The durum wheat line DT696 is a source of moderate Fusarium head blight (FHB) resistance. Previous analysis using a bi-parental population identified two FHB resistance quantitative trait loci (QTL) on chromosome 5A: 5A1 was co-located with a plant height QTL, and 5A2 with a major maturity QTL. A Genome-Wide Association Study (GWAS) of DT696 derivative lines from 72 crosses based on multi-environment FHB resistance, plant height, and maturity phenotypic data was conducted to improve the mapping resolution and further elucidate the genetic relationship of height and maturity with FHB resistance. The Global Tetraploid Wheat Collection (GTWC) was exploited to identify durum wheat lines with DT696 allele and additional recombination events. The 5A2 QTL was confirmed in the derivatives, suggesting the expression stability of the 5A2 QTL in various genetic backgrounds. The GWAS led to an improved mapping resolution rendering the 5A2 interval 10 Mbp shorter than the bi-parental QTL mapping interval. Haplotype analysis using SNPs within the 5A2 QTL applied to the GTWC identified novel haplotypes and recombination breakpoints, which could be exploited for further improvement of the mapping resolution. This study suggested that GWAS of derivative breeding lines is a credible strategy for improving mapping resolution. Fusarium graminearum Schwabe produces mycotoxins that are harmful to human and animal health1. The contaminated grains are downgraded because they are unhealthy for human and animal consumption. Integrated FHB management provides the most effective control strategy and utilizes fungicide application, residue management, crop rotation, and growing cultivars with the highest available level of resistance. Growing cultivars with resistance is an efficient and cost-effective component of the strategy, consequently developing FHB resistant cultivars is one of the major objectives of wheat breeding programs where the disease is a problem. Only partial resistance has thus far been reported in bread wheat germplasm and durum wheat cultivars lack adequate resistance to FHB. The durum wheat line DT696 is a source of moderate FHB resistance adapted to the western Canadian Prairies2. Durum wheat cultivars derived from DT696 as a source of FHB resistance, such as Brigade, Transcend, and CDC Credence show improved FHB resistance relative to other elite durum wheat cultivars in production4. Using resistance available in adapted sources such as DT696 has the advantage of minimizing detrimental effects of linkage drag since DT696 is an advanced elite breeding line adapted to Canadian growing conditions.Fusarium head blight (FHB) is a devastating disease of wheat causing severe reduction in grain yield and quality in warm and moist growing regions of the world. The causal pathogen 2. The QTL on chromosome 5A (5A1 and 5A2) were consistently expressed over different field locations and years. The 5A1 QTL explained up to 20.8% and 5A2 up to 25.7% of FHB phenotypic variance, suggesting a moderate effect of these QTL. The same QTL were also reported in a backcross recombinant inbred line population from T. turgidum ssp. dicoccum line BGRC3487 and a derivative of DT696 (DT735)5. The QTL interaction analysis of the DT707 × DT696 population indicated the presence of an additive effect between the 5A1 and 5A2 QTL2. The 5A1 QTL co-located with a moderate plant height QTL and 5A2 with a major maturity QTL, providing supporting evidence for associations between these developmental traits and FHB resistance2. Three hypotheses are postulated for the co-localization of 5A1 with plant height and 5A2 with maturity QTL. FHB resistance may be the result of disease escape in taller and later-maturing plants as a consequence of height and maturity genes per se. A second possibility is that height or maturity loci have both disease escape and physiological FHB resistance effects, in other words there is a pleiotropic effect of height and maturity genes on FHB resistance. Alternatively, linkage between FHB resistance genes and genes for the developmental traits exist within the QTL intervals. Effective utilization of these QTL in breeding programs and the identification of predictive markers for marker-assisted selection (MAS) will depend on deciphering the type of association with plant height and maturity.High-density mapping of FHB resistance quantitative trait loci (QTL) using a large bi-parental population (number of lines 427) from a cross between lines DT707 and DT696 identified five resistance QTL on chromosomes 1B, 2B, 5A (two loci), and 7A7. The association of an FHB resistance QTL with the major effect plant height gene Rht-B18 suggested the contribution of the dwarfing allele of Rht-B1 to FHB susceptibility10. Buestmayr et al.11 found an FHB resistance QTL on chromosome 5A from T. macha co-located with the Q-gene that controls plant height, and spike traits including anthesis date, and spike density and length. The FHB resistance has also been considered pleiotropic to anther extrusion as the effect on resistance because of the co-localization of Qfhs.ifa-5AS, a FHB resistance QTL from hexaploid wheat line Sumai 3 on chromosome 5A, with an anther extrusion QTL13. These studies further emphasize the importance of greater understanding the relationship of FHB resistance and developmental traits through high-resolution mapping of FHB resistance QTL, designing markers for MAS, and the effective utilization of these QTL for developing resistant cultivars. A recent fine mapping effort that projected the interval of Qfhs.ifa-5AS on the International Wheat Genome Sequencing Consortia (IWGSC) Refseq v1.013 along with the high-resolution mapping of the DT707 × DT696 population2 proposed the co-localization of Qfhs.ifa-5AS with 5A1 QTL.The association of FHB resistance QTL with plant height and maturity is not new. Previous studies highlighted a negative correlation between plant height and FHB severity14. GWAS has been successfully used for the identification and mapping of FHB resistance QTL in a durum wheat diversity15 and a durum wheat breeding panel16. Thus, GWAS is valuable for validating and high-resolution mapping of the FHB resistance QTL previously identified through bi-parental mapping such as the 5A1 and 5A2 QTL.The validation of QTL in breeding lines with relatively uniform plant height and maturity may enable teasing apart the contribution of disease escape mediated by tall alleles of plant height QTL from the potential physiological resistance conferred by the 5A1 and late alleles of maturity from the 5A2 QTL. Since its introduction as a source of moderate FHB resistance in the durum wheat breeding program at the Swift Current Research and Development Centre of the Agriculture and Agri-Food Canada (AAFC SCRDC), DT696 and its derivatives have been used as parents in several hundred crosses. This has provided an opportunity for the application of a genome wide association study (GWAS) for improving mapping resolution of FHB resistance QTL through the identification of novel recombination events at the 5A1 and 5A2 loci. It has also provided the opportunity for validating the 5A1 and 5A2 QTL and deciphering the type of the association of FHB resistance with plant height and maturity reported repeatedly in previous studiesFhb1, an FHB resistance QTL on chromosome 3BS present in the hexaploid wheat line Sumai 3 have been identified19. Candidate resistance genes have been proposed for Sumai 3 FHB resistance loci Qfhs.ifa-5A, on chromosome 5A20 and Fhb2 on chromosome 6B21, and a Wuhan-1 resistance QTL on chromosome 2D22. High-resolution mapping of FHB resistance QTL in DT696 will contribute to the design of predictive markers for MAS, which will allow their rapid and effective utilization in breeding programs.High-resolution mapping of FHB resistance QTL is difficult because of the small effect of resistance loci with quantitative expression, variable expression across environments, their epistasis interaction with other loci, and their interactions with genetic background and developmental traits. To date, only resistance genes associated with 23. A subset of 223 lines representing the recombinants at the 5A1 and 5A2 QTL were phenotyped in multiple environments and subjected to GWAS using the 90 K SNPs. This reduced the size of 5A2 QTL interval to 15.8 Mbp. An FHB resistance QTL in a low recombination region of chromosome 6B was also identified. A panel of 523 contemporary breeding lines from the AAFC SCRDC breeding program with relatively uniform plant height and maturity was genotyped with SNP markers associated with the 5A2 interval, assessed for FHB resistance and subjected to haplotype-FHB trait association analysis. Significant association between markers with the DT696 allele and FHB traits validated the markers for MAS and demonstrated that the contribution of 5A2 QTL to FHB resistance is partially independent of plant height and maturity. DT696 haplotype and novel recombination events were present in 1022 lines of the Global Tetraploid Wheat Collection (GTWC). The novel recombination events constitute valuable resources for further refining the genetic interval and identifying candidate gene. The presence of the DT696 haplotype in the diversity panel will provide opportunity for the utilization of the resistance in other regions of the world from which the diversity panel originated.The objectives of this study were to evaluate lines derived from DT696 from the AAFC SCRDC breeding program for FHB resistance, to sample recombination in the chromosome 5A region for high-resolution mapping of the FHB resistance QTL, to further elucidate the genetic relationship of height and maturity to FHB resistance, and to identify additional loci contributing to FHB resistance in the breeding lines. We genotyped 401 DT696 derivatives available from the AAFC SCRDC durum wheat breeding program with the 90 K iSelect SNP assay2, covariance analysis conducted with either plant height or maturity assigned as a covariate to test the dependency of FHB resistance expression on these traits. The genotypic variation for FHB severity and index remained significant when either plant height or maturity was assigned as covariate.Variance analysis was conducted for the FHB traits  as well as plant height and maturity for 223 DT696 derivatives phenotyped in Morden and Brandon FHB nurseries in Manitoba, Canada in 2016 and 2017. The line variation for FHB severity and index, and plant height was significant in all environments except at Morden in 2016  plotted against the pair-wise genetic distance on average was 1.6 cM in the DT696 derivative lines  decay calculated from pair-wise SNP LD squared allele frequency correlation . A two tailed t-test of plant height between lines carrying alternate alleles at Tdurum_contig27834_260 was significant (P = 2.78E-5) and lines carrying the dwarfing allele (17 out of 223 lines) were on average 10.6 cm shorter than those with the alternate allele .Single nucleotide polymorphisms associated with plant height were revealed on chromosomes 1A (two SNPs), 2A (four SNPs) and two physically distant loci on chromosome 4B (23 and six SNPs) in the Morden 2016 environment, and on chromosome 3A (one SNP) at the Brandon location of the same year  adjusted P-value varied between the SNPs within this interval and was often lower in the 2017 than the 2016 environment  and a 3.7 Mbp interval between SNPs wsnp_Ex-c621_1231298 and Tdurum_contig_69612_781 (5A2–2) was lower (more significant) than their flanking markers in the 2016 environment. A large drop in LD r2 at wsnp_Ex-c621_1231298 suggested the presence of a frequent crossover in the DT696 derivatives at this locus. The physical position of the vernalisation gene VRN1 (TraesCS5A01G391700) inferred from the IWGSC Refseq. v1.0 annotation was between the 5A2–1 and 5A2–2 intervals  at Morden in 2017  was detected in DT696 derivatives between SNPs wsnp_Ex-c621_1231298 and wsnp_Ex-c621_1231444, however, rare recombination events were detected between the other SNPs spanning the 5A2–2 interval  in FHB index at Brandon in 2016 and 2017, and at Morden in 2017 but not in 2016  and Brandon  nurseries, while the difference among other haplotypes remained insignificant.A