J. regia 700 K SNP array, with phenological data from 2018, 2019 and legacy data. These accessions come from the INRAE walnut germplasm collection which is the result of important prospecting work performed in many countries around the world. In parallel, an F1 progeny of 78 individuals segregating for phenology-related traits, was genotyped with the same array and phenotyped for the same traits, to construct linkage maps and perform Quantitative Trait Loci (QTLs) detection.Unravelling the genetic architecture of agronomic traits in walnut such as budbreak date and bearing habit, is crucial for climate change adaptation and yield improvement. A Genome-Wide Association Study (GWAS) using multi-locus models was conducted in a panel of 170 walnut accessions genotyped using the Axiom™ Using GWAS, we found strong associations of SNPs located at the beginning of chromosome 1 with both budbreak and female flowering dates. These findings were supported by QTLs detected in the same genomic region. Highly significant associated SNPs were also detected using GWAS for heterodichogamy and lateral bearing habit, both on chromosome 11. We developed a Kompetitive Allele Specific PCR (KASP) marker for budbreak date in walnut, and validated it using plant material from the Walnut Improvement Program of the University of California, Davis, demonstrating its effectiveness for marker-assisted selection in Persian walnut. We found several candidate genes involved in flowering events in walnut, including a gene related to heterodichogamy encoding a sugar catabolism enzyme and a cell division related gene linked to female flowering date.This study enhances knowledge of the genetic architecture of important agronomic traits related to male and female flowering processes and lateral bearing in walnut. The new marker available for budbreak date, one of the most important traits for good fruiting, will facilitate the selection and development of new walnut cultivars suitable for specific climates. Juglans regia L.) is one of the oldest food sources known [www.fao.org). At more than 22,000 ha, Persian walnut is the second leading tree crop in France, after apple. In the last 3 years, France has oscillated between 7th and 9th position for in-shell walnut production (circa 40 kt) [Persian walnut  . The abi.a 40 kt)a 40 kt) .Climate change, particularly global warming, is no longer to be proven within the scientific community , and res2 progeny in the United-States [Genetic control of phenology-related traits is fundamental for the development of new, resilient cultivars, able to adapt to changing climatic conditions. Many studies have focused on genetic dissection of phenological traits  in diverse fruit crops, such as peach, apricot and sweet cherry , 24. In d-States , but hasJ. regia 700 K SNP genotyping array [Release of the first walnut genome sequence  facilitang array . Applicang array  and wateng array , 34. A rng array .J. regia 700 K SNP array to genotype both a panel of 170 walnut accessions of diverse geographical provenience and an F1 progeny segregating for these traits. This study sought to identify candidate genes for both female and male flowering dates and to develop the first Kompetitive Allele Specific PCR (KASP) marker for phenology in walnut. This will be useful for walnut breeding programs in selecting of new resilient varieties to climate change.Here, we studied for the first time in walnut, the genetic control of budbreak date and female/male flowering dates, using the Axiom™ 1 mapping progeny of 78 individuals resulting from a bi-parental controlled cross between ‘Franquette’ (late flowering), and ‘UK 6–2’ (intermediate to early flowering). Both populations were maintained at the INRAE of Bordeaux field station and phenotyped during 2018 and 2019. For the GWAS panel, we also used previously collected (legacy) phenotypical data taken between 1989 and 2011.Two populations were used in this study: a GWAS panel of 170 diverse accessions of worldwide origin and an F1 progeny in 2019 exhibited a smaller range of 76 to 102 Julian days  than in 2018 .For the GWAS panel, the 2018–2019 data exhibited high variation in phenology-related traits, particularly for budbreak which ranged in 2019 from 57 Julian days for ‘Early Ehrhardt’ to 128 for ‘Fertignac’ (Feb 27th to May 9th) Figures  and S2. s Figure  and S4. 