- and time-dependent profiles of CCL26 production after incubation with IL-4. We measured CCL26 production in A549 cells exposed to IL-4 at a range of concentrations from 0.25 to 50 ng/mL for 24 h, as well as determined the time course of CCL26 production after exposure to 5 ng/mL IL-4 for times ranging from 1 to 72 h. Experiments used A549 cells seeded at 5 × 10v/v) DMSO, respectively, for cultures exposed to 5 μM (2.88 μg/mL) and 10 μM (5.765 μg/mL) procyanidin A2. These represented the negative controls.Procyanidin A2 was dissolved in 100% DMSO to 10 mg/mL and stored in aliquots at −80 °C until experimental use. Procyanidin A2 was then brought to the desired concentration in serum-free media and added immediately to cell cultures. Final DMSO concentrations were 0.03% and 0.06% , LDH substrate consisting of 3.8 mM pyruvate and 0.8 mM NADH was added to one well at a time and absorbance of NAD+ read at 340 nm for 2 min.The potential cytotoxicity of procyanidin A2 was evaluated by determining effects on cellular plasma membrane integrity through the measurement of the release of the cytosolic enzyme LDH. Experiments used A549 cells seeded at 9 × 105 per well in 12-well plates with culture and growth arrest as described above. Cells were then incubated with media containing procyanidin A2 at final concentrations ranging from 0.0001 to 10 μM, or corresponding DMSO concentrations (max 0.06%) for 6 h, washed briefly with PBS, fresh media replaced and then stimulated with 5 ng/mL IL-4 for 24 h to induce CCL26 production. Extracellular media were collected and frozen at −80 °C until analysis for CCL26 production by ELISA. A similar protocol was used for experiments using low (0.5 nM) and high (5.8 nM) IFNγ. A time course of CCL26 inhibition was determined for 5 μM procyanidin A2, low concentration (0.5 nM) and high concentration (5.8 nM) IFNγ, by exposing A549 cells for 1–24 h prior to the inflammatory insult of 5 ng/mL IL-4. CCL26 production was also investigated following a 6 h concomitant exposure to 5 μM procyanidin A2 added to low (0.5 nM) and high (5.8 nM) IFNγ. Procyanidin A2 and IFNγ concentrations were prepared separately and added together in the cell culture well.In experiments examining effects of procyanidin A2 on inflammation, A549 cells were seeded at 5 × 10t-tests with a Welch’s correction. Data that did not reach a p < 0.05 threshold were listed as not significant (NS). Statistical significance in the figures is denoted as * p < 0.05, ** p < 0.01, *** p < 0.001.Statistical analysis of data was performed using GraphPad Prism 5  or GenStat: 14th Edition . Data in the IL-4 concentration response A and timOur data provides evidence that pre-exposure to procyanidin A2 is a prompt yet transitory method to inhibit CCL26 production in A549 human lung epithelial cells. The temporal profile of inhibition is observed to be different from that of IFNγ. These data are encouraging, but further investigation is needed in an animal model or whole body system to determine if procyanidin A2 could be utilized as a preventative approach to help manage airway inflammation.


The purpose of this study is to use the anatomical information from the 4D flow sequence to assess the global left ventricular function and compare these results with the ones obtained from standard cine acquisitions.Between September 2014 and February 2015, we prospectively included 22 consecutive adult patients  planned for CMR with a clinical indication for contrast administration. The 4D flow raw data sets were uploaded to a dedicated web-based software application .Images were reconstructed in 20 cardiac temporal phases separately with a compressed sensing algorithm. The end-diastolic, end-systolic and stroke volumes and ejection fraction were measured by CMR 4D flow and compared against cine CMR measurements.The mean end-diastolic, end-systolic stroke volumes and ejection fraction were 163( ± 30) ml, 77( ± 34) ml, 85 ( ± 17) ml and 54 ( ± 11)% respectively for CMR 4D flow and 182 ( ± 50) ml, 89 ( ± 42) ml, 93 ( ± 18) ml, 53 ( ± 10)% respectively for cine CMR. The Pearson's correlations between CMR 4D flow and CMR were 0.93, 0.96, 0.77 and 0.93 for end-diastolic, end-systolic, stroke volumes and ejection fraction respectively.When applying a threshold of 50% for ejection fraction, 21 out of 22 patients were correctly classified.In this study we showed that global left ventricular function can be quantified accurately using CMR 4D flow imaging analysed using a cloud based software.