few SNPs spanning the 5A2 FHB resistance QTL interval inferred from the QTL mapping of the DT707 × DT696 population were used to genotype 523 contemporary breeding lines developed at AAFC SCRDC. According to the physical positions, the interval formed by these SNPs covered the entire 5A2–1 and a portion of 5A2–2 interval. All lines carried the DT707 allele at the interval between SNPs RN1 Fig. . The phedex Fig. . This haKukri_c33022_198 and wsnp_Ex_c621_1231298; this was also a common breakpoint in the lines of GTWC  was lowered by high disease pressure, which could be similar to what we observed for 5A-2. Moreover, a genotype by environment interaction was expected based on the moderate heritability of FHB resistance in DT696 which was previously inferred from phenotyping the DT707 × DT696 population at multiple field nurseries including Morden where the DT696 derivatives were phenotyped in the present study2. Such a genotype × location interaction had lower impact on the expression of 5A2 QTL in the DT707 × DT696 population than the derivatives as the 5A2 QTL was consistently expressed over environments in the previous mapping study on the DT707 × DT696 population2, but was expressed only at Brandon location for the derivatives  was approximately 50% higher at Morden than Brandon. The effect of disease pressure on the expression of FHB QTL was observed by Clark et al.2, associated with both FHB resistance and plant maturity2. Such linkage decay helps to explain the rare occurrence of lines moderately resistant yet earlier maturing than DT696 within the DT696 derivative set. The association of developmental and FHB resistance genes at a single locus is not new. The co-localization of the plant height gene Rht8 and an FHB resistance QTL on chromosome 2D was previously reported26. Subsequently, Handa et al.14 identified genes associated with DON degradation as the basis for FHB resistance in this interval, demonstrating that Rht8 was not the sole contributor to FHB resistance. The availability of additional recombination events within the 5A2 QTL interval in the GTWC panel is an opportunity to validate further the possibility of a similar disassociation in the 5A2 QTL.The presence of DT696 derivative lines with equal and occasionally higher FHB resistance, but shorter and earlier maturity than DT696 suggests that the resistance may not be solely due to disease escape of the taller and later-maturing plants. Moreover, when plant height and maturity were assigned as covariates in the variance analysis of FHB trait means, the variance of lines remained significant, further supporting the minor effect of plant height and maturity on expression of FHB resistance in the derivatives. Nevertheless, moderate to low correlations of FHB traits with plant height and maturity indicated that FHB resistance is partially associated with the developmental traits in the DT696 derivatives. However, there is no evidence to suggest that the FHB resistance genes segregating in the population are inextricably associated with plant height and maturity. In fact, higher numbers of recombinants in the derivatives could allow the decay of linkage between genes for the developmental traits and those for FHB resistance within the 5A2 region, that was according to Sari Rht-B1 (Tdurum_contig27834_260) was associated with plant height only at Morden in 2016. The effect of the locus partially explains the shorter plant height of the DT696 derivatives at this location compared to Brandon. Despite a relatively large association of Tdurum_contig27834_260 (GWAS R2 = 0.29) with plant height, it was not detected as an SNP significantly associated with FHB susceptibility in the GWAS. This is different from the results of He et al.10 that suggested that the Rht-B1 tall allele is associated with Type I FHB resistance , reflecting lower FHB incidence in taller plants. Even though Rht-B1 was not detected as an FHB resistance QTL in the GWAS, a two-tailed test found significant association between the SNP within this gene and FHB index at the Morden nursery in 2016  between lines with alternative alleles of Tdurum_contig27834_260. It could also be a consequence of the low frequency (8%) of the dwarfing allele of Rht-B1 in the derivative population. Nevertheless, the Type II resistance which is independent of Rht-B1 seems to be the major determinant of FHB resistance in the derivatives. This is supported by the non-significant variance of lines for FHB incidence across all four environments and the identification of the 5A2 and 6B FHB resistance loci only for FHB severity and index ratings, but not for FHB incidence.The SNP within Fhb1, which in a previous study was expressed in a wider range of winter wheat genetic backgrounds than the FHB resistance QTL QFhs.nau-2DL25. The consistency of expression in a diversity of genetic backgrounds improves the success of recovering the 5A2 FHB resistance QTL during breeding of elite durum germplasm and explains the success achieved with the improved FHB resistance in durum varieties, derived from DT696, such as Brigade, Transcend and CDC Credence4.The presence of four sub-populations in the DT696 derivatives suggested the presence of substantial genetic variation in the population. This variation would have originated from genetic diversity of the founders used for crossing with DT696  impeding further mapping resolution. In the present study, we genotyped 716 DT696 derivatives in order to select recombinant representatives at the 5A1 and 5A2 QTL interval for phenotyping. This likely allowed us to capture all existing recombination events in the DT696 derivatives. Additional recombinants at the 5A2–2 interval could be identified in future studies by stimulating recombination using biotic and abiotic treatmentsVRN1 from other genes conferring FHB resistance in the interval due to homogeneity at the VRN1 locus. The breeding populations were subjected to phenotypic selection for lines with reduced levels of FHB by the breeders. Two thirds of selected lines had either Hap 1  or Hap 4 , indicating that genotypic selection agreed with phenotypic selection in 75% of cases. Combining genotypic with phenotypic selection could enhance selection gain in the breeding programs31 over the 75% obtained using phenotypic selection alone in the present study. We were able to rule out an association of inoculation method with the expression of FHB resistance, by comparing the FHB index of haplotypes at Carman nursery with spray inoculation at 50% anthesis and Brandon with corn spawn inoculation. Because Hap 4 was significantly different from Hap 5 at both nurseries despite different inoculation methods, the inoculation method was not the source of variation. The resistance mediated by Hap 4 is not solely due to developmental traits as spray inoculations at 50% anthesis minimizes the possibility of disease escape associated with plant height and maturity33.SNPs associated with the 5A2 FHB resistance QTL were validated for marker-assisted selection on breeding lines of 13 AAFC SCRDC crosses. Not only did the application to contemporary breeding material allow the validation of SNPs for marker-assisted selection, it also allowed discriminating the effect of Forty-six GTWC lines with the DT696 haplotype provide opportunity at the international level for breeding resistance to FHB. Additional research is required to validate that these lines express resistance to FHB in their target environment. As expected, population size raised the recombination frequency at 5A2–2 interval. Assessing the phenotype of germplasm with the recombination events at 5A2–2 interval will be valuable to refine the interval towards identifying candidate genes.T. turgidum ssp. carthlicum line Blackbird34. A pedigree search of DT696 derivatives lines with resistance alleles in the 6B QTL indicated that all were derived from a single breeding population named A0580 (DT696/A0200H-056/3/Langdon-Dic3A (a resistant recombinant substitution line derived from crosses of T. turgidum spp. durum cv. Langdon with wild emmer wheat T. turgidum spp. dicoccoides)//SC9475-CX1*2/ND2710 (a hexaploid source of FHB resistance presumably derived from line Sumai 335) and most of them also carried the DT696 haplotype at 5A2 QTL. Considering the field location-dependent expression of both the 5A2 and 6B QTL in the DT696 derivatives, lines of A0580 with resistance alleles of both QTL are valuable source for developing cultivars with consistent expression of resistance over multiple environments.The FHB resistance locus on chromosome 6B in the DT696 derivatives was not detected from the previous QTL mapping study of the DT707 × DT696 population. This suggests that resistance alleles are contributed from founder lines crossed with DT696 and its derivatives. Comparative mapping indicated that the interval defined by SNPs on chromosome 6B in our current study correspond to the Type II FHB resistance QTL detected from 2 minimized the possibility of environment affecting the expression of this QTL. Unlike the 5A2 QTL, the expression of 5A1 seems to be dependent on other loci or certain genetic backgrounds that were rarely present in DT696 derivatives. Weak expression of 5A1 QTL below a level detectable by the GWAS algorithm could be another reason the 5A1 locus was not detected in the derivatives.GWAS analysis of DT696 derivatives could not associate any SNPs within the 5A1 QTL interval with FHB resistance. Consistent detection of the 5A1 QTL in the QTL mapping study using the DT707 × DT696 population in the same field locations used for GWAS of the derivatives (Morden nursery in 2016 and 2017)More work is needed to further resolve the 5A2 FHB resistance QTL. Efforts are underway to develop Near Isogenic Lines (NILs) carrying recombination break points in the 15.8 Mb interval of the 5A2 FHB resistance QTL. Recombination break points will be mapped with the SNP markers identified in the present study and selected recombinants will be subjected to intense phenotyping to improve the mapping resolution. The NILs will also be subjected to further reverse genetic analysis to identify reliable markers required for high-precision MAS.Fhb1 and Qfhs.ifa-5AS, a complex genetic architecture is proposed for the 5A2 FHB resistance QTL, with the presence of at least two distinct loci (5A2–1-and 5A2–2) in the interval. GWAS along with haplotype analysis in the contemporary breeding lines enabled the validation of the 5A2 FHB resistance QTL in multiple genetic backgrounds, lending support to the transferability of resistance conferred by this locus to elite germplasm. This study led to the identification of the SNP markers desired for marker-assisted selection that will contribute to the speed and precision of gene pyramiding and should pave the way for improving FHB resistance in durum wheat.In conclusion, phenotyping of DT696 derivatives that carried historical recombination from 72 breeding populations, enabled selecting for early-maturing semi-dwarf lines with FHB resistance equal to or better than DT696 as candidates for developing resistant cultivar. GWAS resolved the 25.8 Mbp 5A2 interval obtained from the bi-parental QTL mapping into a 15.8 Mbp interval. Like 2. Thirteen contemporary breeding populations active in the breeding program were used for marker validation. These populations totalling 523 lines were developed from crosses between advanced breeding lines and the cv. Transcend which is a moderately susceptible durum variety derived from DT6964.A search was conducted within pedigrees of AAFC SCRDC durum wheat lines to identify DT696 derivatives among advanced breeding lines using an in-house script of Statistical Analysis System (SAS) software version 9.3  that enabled retrieval of pedigrees from crosses made with DT696 and its secondary and tertiary derivatives. This identified 72 populations with a total number of 716 lines derived from DT696  and 15-acetyl-deoxynivalenol (15ADON) producing strains of F. graminearum. The Morden nursery was inoculated approximately 2–3 weeks prior to heading, while at the Brandon nursery the first application of corn inoculum was done at six weeks after planting followed by another application two weeks after the first application. Colonized