1 progeny . However, we found low values for male flowering duration (H2 = 0.22) and female flowering duration (H2 = 0.26) within GWAS panel (using the recent two phenotyping years), while no genetic effect was found for the F1 progeny. Therefore, we did not consider both male and female flowering durations in the GWAS and QTL mapping analyses.High broad-sense heritability values were observed for budbreak date, with Hta Table . OverallA total of 364,275 SNPs were retained after filtering for high resolution SNPs categories (Poly High Resolution and No Minor Homozygotes), for genotyping rate > 90%, and minor allele frequency > 5% Table . At K = PCA shows similar clustering of our germplasm collection as fastSTRUCTURE Figure . PC1, wh1 progeny  with the KING method. To identify first-degree relationships and differentiate “parent-offspring” from “full sibling” pairs, we used the estimates of k and the proportion of zero identical-by-state (IBS0) observed in the Fy Figure . In party Figure . Moreovep-value: 2.98E-14), and two additional associations are also found on Chr6  and Chr8 , according to the false discovery rate (FDR) threshold (≥ 0.05).For bearing habit, we found no influence of population structure , leading to an increased yield.The boxplots show the bearing habit phenotypes of 2019 for the different alleles of the three associated SNPs Fig. . For the1 progeny data. The allele T of this SNP is linked to a late budbreak date   on Chr1 was systematically found associated with all stages of female flowering , linked with a delayed budbreak date. Since we designed the KASP primers using the complementary strand, we expect to find the allele A (versus C) as involved in late leafing genotypes. The three different genotypes were clearly separated for 95 accessions, whereas one of them could not be determined . Then, we ran a pairwise comparison using Dunn’s test with Bonferroni p-value adjustment. Results of pairwise comparisons showed that A/A vs. C/A, and A/A vs. C/C have a significant effect on leafing date , contrary to C/A vs. C/C genotypes . The homozygous accessions A/A have a median leafing date of almost 110 Julian days, while genotypes C/A and C/C have a median leafing date of only 76 Julian days  were developed. These primers were used to genotype 96 unreleased breeding line accessions of the Walnut Improvement Program of the University of California, Davis. Among them, 48 are early leafing accessions and 48 are late leafing accessions. Leafing date occurs 3 days after budbreak date. Our GWAS results showed that the allele T of the SNP ‘AX-171179714’ (versus G) has an allelic estimated effect of 5.9  the loci identified in the GWAS likely are not segregating in the F1 progeny, and (ii) the QTLs found in the linkage mapping analysis have rare alleles in the association panel. For both GWAS results and QTLs mapping, the minor trait-loci associations were not stable across years, suggesting they are more affected by environmental conditions. For this reason, we decided to focus mostly on the GWAS results based on the two-year BLUPs. Nevertheless, results based on the legacy data allowed confirmation of the major SNP associated with budbreak date.While the major loci controlling the traits studied were identified with both GWAS and linkage mapping, this did not happen for the minor associations. For instance, for budbreak date, GWAS found a second trait-locus association on Chr2, while one minor QTL was detected on LG 6 of the ‘UK 6–2’ map. This finding can be explained in two ways:  [1 progeny, due to monomorphism in the parents, likely preventing accurate QTL detection. This is the case, for example, of the QTLs detected on LG 2 for both parental maps. By checking the physical position of the associated markers, we observed that the beginning of the Chr2 lacks regions from 0 to 5 Mbp for the ‘Franquette’ map, and from 0 to 7.7 Mbp for the ‘UK 6–2’ map.We observed a different scenario for all the stages of male flowering date: while the major QTL found on LG 1 co-localizes with those for female flowering dates and budbreak date, these results were not confirmed in the GWAS panel. Here, instead, we identified the most significant associations on chromosomes 11 and 4. Then, since heterodichogamy is the measurement of how the male and female flowering dates overlap, it is not surprising to find a marker-trait association on Chr11 with more consistency across years. Since the F effect) , we