Nucleophosmin (NPM1) is a multifunctional protein that is implicated in the pathogenesis of several human malignancies. To gain insight into the role of isolated fragments of NPM1 in its biological activities, we dissected the C-terminal domain (CTD) into its helical fragments. Here we focus the attention on the third helix of the NPM1-CTD in its wild-type (H3 wt) and AML-mutated (H3 mutA and H3 mutE) sequences. Conformational studies, by means of CD and NMR spectroscopies, showed that the H3 wt peptide was partially endowed with an α-helical structure, but the AML-sequences exhibited a lower content of this conformation, particularly the H3 mutA peptide. Thioflavin T assays showed that the H3 mutE and the H3 mutA peptides displayed a significant aggregation propensity that was confirmed by CD and DLS assays. In addition, we found that the H3 mutE and H3 mutA peptides, unlike the H3 wt, were moderately and highly toxic, respectively, when exposed to human neuroblastoma cells. Cellular localization experiments confirmed that the mutated sequences hamper their nucleolar accumulation, and more importantly, that the helical conformation of the H3 region is crucial for such a localization. Nucleophosmin 1  is an abundant multifunctional protein belonging to the nucleoplasmin family of nuclear chaperones . The C-tThe presence of several nuclear import/export signals in the NPM1 sequence indicates its propensity to act as a nuclear and/or nucleolar shuttling protein. Remarkably, it is involved in several biological processes such as ribosome biogenesis, tumor suppression and nucleolar stress response , 8. WhilA so-called nucleolar localization signal (NoLS) is present in the three-helix bundle of NPM1. Importantly, although most of the identified NoLS are formed by sequence motifs enriched in consecutive basic residues, which are thought to be recognized by specific nucleolar hub proteins (including NPM1), the NoLS of NPM1 is unique and constituted by the two tryptophan residues in helix H3 . AML-assin vitro cell viability assays. Moreover, the extension of the H2 sequence beyond its N-terminus, including the linker loop connecting H2 with H1, delayed aggregation and its associated cytotoxicity  NMR spectroscopic analyses were performed for H3 wt, H3 mutE and H3 mutA peptides in 10 mM sodium phosphate, pH 7.2. Comparison of TOCSY and NOESY experiments allowed to get resonance assignments for most of the proton atoms  appeared small, but negative for most residues, thus indicating a certain propensity towards α-helical conformations Figure . The perWe evaluated CSD values for H3 mutA and H3 mutE peptides as well  experiments over time at a peptide concentration of 2 mM. DLS is a quantitative, optical method for determining diffusion coefficients of particles undergoing Brownian motion in solution or suspension. The knowledge of diffusion coefficient of molecules allows the determination of size and flexibility of molecules. DLS is well suited for the detection of large aggregates such as amyloid since the intensity of the scattered light is proportional to the square of the particle mass . As repoThe H3-derived peptides were also evaluated for their ability to impair cell viability. We first analyzed the effects of the aggregates formed from the H3 peptides on the mitochondrial status of human SH-SY5Y neuroblastoma cells, using the MTT reduction assay. The H3 wt, H3 mutA and H3 mutE peptides were incubated at a concentration of 1 mM in 50 mM phosphate buffer, pH 7.2, at 25°C, at 3 different times  and then added to the cells at a concentration of 100 μM. The three peptides added without pre-incubation (0 h) did not affect cell viability at this time-point Figure . Followi2+ homeostasis is one of the earliest biochemical consequences of the interaction of prefibrillar aggregates with cell membranes  TOCSY  (70 ms mixing time)  experiments were usually recorded with 32–64 scans, 128–256 FIDs in t1, 1024 or 2048 data points in t2. Water suppression was achieved by Excitation Sculpting  × 100 where [(CSD = ΔδHα= δHαobserved− δHαrandom-coil)] was averaged over all residues in a helical conformation [Helical populations were estimated from CSD data with the equation: [Δδormation . Peptideormation  (versionormation .The ThT solution (6.48 μL at 38.5 mM ThT in water) was added to 2.5 mL of the peptide stock solutions  at 25°C. For H3 mutA a similar experiment at 2 mM was also carried out and the sample was previously centrifuged. ThT fluorescence was measured using a Varian Cary Eclipse spectrofluorimeter  and a cell consisting of a 10 × 10 mm path-length quartz cuvette, under magnetic stirring. Measurements were collected every day during 25–30 days, by using excitation and emission wavelengths of 440 and 450–600 nm, respectively.H3 derived peptides , were kept under stirring in 50 mM phosphate, pH 7.2, at 25°C. The measurements were performed using a Zetasizer Nano S DLS device from Malvern Instruments  with 633 nm laser, dual scattering angle mode, thermostated with a Peltier system and using a low-volume (45 μL), ultramicro cell. Size distributions by intensity and total light scattering intensity were determined in automatic mode at regular time-intervals over a period of 10 min for each measurement. Thirteen acquisitions were recorded, each 10 seconds in duration.3 (1:1) and supplemented with 10% vol/vol FBS, 1.0 mM glutamine and