corn grains were broadcasted between the rows at the rate of 40 g m−2 at Brandon, and at 8 g per row performed twice, one week apart at Morden. The nursery at Brandon was irrigated three times a week with an overhead low-pressure mist irrigation system immediately upon completion of inoculation to provide moisture for inoculum development in the corn and to provide moisture on the spikes for disease development. The nursery at Morden was irrigated three times a week using Cadman Irrigation Travellers with Briggs booms.Phenotyping 223 DT696 derivatives for FHB resistance, plant height and relative maturity was conducted following the method described by Sari 2 pressurized backpack sprayer calibrated at 2 kPa at 50% anthesis with 50 ml per row conidia suspension composed of a mixture of aggressive 3ADON and 15ADON producing strains of F. graminearum. The concentration of the conidial suspensions was adjusted to 5 × 104 conidia mL−1 using a hemocytometer and Tween 20 (1 drop per 100 ml) was added to the suspension. Inoculation was repeated 4 d after the first inoculation. The nursery was mist irrigated in the evening, and the morning after each inoculation.Phenotyping of the 523 contemporary breeding lines was conducted in FHB nurseries located at Brandon and Carman in 2017. Lines at the Brandon nursery were subjected to the same inoculation protocol as DT696 derivatives lines with the experiment conducted as an unreplicated alpha-lattice field designs with 12 entries per incomplete block. At the Carman nursery, the spikes of the plots were spray-inoculated using a COFHB incidence (percentage of spikes showing symptoms) and severity (percentage of spike area infected) were recorded for each plot. FHB index was calculated from the incidence and severity rating data using the formula (incidence × severity)/100. Plant height was measured on a representative plant from the soil surface to the tip of spikes excluding the awns. Relative maturity was rated using a 1–4 scale (1 = latest and 4 earliest maturity) concurrent with the FHB rating at three weeks post anthesis, by pinching the seeds and comparing their moisture levels with the parents.et al.23. The SNP clustering was performed in Genomestudio software v. 2011.1  using the default clustering algorithm and following the workflow described by Cavanagh et al.36. Approximately 10% of total SNPs  were scorable. The order of markers on the physical map of the IWGSC Refseq v1.037 and durum wheat cv. Svevo38 and wild emmer wheat accession Zavitam reference assemblies39 was determined by aligning the 90 K SNP source sequences using the Nucmer program available in Mummer v.3 software40. Only SNPs with missing data less than 10% and minor allele frequency of higher than 5% were retained. The retained SNPs were then used to calculate an identify-by-state genetic similarity matrix for all possible pairs of lines using Plink v1.0741. For lines showing ≥ 0.99 genotypic similarities, only one representative line with the lowest number of missing SNPs was maintained.A subset of DT696 derivatives (401 lines) were genotyped using the wheat iSelect 90 K SNP genotyping assay following the method described by Wang 42. Lines were then genotyped with a Fluidigm  high-throughput genotyping system using nine KASP markers within the 5A1 and seven KASP markers within the 5A2 QTL interval. The genotypic data of these lines were then combined with those of the 401 lines obtained from the iSelect 90 K SNP genotyping assay and a similarity matrix was generated using Flapjack v. 1.16.10.31 software43 to select 359 representative recombinant lines for phenotyping. Lines from the contemporary breeding populations used for validation of SNPs for MAS were genotyped using the same KASP markers with the Fluidigm high-throughput genotyping system.To select recombinant representatives at the 5A1 and 5A2 FHB resistance QTL on chromosome 5A, SNPs within the QTL interval were used for genotyping 374 additional DT696 derivatives. SNPs within the 5A1 and 5A2 interval were converted to Kompetitive Allele-Specific PCR (KASP) markers using the Polymarker pipeline44. The population structure was inferred from the model-based quantitative assessment of sub-population available in Faststructure v1.0 software45. Linkage disequilibrium (LD) squared allele frequency correlation (r2) was calculated for each pair of SNPs within each chromosome using Haploview software46. The overall pattern of LD decay in the population was estimated following the method described by Maccaferri et al.27 from pairwise LD r2 estimates of all chromosomes plotted against the genetic distance between SNPs inferred from the tetraploid wheat consensus genetic map24. A non-linear regression model was then fitted using the nonlinear least squares method in R v. 3.5.3 software (https://www.r-project.org/). The genetic distance (cM) at which the r2 was lower than the empirical threshold of 3.0 was considered as the average LD decay of the population.Of 359 derivatives phenotyped at multiple locations, 223 genotyped using iSelect 90 K SNP genotyping assay, were subjected to GWAS. When markers were filtered for missing data and minor allele frequency (using the criteria described above), 6231 SNPs met the criteria for GWAS. Centered identity-by-state kinship matrix was calculated for these SNPs using Tassel v. 547 and Settlement of MLM Under Progressively Exclusive Relationship (SUPER)48 models of R package GAPIT v. 2 software49 with the kinship matrix generated as described above. The following models were tested with each of cMLM and SUPER methods: 1) no correction for population structure; 2) model corrected for population structure using the % membership coefficient generated by Faststructure for number of subpopulations (Q) of 3–7. The model with correction for the population structure of Q4 was superior to other models according to the quantile-quantile (QQ) plot generated by GAPIT, hence the probability (P) values of this model were used for determining the significant SNPs. The experiment-wise P value threshold was either 0.05 when the FDR adjusted P values were reported or based on Bonferroni adjusted P value which was equal to a marker-wise P value of 8.25 × 10−6. Assigning significant SNPs to QTL was implemented through anchoring the SNPs to the tetraploid wheat consensus genetic map24 and durum wheat cv. Svevo genome assembly38, and comparing the genetic and physical position of QTL reported in the previous QTL mapping study2. The FDR adjusted P values were plotted against LD r2 values extracted from Haploview to associate recombination break points with changes in significance of P values.GWAS was conducted with the 6,231 SNPs, FHB traits , plant height and relative maturity data using compressed mixed linear (cMLM)et al.38. The haplotype and recombination break points were compared between the GTWC and DT696 derivatives at 22 SNPs within the 5A2 QTL interval. Since DT696 and cv. Strongfield were common to both datasets and cv. Strongfield had an identical haplotype to DT707 for the 22 SNPs spanning the 5A2 interval, we could reclassify the genotypic data of the GTWC to either a DT696 or DT707 haplotype. Lines with missing records or heterozygous at these 22 SNPs were excluded from analysis. One-dimensional hierarchical cluster analysis and haplotype visualization was performed using the dendextend and ComplexHeatmap packages of R v.3.5.3.Haplotypes and recombination breakpoints in the GTWC were based on the genotypic data generated using the 90 K iSelect SNP genotyping assay described by Maccaferri t-test with correction for heteroscedastic distribution  of data in Microsoft Excel.Variance and covariance analyses were conducted using SAS v.9.3. To estimate the variance due to line, the mixed model was used with genotype assigned as a fixed effect and block assigned as random effect. To estimate the variance due to genotype when plant height and maturity were assigned as covariates, the mixed model was used with line and either plant height or relative maturity assigned as fixed effects, while block was assigned as a random effect. Spearman’s rank correlation analysis was conducted between each of the FHB traits and plant height and maturity using PROC CORR of SAS v.9.3. The significant difference between lines of different haplotypes was inferred using a two-tailed Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.


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The penultimate sentence of the first paragraph should have referred to a 0.8% weight loss among patients receiving placebo. The final sentence should have read that weight loss with bimagrumab was greater than the 1-year FDA threshold of 5% for registration of new medicines for treating obesity and overweight. This article has been corrected.1In the Original Investigation titled “Effect of Bimagrumab vs Placebo on Body Fat Mass Among Adults With Type 2 Diabetes and Obesity: A Phase 2 Randomized Clinical Trial,”


The percentage of patients in the healthy control group who reported worsened quality of life should have read 9.5. This article has been corrected.1In the Research Letter titled “Sequelae in Adults at 6 Months After COVID-19 Infection,”


Scoliosis is an abnormal bending of the body axis. Truncated vertebrae or a debilitated ability to control the musculature in the back can cause this condition, but in most cases the causative reason for scoliosis is unknown (idiopathic). Using mutants for somite clock genes with mild defects in the vertebral column, we here show that early defects in somitogenesis are not overcome during development and have long lasting and profound consequences for muscle fiber organization, structure and whole muscle volume. These mutants present only mild alterations in the vertebral column, and muscle shortcomings are uncoupled from skeletal defects. None of the mutants presents an overt musculoskeletal phenotype at larval or early adult stages, presumably due to compensatory growth mechanisms. Scoliosis becomes only apparent during aging. We conclude that adult degenerative scoliosis is due to disturbed crosstalk between vertebrae and muscles during early development, resulting in subsequent adult muscle weakness and bending of the body axis. Diseases that result in bending of the spine are more common in elderly populations. Camptocormia (a 45 degree anterior bent of the lower joints of the spine) has an average age of onset of 66 years  and frommyoD [The segmentation clock controls an essential process in the vertebrate embryo and leads to the formation of the somites, transient structures which will later contribute to the formation of one of the most well-known and characteristic structure of all vertebrates, the vertebral column. This structure together with the axial muscles act to control axial posture and function. During early stages of development, the pre-somitic mesoderm is segmented from anterior to posterior by the periodic expression of genes from the Notch-Delta family . This exmyoD , 9. In cmyoD , 11. MormyoD , 13. MyomyoD   single, -/-; her7-/- double, and her1-/-; her7-/-; tbx6her1-/- triple mutants). These mutants show varying levels of somite segmentation defects and subsequently mild axial skeletal phenotypes [-/-; her7-/-, tbx6-/- and her1-/-; her7-/-; tbx6-/-her1 at adult and embryonic stages. We found that scoliosis is not coupled to vertebral defects and may correlate with early perturbations of muscle formation during development. Strikingly, -/-; her7-/-her1 double mutants develop scoliosis with the same penetrance as wild type animals, despite showing aberrant vertebral skeletal phenotypes. In contrast, loss of Tbx6 function greatly increased the chance of an animal developing scoliosis with age. Finally, we present evidence that this correlates with severe muscle patterning defects during embryogenesis and propose that vertebrae and muscle segmentation are not directly coupled during development.It has been suggested that human patients with congenital scoliosis may have early abnormalities during somitogenesis . The numenotypes . The str-/-; her7 -/-her1 and -/-; her7-/-; tbx6-/-her1 present mild defects in the segmentation of the axial skeleton (fusion and hemivertebrae) with a 100% incidence. This is also true for 80% of fss (tbx6-/-) mutants [As we have previously shown  zebrafish mutants for  mutants . Later o-/-tbx6 third generation animals developed scoliosis, similar to first generation animals 3 out of 8 (38%) evaluated at a similar age . By the end of the experiment (12 months post fertilization) all -/-; her7-/-; tbx6-/-her1 first generation animals (8/8) had developed scoliosis. Similarly, in the third generation, all animals (7/7) -/-; her7-/-; tbx6-/-her1 animals developed scoliosis at 14 months post fertilization  of the third generation and 38% (3/8) of the time lapse individuals developed visible scoliosis at 12 months post fertilization. All of the -/-; her7-/-her1 mutants had vertebral fusions and hemivertebrae in more than one position within the axial skeleton. This value was the same as the wild type scoliosis recurrence. In wild type individuals, scoliosis was first detected at 5 months post fertilization in 1 out of 22 fish (4.5%) and at 6 months post fertilization in 1 out of 8 (12%). At the end of the analyzed period (1 year) 38% (3/8) of wild type animals had developed scoliosis. These individuals had no vertebral defects. Presentation of scoliosis therefore did not correlate with axial skeletal phenotypes in wild type animals. An additional observation, showed that 64% of the females (9/14 that developed scoliosis during the whole experiment) had the first measurable sign of scoliosis at 3 months post fertilization compared to 0 % of males  ilar age  and 2D. lization . By 12 mcoliosis  and 2D;  mutants . Despiteof males .