antibiotics. Cell cultures were maintained in a 5.0% vol/vol CO2 humidified atmosphere at 37°C and grown until they reached 80% confluence for a maximum of 20 passages.Human SH-SY5Y neuroblastoma cells  were cultured in DMEM, F-12 Ham with 25 mM HEPES and NaHCOHeLa cells were grown in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% v/v of fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin sulfate.5 cells/mL in 24-well plates overnight. The following day, 10 μM of TAT-H3 derived peptides were diluted in DMEM without red phenol and sonicated with a Bioruptor instrument , 30 sec on/off at the highest power for 30 min to avoid aggregates and then incubated over cells for 24 h.For transient peptide transfection, HeLa cells were seeded onto glass coverslip at 1 × 10SH-SY5Y cells were seeded in 96-well plates. Aggregates of the H3 wt, H3 mutA and H3 mutE peptides (1 mM monomer concentration) were incubated in 50 mM sodium phosphate buffer, pH 7.2, 25°C under stirring, for 3 different times . The samples were then diluted into cell culture media at a 100 μM peptide concentration, and then added to the SH-SY5Y cells for 24 h at 37°C. Cell viability was then assessed by the 3--2,5-diphenyltetrazolium bromide (MTT) reduction assay as previously described . Cell viAggregates of the H3 wt, H3 mutA and H3 mutE peptides (1 mM monomer concentration) were incubated in 50 mM sodium phosphate buffer, pH 7.2, 25°C under stirring, for 0 h, 15 days and 30 days. The samples were then diluted in cell culture media at a 100 μM peptide concentration, and then added to the SH-SY5Y cells seeded on glass coverslips for 60 min at 37°C. The cells were then loaded for 30 min at 37°C with 10 μM fluo3-AM , as previously described . The resFor fluorescence analyses, HeLa cells grown on glass coverslips were fixed with paraformaldehyde 4% v/v in PBS for 20 min at room temperature (RT). Fixed cells were washed with PBS and incubated with the TO-PRO-3 for nuclei counterstaining. After washing three times with Tween-20 0.1% v/v in TBS, the coverslips were mounted. Cells were examined under a Leica TCS SP laser-scanning confocal microscope  equipped with a 488-nm argon laser, a 543-nm He-Ne laser, and a 63× oil immersion objective . Data were acquired at room temperature (23°C) using the integrated Leica Confocal Software package; multicolor images were captured through sequential scanning.


SMCHD1 gene segregate with 4qA haplotypes and D4Z4 hypomethylation.The main form of Facio-Scapulo-Humeral muscular Dystrophy is linked to copy number reduction of the 4q D4Z4 macrosatellite (FSHD1). In 5 % of cases, FSHD phenotype appears in the absence of D4Z4 reduction (FSHD2). In 70-80 % of these patients, variants of the SMCHD1 mutation at the protein level and on the NMD-dependent degradation of transcript.We report a family presenting with neuromuscular symptoms reminiscent of FSHD but without D4Z4 copy reduction. We characterized the 4q35 region using molecular combing, searched for mutation in the SMCHD1 gene and determined D4Z4 methylation level by sodium bisulfite sequencing. We further investigated the impact of the DUX4-fl expression. Exome sequencing revealed a heterozygous insertion of 7 bp in exon 37 of the SMCHD1 gene producing a loss of frame with premature stop codon 4 amino acids after the insertion (c.4614-4615insTATAATA). Both wild-type and mutated transcripts are detected.In muscle, we observe moderate but significant reduction in D4Z4 methylation, not correlated with The truncated protein is absent and the full-length protein level is similar in patients and controls indicating that in this family, FSHD is not associated with SMCHD1 haploinsufficiency.The online version of this article (doi:10.1186/s12881-016-0328-9) contains supplementary material, which is available to authorized users. In mostte <25 % , <30 %; te <25 % ). Severaonsenses .SMCHD1 gene encodes a 226KDa protein containing a GHKL-type ATPase domain and a hinge domain. The SMCHD1 protein belongs to the “Structural Maintenance of Chromosomes” family, which includes seven members . In mice, this protein binds to the PRC2 polycomb complex and colocalizes with trimethylH3K27 or associates with Dnmt3B for X chromosome inactivation and variegation of gene expression [DUX4 transcript (DUX4-fl) encoded by the last D4Z4 repeat and the flanking 4qA region [The pression –16. In FA region .SMCHD1 gene. We determined the segregation of the mutation in the family, its functional consequence at the mRNA and protein levels in peripheral blood mononuclear cells (PBMCs), fibroblasts and muscle together with association with D4Z4 methylation and DUX4 expression.We identified a family in which the proband carries a 7-nucleotide insertion in exon 37 of the Individuals were clinically assessed by neurologists with expertise in neuromuscular diseases who defined the presence or total absence of clinical signs and evaluated the involvement of the typical groups of muscle usually affected in the disease . Based on the recent CCEF classification of FSHD patients, the proband was classified in category A2  . Researc2.Skin biopsy was obtained using standard procedures. Primary fibroblasts were obtained by placing the skin biopsy in a culture dish containing DMEM medium supplemented with 4.5 g/L of glucose, 2 mM glutamin, 10 % fetal calf serum (FCS) and 1 % Penicillin/Streptomycin for 15 days. At subconfluence, primary cells were collected by addition of 0.25 % trypsin and 1 mM EDTA, resuspended in fresh DMEM medium, plated and incubated at 37 °C in 5 % COTotal DNA was extracted from peripheral blood mononuclear cells (PBMCs) using the Qiagen DNeasy Blood & Tissue Kit, following manufacturer’s instructions. Total RNA was extracted from peripheral blood with Trizol-Chloroform (Life Technologies) following manufacturer’s instructions. After DNAse treatment (Ambion), total RNA was converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) with random primers (Life Technologies). Whole protein extracts were obtained from cells disrupted in 200 μL extraction buffer .Proteins were separated by electrophoresis and transferred onto a PVDF membrane following the protocol recommended by the supplier for the Life Technologies NuPAGE system for Bis/Tris 4-12 % gels. After transfer, PVDF membranes were blocked for 1 h in 5 % (w/v) non-fat dry milk in PBS-T (0.1 % Tween-20 in PBS) and incubated for 90 min with either Lamin B , N-terminal SMCHD1  or C-terminal SMCHD1 . After 4 washes in PBS-T, membranes were incubated for 90 min with anti-mouse IgG secondary antibody coupled to HRP . Signals were revealed using enhanced chemiluminescence  using a ChemiDoc XRS system (Bio-Rad).Experiments were performed as described by Nguyen et al,  on peripIn silico predictions were performed with HSF3.0 [Exome capture with Agilent SureSelect All Exon kit V5 followed by paired-end sequencing with HiSeq2000 was applied to DNA samples from patients II.1 and I.1. Sequencing was performed by IntegraGen SA . Other muscular gene variants were selected from a restricted list of 45 genes currently used for neuromuscular disease diagnosis. All variants, listed in Additional file h HSF3.0 . Finallyhttp://frodo.wi.mit.edu/) and checked by BLAST against the human genome to ensure specificity and SNPcheck  to avoid allele drop-out. Primers used for SMCHD1 exon 37 amplification and Sanger sequencing are the following: forward 5’- TGC-CTG-TGG-AAC-ACT-CAA-AC-3’ and reverse 5’-GCT-GAC-TTC-CCA-ATT-TAG-TGC-3’. Reactions  were run for 35 cycles. PCR products were purified by Exonuclease I (New England Biolabs) and Rapid Alkaline Phosphatase  digestion, subjected to Big Dye Terminator v3.1 sequencing reaction (Applied Biosystems) and analyzed by 3130xl Genetic Analyzer (Applied Biosystems) following manufacturer’s instructions.All primers were designed using the Primer3 software (®-T Easy Vector cloning kit (Promega). After overnight incubation at 37 °C with antibiotic selection, at least ten randomly selected clones were PCR amplified for each sample using T7 and Sp6 primers and sequenced using by Sanger’s method  with either Sp6 or T7 primers. Sequences were analyzed using the BiQ Analyzer software [For bisulfite modification, 2 μg of genomic DNA was denaturated for 30 min at 37 °C in NaOH 0.4 N and incubated overnight in a solution of sodium bisulfite 3 M pH5 and hydroquinone 10 mM using a previously described protocol . Convertsoftware  and the XNP transcript was amplified for all cDNA samples in order to check for genomic contamination using the following primers: forward 5’- AGG-AAA-GGC-AGG-TGC-AAA-GC-3’ and reverse 5’- CGG-AGC-TTA-AAC-TCA-TGG-AGG-3’. SMCHD1 transcript was amplified for all cDNA samples with forward 5’- AAT-GTT-CGC-TCA-GTT-GCC-AG-3’ and reverse 5’- AGG-ACT-ACT-TTC-TGC-CAG-CA-3’ primers for 35 cycles . PCR products from sample II.1 and controls were subcloned in the pGEM®-T Easy Vector cloning kit following manufacturer’s instructions (Promega) and Sanger sequenced with Sp6 and T7 forward specific primers. DUX4 expression was analyzed as described [The escribed .2 in DMEM medium supplemented with 15 % fetal bovine serum and 1 % antibiotics (Thermo Fisher). NMDi14 (Merck Millipore) was dissolved and diluted in dimethylsulfoxide (DMSO) to a 48 mM final concentration. NMDI14 treatment on fibroblasts was performed at a 50 μM final concentration for 2 h and RNA was extracted by Trizol-Chloroform (Invitrogen) following manufacturer’s instructions. ATF3 positive control as well as NMDI14 treatment conditions were chosen based on previous report [SMCHD1 expression was quantified by RT-QPCR using the 480 Light Cycler real-time quantitative PCR (Roche) and SYBR Green mix . Primer sequences were picked in order to amplify simultaneously WT and mutated allele transcripts, while sequence similarity did not allow performing allele specific amplification. Differences in gene expression levels were determined as previously described [Skin fibroblasts from proband and healthy controls were grown at 37 °C and 5%COs report . SMCHD1 escribed .orbiculis occuli. In addition, the proband displayed shoulder girdle involvement and scapula alata  is a 68-years old woman who presented first signs of FSHD at the age of 56 Fig.  with lowata Fig. , abdomin is a 68-ata Fig. .Fig. 1ClThe 12 RU-4qA allele has been transmitted by her mother (I1), also affected. In this second patient, first signs appeared at the age of 45. She showed stepping at the age of 56 and became wheelchair-dependent at the age of 74. She was diagnosed at the age of 81 and displayed asymmetrical facial and upper limb weakening. The 12 RU-4qA allele has been transmitted to her son  who is not affected. Interestingly, her unaffected daughter  carries a complex pattern with 3 different 4q35 alleles with one qA allele