-/-tbx6 mutants, the muscle volume at the pelvic fin and the anal fin was also statistically different from wild type  beginning of the pectoral fin, (2) end of pectoral fin, (3) pelvic fin and (4) anal fin. The first and second positions were selected as the majority of the scolioses were observed in the fish at these levels. The musculature of the fish changes in volume significantly at different anterio-posterior positions, such that anterior myomeres are larger than those in posterior region. By standardizing the locations of all measurements of muscle volume for each animal, we could compare absolute muscle volumes between animals. This allowed us to ensure that any differences in muscle volume were not due to inherent differences in muscle volume along the trunk or due to differences in animal size –3E. A twild type  and 3I. Three-way correlation analysis was performed in order to establish if the length of the fish had an effect on the muscle volume or the angle of deviation from the body axis. It was found that the length of the fish has a negligible correlation with the angle of deviation of the body axis . There is a low positive correlation between muscle volume and fish length  and a low negative correlation between muscle volume and angle of deviation . An interpretation of these correlation scores is that severity of scoliosis correlates with a lower muscle mass but is independent of the length of the fish.-/-; her7-/-; tbx6-/-her1 or -/-tbx6 mutants    to deterFM1954A) . Therefo-/-tbx6 and the triple -/-; her7-/-; tbx6her1-/- mutants cavities can be seen in the fast muscle  could be measured with an average size of 1.105μm ± 0.66. In -/-; her7-/-; tbx6-/-her1 cavities were found in 18 out of 20 optical sections, 40 cavities (between 1 and 5 per section) were measured with an average size of 1.407μm ± 0.93. In order to determine whether the cavities contained cells that do not express muscle fiber markers, whole mount DAPI staining was performed. In all -/-; her7-/-; tbx6-/-her1 and -/-tbx6 embryos imaged at 32hpf (n=8 for each genotype), the cavities did not contain any cells  embryos were analyzed. In all mutants the fast  and slow (F59 positive) muscle fibers lose their metameric organization –4L. In at muscle ´, 4D´. Tt muscle ´. In tbxny cells . In addi regions ´. The sl present ´ or the  present ´ as prev mutants .-/-; her7-/-her1 mutants have a significantly smaller region of fast muscle compared to the wild type at 32 hpf , but only start to develop scoliosis as they age (after six weeks of age). In the -/-; her7 -/-her1 mutants there is no correlation between the position or amount of vertebral defects and the risk of developing scoliosis. If vertebral defects were correlated with scoliosis, we would predict that all animals with significant vertebral aberrations would present scoliosis. There is a high incidence of vertebral defects in -/-; her7-/-her1 mutants ranging from 1-12 defects per animal at 25 dpf [-/-;her7-/-her1 mutants should show a high incidence of scoliosis. However, the incidence of scoliosis was the same as in the wild type animals examined at a similar age. When evaluating whether muscle morphology in -/-; her7-/-her1 mutants was related to where scoliosis occurred along the body axis we did not observe any difference in muscle volume at regions where scoliosis occurred relative to other axial positions. Furthermore, there were no lesions in fast or slow muscle during embryogenesis in -/-; her7-/-her1 mutants. In contrast, -/-; her7-/-; tbx6-/-her1 mutant animals showed a lower muscle volume in regions in which scoliosis was observed and also had perturbed slow and fast muscle development.In order to understand more about the link between the somite clock and adult degenerative scoliosis, we analysed zebrafish mutants defective for three clock segmentation genes: t 25 dpf  and betw-/-; her7-/-her1 and -/-; her7-/-; tbx6-/-her1 mutants this was unrelated to the skeletal phenotype. The higher scoliosis incidence in the -/-; her7-/-; tbx6-/-her1 mutants is probably due to the muscle defects caused by the loss of Tbx6 activity. It is possible, that -/-; her7-/-her1 mutants recover from early muscle patterning defects by compensatory growth, but in an absence of Tbx6 function, this cannot occur. In -/-tbx6 and -/-; her7-/-; tbx6-/-her1 mutants the muscle contains many cavities that originated from embryonic stages. There is no recovery of the early muscle defects in these mutants, potentially due to a deficit in the myogenic precursor population at embryonic stages, in turn due to the role of Tbx6 in regulating differentiation of dermamyotome progenitor cells [her1 and her7, results in long lasting muscle phenotypes that appear to be correlated with scoliosis. The occurrence of muscle phenotypes in compound -/-; her7-/-; tbx6-/-her1 mutants occurs independently of skeletal abnormalities as there is no correlation of skeletal abnormalities relative to the presence of scoliosis between genotypes.Despite differences in adult muscle phenotype and embryonic muscle development between or cells . In the or cells . Thus, p-/-tbx6 mutant individual in this study that did not develop scoliosis had higher muscle mass than its scoliotic siblings  implying that muscle mass and scoliosis are directly correlated. In human patients, differences between the left and right-side muscles at the position of axial curvature have been documented. In the bent side the muscle becomes overstretched, while in the opposite side the muscles are shorter and tighter [In humans, spinal muscles are essential for stabilising the spine, and changes in muscle mass have been linked to axis curvatures. It is known that upon spine curvature, muscles adjacent to the curvature are generally weaker and progressively deform , 19. Our tighter . We did  tighter , 37.-/-; her7-/-; tbx6-/-her1 and -/-tbx6 individuals can be explained by differences in total number of individuals quantified. Regardless, in both generations, there is a trend towards scoliosis, which is much higher than in wild type.In elderly patients there is no difference in scoliosis prevalence between males and females . In our tbx6 function leads to the strongest scoliotic phenotype. The -/-tbx6  mutant zebrafish and human patients with mutations in TBX6 have similar skeletal phenotypes including hemivertebrae, butterfly vertebrae and rib abnormalities [TBX6 is available for comparison. Our findings from zebrafish intriguingly suggest that scoliosis-associated genes may be needed for normal muscle patterning at early embryonic stages and it is the disruption of early muscle organisation that may underlie scoliosis onset later in life.Our data show that loss of malities . In humamalities . In zebr-/-; her7 -/-her1 mutants but not in -/-tbx6 or -/-; her7-/-; tbx6-/-her1 mutants. Nonetheless, in -/-tbx6 and the triple -/-; her7-/-; tbx6-/-her1 mutant embryos, the slow and fast muscle fibers show cavities depleted of nucleated cells , is a similar phenotype described [Him is regulated by Notch signalling [-/-tbx6 mutants have a more than 50% reduction in the number of Pax3+ Pax7+ muscle progenitor cells at embryonic stages and unusually large fast muscle fibres that persists until later larval stages [tbx6 interacts with Mesp-b and Ripply1 to regulate myogenesis in zebrafish [The fast muscle domain and fast fiber cross sectional area were decreased in ed cells , which ced cells . Adult hreported . This phescribed . Him inhescribed . It is kgnalling . Interesgnalling , 46. Thil stages . In addiebrafish . Hence, -/-; her7-/-, tbx6-/-her1 and triple -/-; her7-/-; tbx6-/-her1 can serve as a model for adult scoliosis, because they reproduce the muscle and bone indicators present in clinical patients. Previously, it has been proposed that human patients with congenital scoliosis, may have early abnormalities during somitogenesis [In summary, we have shown that the zebrafish mutants for 2, 0.33 mM MgSO4) at 28°C. For anesthesia, a 0.2 % solution of 3-aminobenzoic acid ethyl ester (Sigma), containing Tris buffer, pH 7, was used.The work was carried out at the Hubrecht Institute (the NL), according to local laws. Ethical approval was obtained through the relevant DEC committee (HI 10.1801). Standard husbandry conditions applied, according to FELASA guidelines . Embryossa38869fss and hu2526her7 mutants were provided by Jeroen den Hertog (Hubrecht Institute). The gSAIzGFFM1954A line was obtained by a gene trap method [her1 in the her7hu2526 background, were generated as described in Lleras-Forero et al. [The p method . Double o et al. . DNA waso et al. .Olympus SZX16 stereoscope with a Leica DFC450C camera.Hematoxylin/eosin (H&E) was performed on sagittal and transverse cryo-sections (15μm) of adult zebrafish according to standard procedures. H&E sections were photographed on a Nikon eclipse NI with a DS-Ri2 camera and 4x Plan Fluor objective. Alizarin Red staining of bone was performed as described previously . Whole mTM plus Cyanine 3 system . For detection of nuclei DAPI was used in which embryos were fix at 32 hpf as described above and then washed twice for 10 minutes in PBS. Staining was performed with DAPI solution  in Eppendorf tubes, at 4 degrees in the dark, over two nights.32 hpf embryos were dechorionated and fixed in 4% PFA overnight. Phalloidin (Invitrogen Alexa Fluor 546) staining was performed according to Goody, 2013 . StaininImage acquisition was performed by embedding embryos in 0.8% agarose and visualising on a Leica SP8 confocal microscope using a 20X objective  for the whole mounts and a 40x objective  for the cloacal area acquisitions.Olympus SXZ16 stereomicroscope (1.5X PlanApo objective) connected to a DFC450C Leica camera. Immediately afterwards, the individual was returned to warm E3 media without anesthesia. Test subjects were returned to their specific tank in the animal facility only when they were completely awake and moving. This procedure was repeated at 3, 6, 9 and 12 months after fertilization. From 3 months onwards, a picture was taken using a Sony Xperia mobile. Sedation and photography did not take more than two minutes per animal and did not compromise survival.Embryos from mutant crosses were kept in E3 at 28°C until 7 dpf. They were then housed at a density of 50 embryos in a 3 Liter tank. At six weeks post fertilization, eight juvenile animals, with fully developed swim bladder, from either mutant or wildtype embryos were housed individually in the animal facility. Individuals were fed tetrahymena in combination with Gemma 75 for the first two weeks, followed by Artemia and Gemma 150 for the following two weeks. The same day each individual was anesthetized (as described above) and photographed using an ° rotation, with 400 ms exposure time. Reconstructions were performed using NRecon (Version 1.7.1.0). Each individual scan overlapped its neighbouring scans in its respective batch by 200 mm, to ensure robust concatenation. For One-year-old fish were fixed in 4% paraformaldehyde overnight at 4 degrees; afterwards an incision was made in the abdomen and the organs were removed. Staining for soft tissue visualization with Micro CT was performed according to Descamps et al., 2014  for 9 daReconstructions were processed using ImageJ/Fiji . ConcateUsing a Leica SP8 confocal with a 40x plan Apo objective  and a Z-step size of 0.27μm, an image from the cloacal region from six 32 hpf embryos , was taken. Five slides within each image were randomly chosen and the area was measured manually using Fiji. The data point plotted in the graph is the mean of the 5 slides within one embryo.Cross-sectional area quantification was done in the images of the cloacal region described above. Using Fiji, 5 randomly selected fibers were manually measured in the slides described above. Each point in the graph represents one embryo (25 different fibers). The statistical significance of the data was measured using a two-tailed Student’s t-test with unknown variance in Excel 2016 (Microsoft).