with 6 RU and two other alleles with more than 11 D4Z4 units  suggesting respectively post and pre-zygotic mosaicism  and the different members of the family at four different positions along the D4Z4 repeat Fig.  followinIn affected members (I1 and II1), hypomethylation in the DR1 (12.1 % and 11.3 % respectively) and 5’ (52 % and 35.5 % respectively) sequences segregates with the symptoms whereas unaffected member display higher methylation level  Figs.  and 3b. In addition, we analyzed the D4Z4 methylation pattern in DNA from primary fibroblasts and quadriceps muscle biopsy for the index case and observed that hypomethylation is more pronounced for the DR1 and 5’ regions in these two tissues compared to blood  Fig. . These dSMCHD1 gene (c.4614_4615 insTATAATA) . By in silico analysis [in silico predictions are detailed in Additional file In order to search for other muscular gene variants, which may contribute to the neuromuscular or FSHD phenotype, we selected variants among a list of 45 genes screened in the diagnosis of neuromuscular disorders  indicating the absence of splicing defect  with a predicted size of 1543 residues and an expected weight of 173.57 kDa, we could not detect it by western blotting with an antibody directed against the N-terminal part of the protein either in fibroblasts or PBMC from the II1 index case. In order to determine whether the SMCHD1 mutated allele was degraded by non-sense mediated mRNA decay (NMD), we treated fibroblasts from the index case and controls for 2 h with 50 μM NMDi14 inhibitor, following previous publication [ATF3 gene was used as a positive control of NMD inhibition [By Western blotting, we also demonstrate that the expression of lls Fig. . Howeverlication . Expresshibition . Using dSMCHD1 gene (c.4614_4615 insTATAATA) in a family with FSHD. The proband (II1) and her mother (I1), both showing clinical signs of FSHD, carry this SMCHD1 mutation, together with an 12 RU D4Z4 array associated with a qA haplotype. SMCHD1 insertion is not present in the other unaffected members of the family (II2 and II3), whereas the 12 RU D4Z4 qA allele is carried by the brother (II2). Interestingly, the unaffected daughter (II3) carries three different 4q alleles, two with more than 11 repeats and one with 6 RU and a qA haplotype suggesting a complex 4q mosaicism. Nevertheless, she did not show any sign of FSHD at the time of collection.We describe here the identification of a new mutation corresponding to the insertion of 7 nucleotides in the exon 37 of the SMCHD1 mutation and D4Z4 hypomethylation, rather than D4Z4 copy number.We analyzed CpG by CpG the D4Z4 methylation level at four different positions using previously described primers  , 7. As oSMCHD1 mutation has been transmitted from the mother to one daughter and segregates with the presence of the symptoms. No transmission has been observed in the second daughter and brother who are not affected. Interestingly, we analyzed if other muscular gene variants might contribute to the FSHD phenotype in this family. Rare or unreported variants with a genetic status in proband and mother consistent with the age of disease appearance and/or severity are detailed in Additional file TTN gene  which might act as a phenotype modifier.Both wild-type and mutated transcripts were detected by RT-PCR. The 7 bp insertion is predicted to activate an exonic cryptic acceptor site, however such altered splicing was not evident in cells available from the patient. At the protein level, the insertion would cause a frameshift with the presence of premature stop codon 4 amino acids after the insertion. In western blot, the truncated protein was undetectable and the quantity of SMCHD1 protein was comparable between the patient and controls thus making haploinsufficiency improbable. The SMCHD1 gene. Most of the variations described so far are single base deletions, insertions, missense or nonsense mutations and splice site variants [In 80 % of the FSHD2 patients, the pathology has been associated with mutation in the variants . Larger variants , 11. Onlvariants , 11. TheSMCHD1 mutation, together with methylation level at D4Z4 segregates in the affected members of the family thus representing the strongest genetic candidates for FSHD in this family. Nevertheless, for the proband, the DUX4-fl transcript, which is only amplified in approximately 50 % of the muscle biopsies tested [In this study, we demonstrate that insertion of a TATAATA DNA motif within the exon 37 of SMCHD1 leads to the production of a mutant transcript detectable in the different tissues analyzed that is not specifically degraded by NMD. However, at the protein level, we did not observe a clear decrease in the protein level, which was comparable between affected and control individuals. s tested , 27 is nSMCHD1 haploinsufficiency is associated with D4Z4 hypomethylation. However, how this haploinsufficiency triggers disease onset remains to be established. Although speculative, we cannot exclude a phenotype modifier effect by other muscle gene variants, like the TTN heterozygous p.D2723A variant. In addition, besides its role in D4Z4 methylation, SMCHD1 might contribute to the 4q35 higher-order chromatin conformation and long-range regulation of other cis-acting genes such as FRG1 [FAT1 [SORBS2 [In this family,  as FRG1 , FAT1 [2G1 [FAT1 , 30 or S [SORBS2  or activ [SORBS2 , 33 at e [SORBS2 , 32.


Repeated measurement analysis of variance revealed a significant effect of time period on selected fitness parameters (p < 0.05). Post hoc analysis for test A505DL revealed significant improvements of performance at t3 (2.71 ± 0.08 s) and t4 (2.72 ± 0.06 s) compared to t1 (2.81 ± 0.09 s). A505NL was significantly different between t1 (2.83 ± 0.09 s) and t2 (2.76 ± 0.09 s), t3 (2.7 ± 0.07 s) and t4 (2.71 ± 0.09 s). Performance of CODS at t1 for the IAT (18.82 ± 0.56 s) was significantly lower (p < 0.05) than any other time period . The power of LRS was significantly different at t3 (4.99 ± 0.15 s), and t4 (4.98 ± 0.17 s) compared to t1 (5.15 ± 0.21 s), and t2 (5.07 ± 0.14 s). For the Yo-Yo IRT1 test, we observed a significant increase in performance between t1 (625.26 ± 170.34 m), t2 (858.95 ± 210.55 m), and t3 (953.68 ± 229.88 m). These results show the impact of soccer season time period on young soccer player performance and may further serve as a basis for comparison with similar research conducted by peers. These results may aid sports practice for clinicians, conditioning coaches, soccer coaches and physiotherapeutic coaches.The aim of this study was to investigate changes in the linear running speed (LRS) for 30 m, change of direction speed (CODS), and endurance in young elite Czech soccer players. The following tests were conducted to assess CODS and endurance: Agility 505 turning toward the dominant (A505DL) and non-dominant lower limb (A505NL); Illinois Agility Test (IAT); and intermittent test (Yo-Yo IRT1). During the soccer season, we investigated performance at the following time periods: the start (t In soccer, there are demands imposed on soccer players in terms of fitness readiness requirements to produce power, explosiveness, speed, agility, balance, body stability, flexibility and an adequate level of endurance ,2,3. MaiHigh-intensity physical activities are similar in terms of morphological and biochemical determinants and are believed to be interconnected , howeverPrevious studies dealing with variability of performance during the season have been focused on professional ,19 and sThe aim of this study was to investigate changes in performance of linear running speed, change of direction speed and endurance throughout the season of young soccer players.The sample consisted of 28 players from the U13 category of the highest level Czech league during the 2015–2016 season . Players completed five trainings and one match during the week microcycle. The average soccer experience of players was 5.59 ± 0.89 years. During testing, all subjects were healthy, and none was recently injured.The pre-season period started 20 January and ran until 28 March. There were 69 training units (TU) and 10 matches (M) during this period. The first half of the competitive period lasted from 28 March until 9 May with 23 TU and nine M. The second half of the competitive period was from 9 May until 20 June and included 23 TU and 11 M. 1), the start of the competitive period (t2), the middle of the competitive period (t3) and the end of the competitive period (t4) . All the tests were performed wearing football boots. A controlled warm-up by the trainer that lasted 25 minutes and consisted of jogging (5 min), static stretching (5 min), dynamic stretching (6 min), athletic alphabet (5 min) and four runs (40 m with speed change), occurred before testing. Tests we carried out during the spring season of 2015–2016. Testing occurred at the beginning of the pre-season training period (tiod (t4) . The dayNo subjects performing physical testing had undergone any surgery on the knee joint, and within two days prior to testing, they had not undergone any exhausting physical activity. All test subjects were notified of the content and implementation of testing procedures and consent was obtained by signatures from their parents. This research was approved by the ethical committee of the Faculty of Physical Education and Sport, Charles University in Prague (Czech Republic). Measurements were carried out in accordance with ethical standards of the Declaration of Helsinki and ethical standards in sport and exercise science research .The ability to perform single direction changes was investigated using the Agility 505 test, turning towards the dominant (A505DL) and non-dominant (A505NL) lower limb . On eachThe ability to perform repeated quick direction changes was evaluated through the Illinois agility test (IAT) . PlayersLinear running speed was measured by running 30 m from a standing position. Running time was recorded using two pairs of photocells . Players initiated the start on their own impulse. Players had two attempts and the better performance was kept for analysis.