-/-tbx6 and -/-; her7-/-; tbx6-/-her1 were measured in the optical sections stained by Phalloidin as described above. Using Fiji, the longest vertical distance of each cavity was measured in μm. The average and standard deviation were calculated for each mutant.Cavity size in In order to measure accurately if individual zebrafish had developed scoliosis and if the scoliosis progressed over time, the individual pictures from the time lapse were used. A straight line was drawn from the tip of the premaxilla, through the middle of the eye to the caudal fin. In fish lacking scoliosis or with a perturbation of the body axis the line ends exactly at the apex of the caudal fin. In a fish with a bent body axis, the line deviates from this position. The angle between the position of the line coming from the premaxilla to the new position of the apex was manually measured. In humans, the spine is considered deformed when the Cobb angle is ≥ 10 degrees . The samSupplementary FiguresSupplementary Custom Macros


Our aim was to determine whether carbon dioxide (CO2) levels in the first 3 days after birth reflected abnormal EtCO2 levels in the delivery suite, and hence, a prolonged rather than an early insult resulted in IVH. In addition, we determined if greater EtCO2level fluctuations during resuscitation occurred in infants who developed IVH. EtCO2 levels during delivery suite resuscitation and CO2 levels on the neonatal unit were evaluated in 58 infants . Delta EtCO2 was the difference between the highest and lowest level of EtCO2. Thirteen infants developed a grade 3–4 IVH (severe group). There were no significant differences in CO2 levels between those who did and did not develop an IVH (or severe IVH) on the NICU. The delta EtCO2 during resuscitation differed between infants with any IVH (6.2 (5.4–7.5) kPa) or no IVH (3.8 (2.7–4.3) kPA) (p < 0.001) after adjusting for differences in gestational age. Delta EtCO2 levels gave an area under the ROC curve of 0.940 for prediction of IVH.Abnormal levels of end-tidal carbon dioxide (EtCOConclusion: The results emphasize the importance of monitoring EtCO2 levels in the delivery suite. Our aim then was to study EtCO2 levels in the delivery suite during resuscitation of preterm infants and their relationship to the subsequent CO2 levels within the first 3 days after birth and the development of IVH.Improvements in neonatal intensive care have resulted in decreased mortality rates of preterm infants. The development of intraventricular haemorrhage (IVH), however, can result in long-term adverse outcomes. Several studies of preterm infants have demonstrated that abnormal levels of carbon dioxide  were associated with severe IVH development, as was a greater degree of fluctuation in pCO2 levels within the first 4 days after birth [2 variability [2 can adversely affect cerebral oxygenation and perfusion and the electrical activity of the brain [2 levels during resuscitation in the delivery suite were associated with IVH development but also inflations resulting in larger tidal volumes and lower EtCO2 levels [2 levels during resuscitation in the delivery suite would be at greatest risk of developing an IVH.In one study of very low birth weight infants, not only high but also low partial pressures of carbon dioxide pCO were assiability . Furtherhe brain ; such flhe brain . We have2 levels . Those f2 levels . A furthWe reviewed the records of infants born at less than or equal to 33 weeks of gestational age who required resuscitation in the delivery suite between March 2010 and March 2014. A certain part of their data was reported in a previous paper [2 levels.The resuscitation protocol, together with the monitoring methodology, has previously been described . The cliTE) of each inflation and the breath-by-breath minute ventilation. The highest EtCO2 was recorded as were the number of inflations with tidal volumes more than 6 mls/kg  or end-tidal CO2 levels of less than 4.5 kPa (low EtCO2 levels) and the number of inflations with both high tidal volumes and low EtCO2 levels as previously described [2 (delta EtCO2) during resuscitation was determined by calculating the difference between the highest and lowest EtCO2 value during resuscitation in delivery suite.The resuscitation recordings were analysed to determine the duration of resuscitation, the number of inflations, the inflation pressure, the positive end expiratory pressure (PEEP), inflation time (Ti), mean airway pressure (MAP), expiratory tidal volume  during each of the first 3 postnatal days was determined by calculating the difference between the highest and lowest CO2 value on each day. Volume targeted ventilation was not used on the NICU during the study period.The unit’s routine policy was to insert arterial lines for blood gas sampling, and in the absence of such, access capillary sampling was undertaken. Blood gases were recorded on the intensive care observation charts every 4 to 6 h depending on the clinical status of the infant, and the hourly ventilator settings were collected at the time as each arterial gas result. Venous blood gases were not analysed in this study. Data on the maximum, minimum and average pCOThe medical records were examined to determine whether the infants developed an IVH which was diagnosed from cranial ultrasounds. As per unit policy, cranial ultrasounds were performed on admission to the unit for all infants born below 34 weeks of gestation. Cranial ultrasounds were repeated on day 3, between days 7 and 10, days 14 and 21 and days 28 and 30. All cranial ultrasounds were reported by a neonatal consultant. The IVHs were reported as grades I to IV using the Papile classification . Severe U test or the chi-square test as appropriate. As infants who develop IVH tend to be more immature, regression analyses were performed to determine if the results remained statistically significant after correcting for differences in gestational age. The strength of any relationships of EtCO2 values during resuscitation with CO2 levels during the first 3 days after birth were assessed by calculating Spearman correlation coefficients. The performance of factors identified as significantly related to IVH development from the regression analysis were assessed by receiver operator characteristic curve analysis and estimation of the corresponding area under the curve (AUC). Statistical analyses were performed using JMP statistical software  and SPSS software 25.0 .The data were tested for normality using the Kolmogorov-Smirnov test and found to be non-normally distributed; hence, differences were assessed for statistical significance using the Mann-Whitney 2 traces which were unsuitable for analysis, a further 24 were excluded due to a large leak throughout and 12 excluded due to missing neonatal unit observation charts. The remaining 58 infants were included in this study and had a median gestational age of 27.3 (interquartile range (IQR) 24.9–29.0) weeks and a birth weight of 0.90 (0.72–1.21) kg. The infants who were and were not included in this study did not differ significantly with regard to birth weight (p = 0.893) or gestational age (p = 0.785). Fifty-three of the 58 infants were ventilated on day 1 after birth. Thirty infants developed no IVH with 28 infants developing an IVH. Seven infants developed a grade 1, eight infants grade 2, five infants grade 3 and the remaining eight infants a grade 4 IVH, that is 13 infants developed a severe IVH. All infants who developed an IVH did so in the first 3 days after birth. Eleven infants developed an IVH on day 1 (9 grade 1–2), nine on day 2 (5 grade 1–2) and eight on day 3 (1 grade 1–2). There were no significant differences in CO2 levels on the NICU and IVH development.Three hundred and seventy infants were born at less than or equal to 33 weeks of gestation within the study period. There were 150 infant recordings available for analysis, but 56 had recordings of tidal volume or EtCO2 during resuscitation in the no-IVH group was 8.0 (7.1–8.4) kPa and 10.0 (8.9–11.2) kPa in the IVH group; the difference remained statistically significant after adjusting for differences in gestational age between the two groups (p = 0.0002). The highest EtCO2 during resuscitation in the no/non-severe group IVH was 8.2 (7.4–9.9) kPa and 10.2 (8.6–11.1) kPa in the severe IVH group; the difference remained statistically significant after adjusting for differences in gestational age between the two groups (p = 0.037). There was no significant difference in the mean tidal volume during resuscitation between infants in the no/non severe IVH group and the severe IVH group (p = 0.621).The highest EtCO2 during resuscitation in the no-IVH group was 4.2 (3.2–5.2) kPa and 3.5 (3.0–4.5) kPa in the IVH group, but this was not statistically significant after adjusting for the difference in gestational age between the two groups (p = 0.11). The lowest EtCO2 during resuscitation in the no/non-severe IVH group was 4.0 (3.2–5.1) kPa and 3.1 (2.7–3.5) kPa in the severe IVH group; the difference remained significant after adjusting for the difference in gestational age between the two groups (p = 0.041).The lowest EtCO2 on delivery suite and the highest pCO2 on the neonatal unit on days 2  and 3 .There were weak correlations between the highest end-tidal CO2 during resuscitation was significantly different between the no/non-severe IVH (4.3 (3.1–5.6) kPa) and severe IVH groups (7.2 (5.9–7.6) kPa) (p = 0.003) and between infants with any IVH (6.2 (5.4–7.5) kPa) or no IVH (3.8 (2.7–4.3) kPa) (p < 0.001) after adjusting for differences in gestational age. The delta CO2 level during resuscitation weakly correlated with the delta CO2 level on the neonatal unit on days 2  and 3 .The delta EtCO2 on the delivery suite had an AUC of 0.856 and the delta CO2 during resuscitation had an AUC of 0.940. A delta EtCO2 equal or greater than 4.9 kPa predicted any IVH development with 96.4% sensitivity and 83.3% specificity.The receiver operator characteristic curve analysis demonstrated that in predicting any IVH, the maximum level of EtCO2 and greater degree of fluctuation of EtCO2 levels during resuscitation. In addition, we highlight that the delta EtCO2 level in the delivery suite had both a high sensitivity and specificity in predicting IVH development. Importantly, we have shown that abnormal EtCO2 levels during resuscitation did not necessarily translate into abnormal levels on the neonatal unit. Our findings, then suggest that it is early abnormal carbon dioxide levels in preterm infants that are important in IVH development. Previously, initial post-delivery levels of pCO2 were found to be significantly higher in infants with cerebral haemorrhage than those without, but by 8 h after birth, those differences were no longer significant [2 levels in the delivery suite, but not the neonatal unit, with IVH development. Some studies have