−1 (0–160 m) and another seven runs at 13.5–14 km·h−1 (160–440 m); this continues with stepwise 0.5 km·h−1 speed increments after every eight running bouts  until exhaustion. The test was performed on outdoor running lanes, marked by cones, with a width of 2 m and a length of 20 m. Another cone, placed 5 m behind the finish line, marked the running distance during the active recovery period. All subjects were familiarized with the test from at least one pre-test.Endurance was investigated using the Yo-Yo intermittent recovery test level 1 (Yo-Yo IRT1), which is considered a valid and reliable tool for evaluating specific intermittent endurance in soccer . The Yo-p ≤ 0.05 level. Effect size was assessed using the ”Eta square” coefficient (2η), which explains the proportion of variance of the factor. Effect size was examined as follows: 2η = 0.20 is considered a small effect, 2η = 0.50 is considered a medium effect, and 2η = 0.80 is considered a large effect [For statistical processing of the data, we used descriptive statistics. Measurements were expressed using the arithmetic mean and the measure of variability was expressed using the standard deviation. Parametric procedures were chosen after the verification of normality of the data distribution using the Shapiro–Wilk test. To assess the significance of the independent variables  on the dependent variables , a repeated measures analysis of variances (RM ANOVA) was used. To evaluate the sphericity, an assumption of RM ANOVA, a Mauchly’s test was performed. To compare measurements among many variables, we used a Bonferroni post hoc test. We rejected the null hypothesis at the e effect .® SPSS® v21 . Statistical analyses were carried out using IBM1) to performance during the competitive period (t3) (3.6%), and at the end of the competitive period (t4) (3.2%). In the same test, turning towards the non-dominant lower limb, we found a significant difference between the first time period (t1) and all other measurements  to measurements during the competitive period (t3) (2.2%). Similar results were found using the Illinois agility test where performance from the pre-season period (t1 = 18.82 ± 0.56 s) was significantly lower compared to the performance from other time periods . Post hoc analysis also revealed a significantly lower time at the end of the pre-season training period (t2 = 18.52 ± 0.63 s) compared to that during the competitive period (t3 = 17.94 ± 0.51 s), or at the end of the competitive period (t4 = 17.89 ± 0.66 s). Pre-season performance (t1) was not significantly different for the linear running speed at 30 m distance (p > 0.05) compared to the start of the competitive period (t2). However, for the competitive periods (t3) and (t4), there was a significant improvement compared to the start (t1) and end of the pre-season period (t2).For this test, we also observed significant improvement in the performance from the end of the pre-season period (tp < 0.01). At time period (t3), there was further improvement of this parameter by 9.9% (The pre-season period had the highest effect on players’ endurance (Yo-Yo IRT1), where players managed to increase the efficiency by 27.2%  of the pre-season period on maximum speed during a test run of 50 m with adult soccer players was found by researchers [1 = 5.15 ± 0.21 s) and end of the pre-season training period (t2 = 5.07 ± 0.14 s). This may be explained by the fact that this component was not particularly stimulated during training sessions. Game aspects, movement games and quick change of direction speed in a small space were emphasized during training. Caldwell and Peters [2 = 5.07 ± 0.14 s, t3 = 4.99 ± 0.15 s). These differences may indicate a different adaptation to the training load during the training period between adults and young soccer players. A significant difference was also shown between the time period t1 (5.07 ± 0.14 s), and t4 (4.98 ± 0.17 s), which may be due to the higher number of adaptation initiatives (more matches) during the competitive period, or morphological changes in the player´s body , which could positively affect a player´s motor skills . Ostojic [2max. Changes in running speed in the middle of the season were also observed by Wislof et al. [Changes in performance of linear running speed over time have been considered by several authors. Significant effects , compared to the beginning (t1 = 14.97 ± 0.38 s). This is also confirmed by our research, where players at the end of the pre-season training period (t2 = 18.52 ± 0.63 s) showed significantly better results than at the beginning (t1 = 18.82 ± 0.56 s). The agility 505 test from the pre-season training period was significant only when changing direction towards the non-dominant lower limb . Turning towards the dominant lower limb showed a significant improvement at t3 (2.71 ± 0.08 s) and t4 (2.72 ± 0.06 s), compared to t1 (2.81 ± 0.09 s). Significant improvement (p < 0.01) in the test of repeated quick changes of direction speed (IAT) for the pre-season period (t1 = 16.5 ± 0.4 s vs. t2 = 16.0 ± 0.4 s) was presented by researchers [One of the major differences in agility compared to linear speed is that the player learns to anticipate the next move , which iearchers , who att2max through an incremental running test on a treadmill in adult players in a top Spanish league since the start of the competitive period (50.2 ± 8.0 mL·kg−1·min−1) to the middle of the season (52.7 ± 8.5 mL·kg−1·min−1), but this difference was not significant. According to him, the players probably reached their VO2max early in the season, and it was therefore difficult to raise this level further during the season. Achieving such a high level at the beginning of the season could be due to high aerobic-oriented pre-season training, which has been created in order to improve the aerobic component of