suggested that sicker infants undergo more blood gas analysis than more stable infants, hence, allowing detection of a greater degree of fluctuation in levels of carbon dioxide [2 during resuscitation in all infants included in our cohort, and therefore, the outcomes relating to fluctuation of carbon dioxide are not influenced by the infant’s degree of sickness. We speculate that by the time infants are admitted to the neonatal unit, clinicians have greater access to routine monitoring and would readily re-check abnormal levels of pCO2 by blood gas analysis, hence, limiting the degree of fluctuation between extremes of values and thereby achieving tighter control. This would explain our findings that it is the delivery suite levels of CO2 that are more influential regarding IVH development and why the extreme levels of carbon dioxide seen during resuscitation do not translate to neonatal unit levels of CO2 during the subsequent 72 h. We did not use volume targeted ventilation during the study period and this would have likely reduced fluctuations in CO2 levels on the NICU.We have demonstrated that preterm infants who developed an IVH had a higher maximum level of EtCOnificant , which a2 levels that were associated with IVH development, but this was no longer significant after adjustment for gestational age. Nevertheless, we highlight fluctuations in EtCO2 that are highly predictive of IVH development. Hence, it is important to avoid both high and low levels of EtCO2 during resuscitation. Our results emphasize that respiratory function monitoring should be helpful in the labour suite. It has been previously shown that resuscitators were unable to accurately assess chest wall movement [2 levels during resuscitation.As previously mentioned , we did movement , 12, andmovement . We havemovement . Eye tramovement  suggesti2 levels and fluctuation of EtCO2 levels during resuscitation and how they correlated with both carbon dioxide levels in the first 3 days after birth and IVH development. We monitored EtCO2 levels during resuscitation, yet on the neonatal unit, we have analysed levels of CO2 from blood gas measurements. Previous studies, however, have found EtCO2 to be as accurate as arterial monitoring when trending carbon dioxide levels [Our study has strengths and some limitations. This is the first study to evaluate EtCOe levels . We did 2 levels in the delivery suite was associated with a significantly increased risk in the development of IVH. Those results emphasise the need for EtCO2 monitoring during resuscitation in the delivery suite.In conclusion, a greater degree of fluctuation of EtCO


Mycobacterium tuberculosis. Under the hypoxic condition, phosphorylated DevR interacts with multiple binding sites at the promoter region of the target genes. In the present work, we carried out a detailed computational analysis to figure out the sensitivity of the kinetic parameters. The set of kinetic parameters takes care of the interaction among phosphorylated DevR and the binding sites, transcription and translation processes. We employ the method of stochastic optimization to quantitate the relevant kinetic parameter set necessary for DevR regulated gene expression. Measures of different correlation coefficients provide the relative ordering of kinetic parameters involved in gene regulation. Results obtained from correlation coefficients are further corroborated by sensitivity amplification.The DevRS two-component system plays a pivotal role in signal transmission and downstream gene regulation in  Mycobacterium tuberculosis . Co. CoRv313hspX. The ordering of the parameters related to expression of hspX in terms of correlation values (absolute)  compared to the other synthesis rates associated with hspX. On the other hand, the activation and inactivation rates (kb3 and ku3) related to the formation of active P1 (P1*) show a lesser correlation. These two opposing factors work together to determine the effective contribution of distal binding site P1 in the expression of hspX as reported by Chauhan et al. [KD (= ku/kb) value associated with the distal binding site P1 acts as a bottleneck for its proper functionality. Due to this reason, the mutant pmutP1 (mutation at P1) showed ∼ 30% of the wild type GFP expression, as reported by Park et al. [Next, we check sensitivity of the parameters associated with ute) see  areksm4k et al.  Fig 3C)hspX. Theksm7 which takes care of synthesis of GFP due to the cooperative effect of all the three binding sites  of hspX. The synthesis rates ksm5 and ksm6 due to P2 and S, respectively, come next. As P2 lies close to the transcription start point (TSP) of hspX, the GFP synthesis rate due to P2 is higher than that of S. It is important to note that, mutation at P2 (pmutP2) shows ∼ 53% of the wild type expression. Thus mutation at both primary binding sites P1 and P2 (pmutP1P2) shows only ∼ 12% of wild type expression and agrees with Park et al. [kb4 & kb5) and unbinding (ku4 & ku5) rates with GFP for the activation of P2 and S show that preference of RP is almost equal for P2 and S due to their close proximity.The next parameter that shows high correlation with GFP is k et al. . The cornarK2-Rv1738 system. For these pair of genes we quantify the correlation coefficients  starts producing the transcripts with maximum efficiency. Thus, activation of P1 serves as a bottleneck for narK2 transcripts. On a similar note, the parameter sets  and  controls the activation of the sites P2 and S2, respectively.Both the parameters Rv1738, the ordering of the parameters in terms of correlation values (absolute) are  reduces further and has a weak role in the overall production of transcripts. The weak contribution of P1 is observed in the expression profile of the mutant pBmutP1 [narK2). Transcription rate ksm13 from distal secondary site S2 is least and appears at the end of ordering. In between ksm9 and ksm13, the sensitivity of different binding rates appear.In  are see ksm19>ksRv3134c, hspX and narK2-Rv1738. The parameters with a high correlation coefficient with the output (GFP) indeed show a high level of sensitivity amplification Click here for additional data file.S1 FigThe perturbed parameter is ploted as a function of SA steps. In each panel, the five colored lines originated from different SA simulation. The horizontal dotted lines is for the base parameter value reported in (PDF)Click here for additional data file.S2 FigThe perturbed parameter is ploted as a function of SA steps. In each panel, the five colored lines originated from different SA simulation. The horizontal dotted lines is for the base parameter value reported in (PDF)Click here for additional data file.S3 FigThe perturbed parameter is ploted as a function of SA steps. In each panel, the five colored lines originated from different SA simulation. The horizontal dotted lines is for the base parameter value reported in (PDF)Click here for additional data file.S4 Figksrp and kdrp) and GFP (ksg and kdg). Each panel consists of 103 independent simulation data.The output (GFP in nM) as a function of synthesis and degradation rates of phosphorylated DevR ((PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 Fig(PDF)Click here for additional data file.S1 TableList of all kinetic parameters used in the model.(PDF)Click here for additional data file.S2 Table5 indipendent run for synthesis and degradation rate parameters of four genes.Various correlation coefficient values are obtained by using 10% perturbation and 10(PDF)Click here for additional data file.S3 Table5 indipendent run for all the input parameter with output of Rv3134c.Various correlation coefficient values are obtained by using 10% perturbation and 10(PDF)Click here for additional data file.S4 Table5 indipendent run for all the input parameter with output of hspX.Various correlation coefficient values are obtained by using 10% perturbation and 10(PDF)Click here for additional data file.S5 Table5 indipendent run for all the input parameter with output of narK2.Various correlation coefficient values are obtained by using 10% perturbation and 10(PDF)Click here for additional data file.S6 Table5 indipendent run for all the input parameter with output of Rv1738.Various correlation coefficient values are obtained by using 10% perturbation and 10(PDF)Click here for additional data file.


H3K27me3) as a new immunohistochemistry (IHC) marker for MPNST have recently available to assist pathologists in differentiating MPNST from other histologic mimics. We aim to study the expression pattern of H3K27me3 in MPNST and its histologic mimickers and their association with the clinicopathological data. Diagnosis of malignant peripheral nerve sheath tumor (MPNST) is rather challenging due to its divergent morphologic heterogeneity and lack of specific ancillary test. The emergence of H3K27 trimethylation  for 3 tiers system . The clinicopathological data were retrieved from the patients’ record. A total of 59 benign and malignant spindle cell tumours , 4 leiomyosarcoma, 1 spindle cell liposarcoma, 1 solitary fibrous tumour, 2 low grade fibromyxoid sarcoma and 1 unclassified spindle cell sarcoma), diagnosed from January 1998 to April 2018 in Hospital Universiti Sains Malaysia (HUSM) were tested for H3K27me3 expression which is statistically significant as compared to its histologic mimics (p<0.001). Similar findings (p=0.026) were also observed in high grade MPNST (81.8%), intermediate grade MPNST (100%) and 0% in low grade MPNST. A total of 61.1% (11/18 MPNST) showed loss of H3K27me3, combined with other panel of markers, is useful in MPNST diagnosis to differentiate it from the histological mimickers.  Malignant peripheral nerve sheath tumour (MPNST) is particularly rare with an incidence of 0.001%  in the general population and representing approximately 4% of all soft tissue sarcomas . This soft tissue neoplasm has gained an interest due to its diversity of histologic appearances, overlapping pattern, the rarity of the tumour, and the need of supportive and confirmatory tests which often renders MPNST as diagnostically challenging neoplasm. All these may justify to the delay in diagnosis. MPNST is rare, aggressive soft tissue sarcomas associated with poor prognosis. H3K27me3) as a new immunohistochemistry marker for MPNST have recently available to assist pathologists in differentiating MPNST from other histologic mimics. However, it also remains conflicting results for H3K27me3. Prieto-Granada et al., (2016) found that H3K27me3 immunohistochemistry has good sensitivity and robust specificity for the diagnosis of MPNST, while others noted loss of expression in other type of sarcomas too, which included radiation-associated angiosarcomas  and dedifferentiated chondrosarcoma . In this study we investigated the expression of H3K27me3 in differentiating MPNST from other types of spindle cell tumour and its association with its histological grading and clinicopathological data.The diagnosis of MPNST relies on morphology and IHC interpretation. Differentiating MPNST from other types of spindle cell neoplasm is important for prognosis and management. Several markers have been suggested to distinguish MPNST from other soft tissue tumours. These include S-100 protein, SOX10, epithelial membrane antigen (EMA), cytokeratins, CD34, HMB45 and desmin . None of these markers are specific for MPNSTs and mostly used to narrow down the diagnosis of spindle cell tumour. The emergence of H3K27 trimethylation . The clinicopathological data for all cases which included age, clinical presentation, site and depth of tumour involvement, tumour size, type of sample, patients’progress such as presence of local recurrence and metastasis after diagnosis, were also obtained from patients’ record notes. The study obtained its ethical clearance from the Ethical Committee, Universiti Sains Malaysia (JEPEM Code: USM/JEPeM/17020148).Histological assessment The tissue slides were deparaffinized and hydrated using standard procedures and stained with hematoxylin and eosin (H&E). The slides were viewed using Olympus CX31 light microscopy to assess the morphology of tumour and grading of MPNST. The grading system is based on tumour differentiation, mitotic count and tumour necrosis in FNCLCC grading system. A 3-tiered grading system with low-grade (LG), intermediate grade (IG) and high-grade (HG) MPNSTs is used, with the LG end being characterized by neurofibroma (NF)-like tumors composed of a cellular proliferation of bland spindle cells that appear to be in a continuum with the so-called atypical/cellular neurofibroma. The typical morphologic picture defining a conventional HG MPNST is represented by a fascicular, monomorphic spindle cell proliferation with a “marbled” low-power appearance and areas of geographic necrosis. Finally, examples exhibiting histomorphologic features that deviated from these classic LG and HG patterns were classified under intermediate MPNST .Immunohistochemical (IHC) staining for H3K27 trimethylationH3K27me3 at 1:200 was applied to the sections. Sections of mimickers without primary antibodies served as negative controls, and sections of mimickers stained with primary antibodies for each antibody served as positive controls. All these were run together with each batch of staining. Tissue from colon was used as positive control for H3K27me3 staining.IHC staining was performed to the paraffin-embedded tissue blocks according to standard procedures (Abcam Code Ab192985) using pressure cooker antigen retrieval (Tris-EDTA buffer solution pH 9.0) method. The monoclonal primary antibody Its expression was quantified in the various samples examined using a scoring method adopted from Cleven et al., (2016). A mean percentage of nuclear staining was determined at a magnification of 400x, and scored 0 (0 cell positive), 1+ (1-24% of cell positive), 2+ (25-49% of cell positive) and 3+ (>50% of cell positive). The 0 score is considered loss of expression. Microscopy for immunoreactivity was evaluated by a single independent pathologist who is blinded to the case. Data were statistically analyzed by IBM SPSS version 24, using chi-square test or fisher-exact test. Epidemiological and clinicopathological data A total of 59 participants involved in the present study. A total of 18 MPNST cases and 41 of its histologic mimickers , 4 synovial sarcoma, 3 fibrosarcoma, 2 gastrointestinal stromal tumour (GIST), 4 leiomyosarcoma, 1 spindle cell liposarcoma, 1 solitary fibrous tumour, 2 low grade fibromyxoid sarcoma and 1 unclassified spindle cell sarcoma) were included in this study. The result of the data showed that the mean age of the participants is 40.76 years old (SD=17.21). Our study shows a bimodal distribution of MPNST patients which were most prevalence in the 12-year-old and younger age group and at 40-59 year old age group with mean age of diagnosis was 32.83 (SD=20.54). Ten out of 18 (55.6%) of MPNST cases were sporadic and 44.4% (8 out of 18) were NF-1 associated MPNST.Morphologically 14 cases (77.8%) were classical spindled type, 3 cases (16.7%) were malignant triton tumour and 1 case (5.6%) was epithelioid MPNST. For histologic grades, 77.8% (11/18) of MPNST cases were high grade, 11.1% (2/18) were intermediate grade and low grade cases respectively. In term of patients’ outcomes, 7 out of 18 MPNST cases (38.9%) appeared with no local recurrence or distant metastases. Six out of 18 (33.3%) cases noted to have distant tumour metastasis at diagnosis and less than 5 years after diagnosis. Lungs, intraspinal and bone were the predominant sites of metastases. Three out of 18 cases (16.7%) show disease recurrence and 2 out of 18 cases (11.1%) resulted in death less than 5 years after diagnosis.Pattern of expression of H3K27me3 in MPNSTH3K27me3 in MPNST. Of the 18 tumours evaluated for H3K27me3 expression, 14 are high grade MPNST and remaining 2 each are of intermediate grade and low grade MPNST. Majority of high grade MPNST (9/14) and all of the intermediate grade MPNST showed loss of H3K27me3 expression. None of the low-grade cases show loss of H3K27me3 expression. However, 1 case of high grade MPNST (5.6%) show strong immunoreactivity towards H3K27me3 expression .We evaluated the expression of pression . In our Pattern of expression of H3K27me3 in MimickersH3K27me3   .In general, the malignant peripheral nerve sheath tumour is typically seen in adult life because most tumour occur in patients 20 to 50 years of age with a median age of about 35 . In our study, the median age was 28.5 years old ranged from the youngest age of 3 year old and the eldest was 81 year old. Although MPNST is most prevalent in adolescents and young adults, our study noted 4 cases (22.2%) of the MPNST cases were found in the 12 and younger age group. MPNST in childhood are recognized but rare and it may affect patient in a wide age range from birth to eighth decades. This was supported by Kudesia et al., (2014), in which the incidence of MPNST among paediatric patient, whom age less than 5 months old was only about 1.7%. In this study, we noted that among MPNST cases, 8 out of 18 patients has associated background history of neurofibromatosis 1 (NF1) with skin manifestation. Among patient with NF1, majority are diagnosed with MPNST at slightly earlier age with equal male to female distribution as compared to those with sporadic MPNST .In our study, we found that the commonest site of tumour were upper extremities, followed by the trunk and upper extremities. This findings showed similarity with previous studies . They found this tumour occur predominantly in the extremities, followed by the trunk/retroperitoneum and head and neck region. This findings explain the fact that most MPNSTs arise in association with major nerve trunks, including the sciatic nerve, brachial plexus and sacral plexus. Besides, it has been described at various anatomical sites including intraspinal and various visceral .A tumour can behave like benign, such as in the form of well circumscribed mass, superficial mass with indolent presentation and this may lead to delay in the patients’ management. This type of tumour is either asymptomatic or may cause minimal discomfort. Many patients reported pain and neurological deficits at time of presentation. In our study, this commonly slow enlarging mass may exhibits rapid growth and we found that all of MPNST cases in our study presented with lesions more than 5cm as compared to benign peripheral nerve sheath tumour which in majority of cases presented with tumour less than 5cm at time of diagnosis. In majority of cases the duration of the tumour are less than 1 year upon diagnosis. MPNSTs have a relatively poor prognosis in comparison with other soft-tissue malignancies. The five-year survival for adults and children varies from 35% to 50% . Most MPNSTs are aggressive, high grade sarcomas with a high likelihood of local recurrence (40% to 65%), and distant metastases (40% to 68%.) . From previous case series, they found that MPNST frequently metastasize to the lungs followed by bone whereas lymph node metastases are uncommon . Other sites of metastasis include liver, brain, soft tissue, skin, and retroperitoneum. In our study, nine cases showed distant metastases and local recurrence with majority metastasize to the lung followed by other sites including intraspinal, liver and testes. However, the data from our study were limited because some of the patient had missing data or defaulted follow up. We had difficulty in tracing record of the patient and getting adequate information, especially those who were previously referred case and treated outside of our center. MPNSTs have poor outcome if untreated. Unfortunately, two of our patient developed lung metastases and succumbed to death after diagnosis with advanced MPNST in spite of a wide excision and additional chemotherapy. Although MPNST is rare in the paediatric age group, based on our study, diagnosis should be considered in children without NF1 with a rapidly evolving and painful mass in the distribution of a peripheral nerve. Early diagnosis and referral to multidisciplinary team are important in ensuring the best diagnosis and optimal therapy in this young age. In this study, we would like to emphasize on the important of this marker to help identify MPNST early in order for patient to get appropriate treatment earlier.From our study, the predominant subtype was spindle cell type (77.8%). It is very crucial to differentiate it from other type of spindle cell sarcoma. The diagnosis of spindle MPNST is challenging especially if the mass arising from unusual site. In one of our paediatric cases, it was arising from a pelvic region and the morphological pattern was purely spindle. In this case, synovial sarcoma was one of the differentials and interestingly it showed moderate to strong nuclear immunoreactivity to TLE1 in which rendered more challenges to the pathologist. However, TLE-1 positivity can also be seen in about 15% of MPNST and about 8% in Solitary Fibrous Tumour . Here, we could see the role of H3k27me3 in which it showed loss of its expression thus, it confirmed the diagnosis of MPNST especially when the molecular test is not available in the centre to demonstrate the presence of translocation t in synovial sarcoma.There are several reported cases of a rare spindle MPNST arising in a peripheral nerve, in which the clinical and radiological findings were indistinguishable from benign peripheral nerve sheath tumour, lead to insufficient initial treatment. Based on morphological criteria including high cellularity, nuclear atypia and mitotic activity, pathologist try to differentiate MPNST from atypical neurofibroma. Microscopically the tumours were composed of highly cellular spindle-shaped cells arranged in hypercellular and hypocellular areas with perivascular hypercellularity, characteristic of MPNST .H3K27me3 IHC. MPNST is the malignant counterpart of peripheral nerve sheath tumour and the main differential diagnosis. Logically a greater degree of cellularity, pleomorphism and higher mitotic activity are criteria used to classify the lesion as malignant. A distinctive, rare subtype of MPNST that raises separate differential is characterized by a predominance of large epithelioid cells, such as epithelioid MPNST. These tumours are relatively more common in superficial sites and strongly express S100 protein. In our study only one epithelioid MPNST case encountered and similar to previous study, it expressed diffuse and strong S100 protein and showed weak immunoreactivity towards Although S100 protein is a marker of nerve sheath differentiation, it has limited diagnostic utility as MPNSTs are often negative or very focally positive, (50-90%). Small percentage of MPNST cases (2%-15%) were found to express TLE-1. However the positive expression were either weak (1+) or moderate (2+) staining . Therefore, in practice usually strong staining (3+) for TLE-1 argues strongly against a diagnosis of MPNST. Accurate diagnosis is crucial in choosing the correct treatment given. In this study we found one of the cases was diagnosed and treated as Ewing Sarcoma as initial biopsy of the case was reported as possible Ewing sarcoma. However, the final diagnosis from the excised tumour, it was malignant peripheral nerve sheath tumour. The discrepancy of the diagnoses showed the heterogeneity of the tumour itself which may resembled different morphological features in a small biopsy. Over the past two decades, a few studies have shown that diagnostic agreement in soft tissue neoplasm between primary institutional diagnosis and reviewer diagnosis ranges from 53% to 73% . Overall diagnostic discrepancies range from 28% to 35%, of which minor discrepancies constitute 7% to 16% and major discrepancies 11% to 25% . The rarity of soft tissue tumours and its constantly evolving histopathological criteria, poses a challenge for general pathologists resulting in referral to centres with a soft tissue pathologist. The implication of this is that an inappropriate panel of IHC stains would be performed, with or without IHC misinterpretation . Thus, additional diagnostic markers are needed to aid in this often challenging differential diagnosis .H3K27me3 and altered DNA methylation .In a study on cancer development by epigenetic modifications, they have found that proteins of the polycomb group which are transcriptional repressors that modify chromatin, regulate cell fate, and promote cancer growth actually play an