players. In other studies [2max values of 65.7 ± 4.3 mL·kg−1·min−1 during the season. Bekris et al. [2max of adult players from the top Greek league through measurements that were made at the beginning of the pre-season period (t1), at the beginning of the competitive period (t2), at the end of the first half of the competitive period (t3) and after the last round of the competitive period (t4). Significant differences in values of VO2max were found when comparing the values at t2 (57.16 ± 3.17 mL·kg−1·min−1) to t1 (55.56 ± 3.81 mL·kg−1·min−1) and t4 (56.07 ± 2.84 mL·kg−1·min−1), and a significant difference was also observed when comparing the values of VO2 max at t3 (57.96 ± 2.8 mL·kg−1·min−1) to t1 (55.56 ± 3.81 mL·kg−1·min−1). VO2max values in the first half of the season increased, but at the end of the season were significantly lower. In our research, during the season (t3) we observed further improvement in endurance by 9.9%. A partial drop, however not significant, was observed in our study while comparing the measurements in the middle of the season (t3), and at the end of the competitive period (t4), which could have been affected by the reduced number of matches at the end of the season.We found that the pre-season period had the highest effect on players’ endurance (Yo-Yo IRT1) where we recorded an increase in players’ performance by 27.2% (p < 0.01). We attribute this to the use of interval games in the training process and the competitiveness of players who, after initial measurements, wanted to provide improved performance and be the best of the team. Casajús  reported studies ,19, an i studies  identifis et al.  followedThe results of this study showed the impact of training and match load at different stages of the training cycle for linear running speed, changes of direction speed and endurance in young soccer players. These results may serve as an indicative basis for comparison with peers in similar research studies. We observed that the performance of speed and the ability to quickly change direction of the players improved throughout the observation period. For endurance, we observed a major improvement during the pre-season training period, while at the end of the season, endurance slightly fell. We conclude that the training process in youth plays an important role in the development of young players and positively affects the development of individual components of athletic performance. The study may help sports practice for clinicians, conditioning coaches, soccer coaches, and physiotherapy coaches by comparing these results to other groups.


Compared with dendritic α-Fe2O3 micropines, both a broader effective absorption range—from 10.5 GHz to 16.5 GHz with reflection loss (RL) less than −10 dB—and a thinner matching thickness of 2.0 mm have been achieved in the frequency range 2–18 GHz. The enhanced microwave absorption properties evaluated by the RL are attributed to the enhanced dielectric loss resulting from the intrinsic physical properties and special structures.A novel kind of α-Fe There are also peaks which are not well defined. These peaks may be assigned to the intermediate products when preparing dendritic Fe2O3 micropines, which would not contribute to the absorption properties of the composites. It suggests that α-Fe2O3 dendrites are highly crystalline. However, the α-Fe2O3-filled OMC nanocomposites are relatively amorphous, exhibiting weak diffraction peaks for α-Fe2O3 in the XRD pattern, expect for a diffraction hump of carbon around 15°–35°.Representative SEM images of the as-synthesized products are shown in eratures . As show , and sucrose as the carbon source were purchased from Sinopharm Chemical Reagent Co. Ltd., China.The synthesis of mesoporous silica 6] and 0.50 g for OMC were chosen. For comparison, dendritic α-Fe2O3 micropines were fabricated by the same method, except for the addition of OMC. Then the resulting compounds were filtrated, washed and dried in air at 80 °C overnight. The paraffin wax composites were prepared by ultrasonic agitation to mix a certain weight fraction (50 wt %) of absorbent with paraffin wax matrix.In this work, OMC was synthesized by using mesoporous silica SBA-15 as a hard template, as reported in our previous works ,14,15. O11, S21) were measured by bidirectional transmission/reflection method in a coaxial measurement fixture , scanning electron microscope , energy-dispersive X-ray spectroscopy , and transmission electron microscope . The as-prepared paraffin wax composites were pressed into a toroid with an outer diameter of 7.0 mm, inner diameter of 3.04 mm, and thickness of 3.0 mm. The scattering parameters of the toroidal samples Zin=2O3/OMC hybrid nanorods have been fabricated by the facial hydrothermal method. The unique heterogeneous structure of α-Fe2O3/OMC hybrid nanorods greatly enhance the dielectric loss of the materials, and therefore not only a broader effective absorption range from 10.5 GHz to 16.5 GHz with reflection loss less than −10 dB, but also a lighter weight and thinner matching thickness (2.0 mm) have been achieved. Combining the complementarities with intrinsic physical properties and special structures, the hybrid nanorods can be used as a promising EM wave absorber.In summary, one dimensional (1D) α-Fe