important role in pathogenesis of MPNST . In MPNST, inactivation of polycomb repressive complex II (PRC2) promotes cell proliferation and tumor growth. Inactivation of PRC2 is therefore expected to result in both loss of H3K27me3 could be of diagnostic aid . Other study suggest that PRC2 inactivation in MPNST may occur during progression to higher grades . In our study, immunohistochemistry of H3K27me3 proved to show loss of expression in 61.1% of MPNST, comparable to the findings of a previous study which showed 51% negative for H3K27me3 , and 69% negative for H3K27me3 of all MPNSTs . It showed that MPNST group have the highest number of H3K27me3 negative expression, while mimickers group mostly displayed strong immunoreactivity towards H3K27me3 (H3K27 expression and type of cases (p<0.001). These findings suggest that H3K27me3 loss is definitely a more specific marker for MPNST and useful in differential diagnosis, particularly for tumors that lack expression of supportive Schwann cell markers  and among high grade spindle cell sarcomas. Many studies had shown that inactivation of the polycomb repressive complex 2 (PRC2), has recently been identified in 70–90% of malignant peripheral nerve sheath tumour . The recent discovery of frequent inactivation of PRC2 in MPNSTs suggests that immunohistochemical detection of H3K27me3 . Our finH3K27me3 (61.1%) whereas only 7.3% were loss in non-MPNSTs (histologic mimickers). The findings were also observed by Cleven et al., (2016), who found that 24 out of 341 (7%) cases of other spindle cell tumours were negative for H3K27me3. From this study, we found that the marker also played a role in differentiating benign peripheral nerve sheath tumour and malignant peripheral nerve sheath tumour. In our study, both neurofibroma and schwannoma show moderate to strong expression of H3K27me3 with a p value of <0.001. This indicate that there is a significance difference in expression of H3K27me3 between benign peripheral nerve sheath tumour and malignant peripheral nerve sheath tumour . In our study, 25% of synovial sarcoma (SS) and 33.3% of fibrosarcomatous dermatofibrosarcoma protuberans (DFSP) showed loss of H3K27 trimethylation expression. The 25% of SS cases warrant molecular test for diagnostic confirmation. On the other hand in another study by Cleven et al. (2016) on MPNST and other spindle cell tumours, loss of H3K27me3 was reported in 60% of synovial sarcomas and 38% of DFSP. Our results are almost in agreement with the above study in which loss of expression was also observed in non-MPNST. This highlight the possibility that this antibody is not specific to MPNST. Majority of MPNST showed loss of h tumour . This isHowever, the marker was significantly showed to be useful in differential diagnosis of MPNST when combine with other ancillary test .H3K27me3 expression and MPNST histologic grades is another important finding. In our study, loss of H3K27me3 was observed in 64.3% of high grade, all of the intermediate grade and none of the low grade MPNST. Our study showed there was significant association between the histologic grades and H3K27me3 expression. Most of the H3K27me3-negative MPNST (9/18) were high grade with 3 cases showed local recurrence, 4 cases showed distant metastases and 2 cases end up with death while none of the low grade or intermediate grade having any local recurrence or distant metastases. This is in keeping with previous findings where loss of positivity for H3K27me3 is associated with poor prognosis in MPNST . In this study, there is no significant association between morphologic subtypes of MPNST and the expression of H3K27me3 immunoreactivity. This was supported by Prieto-Granada et al., (2016) where they found no significant differences in staining between different MPNST subtypes. However, all malignant triton tumour showed loss of expression and associated with poor prognosis and more aggressive behavior . We also found that there was no significant difference in the expression of H3K27me3 immunoreactivity between NF1 associated MPNST and sporadic MPNST.The association between H3K27me3 in majority of sporadic MPNST cases as compared to NF1-associated MPNST and conclude that H3K27me3 analysis has good sensitivity and robust specificity for the diagnosis of MPNST, particularly without clinical history of NF-1. Prieto-Granada et al., (2016) found that the sensitivity of H3K27me3 for sporadic MPNST and radiation-induced MPNST are 94 % and 93%, respectively. However, further study needs to be carried out to determine the sensitivity and specificity of this marker for diagnosis of MPNST. For the clinical management of these patients, it may be of value to be able to identify MPNST among these sarcomas to select patients who might be candidates for novel targeted therapeutic approaches .Recent study by Prieto-Granada et al., (2016) showed loss of expression of H3K27me3 is a potentially good immunohistochemical diagnostic marker. Though we found it is not to be a specific marker, but it definitely will give an added value when use in combination with other IHC panels especially in differentiating MPNST from its histologic mimickers. Since most of the panel of IHC in diagnosis MPNST are of limited value, our results had shown that


In the Surveillance Summary “Prevalence of Autism Spectrum Disorder Among Children Aged 8 Years—Autism and Developmental Disabilities Monitoring Network, 11 Sites, United States, 2016,” errors occurred in the Abstract and Table 2 and Table 3.39%. On page 11, the second footnote of Table 2 should have referenced Supplementary Table 7. On page 11, the fourth column header in Table 3 should have been IQ ≤70. On page 1 in Results, the percentage of girls with intellectual disability should have been


Porcine circovirus-associated diseases (PCVADs) cause considerable economic losses in industrial pork production in the field. To minimize the economic losses due to PCVAD, porcine circovirus type 2 (PCV2) vaccines have been developed, and there is widespread vaccination worldwide today. However, limited information is available concerning the current status of PCV2 infection in the field on the Asian continent. The present study aimed to assess sero- and viral dynamics of PCV2 from 12 PCV2-contaminated pig herds with vaccination against PCV2 in Southern and Central Taiwan. In particular, the level of PCV2 load during the window period for seroconversion using real-time polymerase chain reaction and a commercial enzyme-linked immunosorbent assay (ELISA) kit. Our results revealed that pig herds showed slight or no seroconversion after three to four weeks post-PCV2 immunization. The presence of PCV2 was observed during the window period for seroconversion in all herds. In conclusion, natural exposure of PCV2 occurs in the growing to fattening period, and viremia can last until slaughter. Additionally, our findings indicate that using ELISA showed the level of antibodies and aided in the understanding and surveillance of the current PCV2 status in the field. Circoviridae together with the genus Cyclovirus  was observed in the sow vaccination against PCV2 at 2-4 weeks pre-farrowing group, followed by the mass vaccination group , and the difference was not statistically significant between those two groups . The levThe serodynamics of the PCV2 antibody in pigs during the suckling to slaughter stages from different sow vaccination programs were measured by ELISA and are shown in 3 PCV2 genome copies/µl) or low PCV2 loads (<103 PCV2 genome copies/µL) indicated PCVAD or subclinical PCV2 infection, respectively [3 PCV2 genome copies/µL) was detected at 7 weeks old in 2 of 10 pigs in Farm A. Subsequently, ten of ten pigs (100%) and 9 of 10 pigs (90%) had PCV2 viremia at 11 and 15 weeks old, respectively  was detected at 11 weeks old in 4 of 10 pigs  in both Farm E and F, respectively . High PC 10 pigs . Subsequ 10 pigs .1.52 PCV2 genome copies/µL in Farm J (3 PCV2 genome copies/µL)  in Farm K . High PCn Farm J . This man Farm J . Subsequpies/µL) .Natural exposure to PCV2 should be considered starting at 11–12 weeks of age in the most analyzed pig farms. Regarding the production system , naturalPCV2 is one of the major primary infectious agents causing mortality in PRDC in the field. PCVADs have caused enormous economic losses in industrial pork production worldwide . FortunaTo compare the level of PCV2 MDA, it is not surprising that MDA was highly significantly lower in the sow without vaccination against PCV2 group than in the sow with mass or pre-farrowing vaccination groups. However, the highest CV of PCV2 MDA was also observed in the sow without PCV2 vaccination group. These results indicated that gilts and sows in pig herds have been naturally exposed to field viruses. Thus, sow vaccination against PCV2 should be a critical concern in the field. Our results indicated that a high level of PCV2 MDA was observed in the sow vaccination against PCV2 at 2–4 weeks in the pre-farrowing group, followed by the mass vaccination group. Fraile et al.  indicateTo compare the sero- and viral dynamics of PCV2, despite the pig production system, sow and piglet vaccination programs, all pig herds, except the Farm G herd, showed a decrease of PCV2 total antibody levels during 3–4 to 6–7 weeks of age, reaching the lowest levels of 0.25 to 1.24 S/P values. The natural exposure of PCV2 as measured by real-time PCR occurred in all herds, and seroconversion of PCV2 antibodies was subsequently observed. Regarding natural exposure of PCV2 during the growing to fattening period, better protection against natural exposure of PCV2 under different types of vaccine, vaccination timing or two shots of PCV2 immunization worth to further investigated. This result is in accordance with a previous study; natural PCV2 infection can occur late in growing to fattening pigs, and viremia can last very long until slaughter ,31. BaseConsistent with previous field observations ,11,12, nOur study revealed the cross-sectional nature of the sero- and viral dynamics of PCV2 in twelve PCV2-contaminated pig herds in the field. The presence of PCV2 was observed during the window period for seroconversion in all herds. Natural exposure of PCV2 occurs in the growing to fattening period, and viremia can last until slaughter. Additionally, our findings indicate that using ELISA showed the level of antibodies and aided in the understanding and surveillance of the current PCV2 status in the field.


In the original article, there was a typographic error in the introduction regarding the human population and number of pigs in Denmark.The text should read:“In 2017 (3rd quarter), Denmark had 3,226 pig farms with 12.69 million animals (5) compared to a human population of 5.76 million individuals (6).”The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Entropy [1,*, Chi-Fang Chen 1,*, Wei-Chun Hu 1 and Pieretti Nadia 2Shashidhar Siddagangaiah that should be corrected to the following order in this paper [1,*, Chi-Fang Chen 1,*, Wei-Chun Hu 1 and Nadia Pieretti 2Shashidhar Siddagangaiah We have found an error in the order of one author’s name and surname in the article recently published in Entropy :ShashidThe authors would like to apologize for any inconvenience caused to the readers by these changes.


The current investigation provides information on supportive services caregivers said they would be interested in if they were made available. Participants were recruited from the Birmingham, AL metro area and received a $25 gift card for completing a telephone interview. Of the 38 caregivers enrolled, 18 (47.37%) reported being currently employed and working an average of 33.92 hours per week (range = 5 – 60). Participants were caring for individuals with average scores on the AD8 Dementia Screening Scale of 7.3 and 10.4 on the Clinical Dementia Scale Sum of Boxes (above the cutoffs for probable dementia). Thirty-three caregivers (87%) reported being interested in at least one of the services listed with differences observed for services that would be preferred by working vs. non-working caregivers. These data will be utilized to provide initial support for a multicomponent intervention that may be effective in reducing negative outcomes within Black caregivers.