deciCombining multi-locus models for GWAS has become widespread , and theIn this work, classical QTL mapping mostly confirmed the major marker-trait associations identified. We found a major locus on Chr1 for budbreak date and female flowering dates, and a major locus on Chr11 for male flowering dates. Separating the flowering stages  allowed “repeat” phenotyping, generating robust data for identification of the major loci required for marker development purposes. Also, the high complexity of phenology-related traits indicates that many loci may be involved in their expression , making 2 progeny from ‘Chandler’, a lateral bearing cultivar heterozygous for this locus, the genetic basis of lateral fruitfulness was found to be located in the centromeric region of Chr11 [1 progeny from ‘Chandler’ by ‘Idaho’ cross [Lateral bearing is a crucial trait in walnut breeding since it contributes to increased yield . Fruitinof Chr11 . The samo’ cross . Additioo’ cross .1 progeny, this trait did not segregate and all hybrids are lateral bearing. ‘Franquette’ is terminal bearing and homozygous G/G for this locus, whereas ‘UK 6–2’ is lateral bearing and heterozygous G/C for this locus. Maybe because our progeny is too small or because of segregation distorsion, we did not observe any terminal bearing individuals.In the present study, we reconfirmed that walnut bearing habit is controlled by a major QTL in the centromeric region of Chr11. In line with previous studies, we observed that an allelic substitution at the most associated locus, ‘AX-171191765’, causes lateral bud fruiting. We confirm previous results regarding genetic control of lateral bearing by performing GWAS in genetically different plant materials, further supporting the power and high resolution of this method in walnut. In our Fchromosome transmission fidelity (ctf) protein 8 homolog gene as candidate gene for all female flowering dates. Previous studies in Arabidopsis thaliana suggest the involvement of the ctf genes family in the cell division processes [ctf7–1 and ctf7–2 genes resulted in developmental defects, including aberrations in flower morphology and male and female gametophytes. Interestingly, a TPX2 encoding gene required for spindle assembly during cell division process was also found likely to be involved in harvest date determination in walnut [ctf8 protein homolog locus is an excellent candidate gene for female flowering.Availability of the new chromosome-scale reference genome Chandler v2.0  allowed rocesses . In partn walnut . These fJ. regia, during staminate flower development, the tepals differentiate into anthers and filaments shortly before anthesis, enveloping the stamens [trichome birefringence-like 13 protein encoding gene within the LD block surrounding the SNP on Chr4 which is associated with male flowering dates. Also, the SNP on Chr11 associated with heterodichogamy and male flowering dates is in LD with a candidate gene encoding a probable trehalose-phosphate phosphatase D. This dephosphorylating enzyme and other members of the gene family are highly expressed in male flowers of Jatropha, a perennial woody plant, and in transgenic plants of A. thaliana with delayed flowering [probable trehalose-phosphate phosphatase D encoding gene for heterodichogamy.Trichomes consist of only one cell type, appearing as a long, slender appendage . In J. r stamens . Since tlowering , suggestlowering , 49. In A. thaliana, sweet cherry or other species, these mechanisms are regulated by flowering locus, early flowering, and MADS-box homologous genes, among others [agamous-like MADS-box protein AGL11 encoding gene (approx. 2.0 Mbp) and a MADS-box transcription factor 6-like encoding gene (8.8 Mbp), both at the beginning of the Chr1. The first one co-localizes with the major QTL found for budbreak date using the F1 progeny and the second is very near to the major marker-trait association found for female flowering dates (9.3 Mbp). Then, our results showed several marker-trait associations for both female and male flowering dates on Chr7 between 23.0 and 48.0 Mbp. Interestingly, we found the following encoding genes within this window: a flowering time control protein FCA-like (33 Mbp), a flowering-promoting factor 1-like protein 3 (34.4 Mbp), a dormancy-associated protein homolog 4 (42.9 Mbp), a protein early flowering 3-like (44.3 Mbp), a MADS-box protein SOC1-like (48.0 Mbp), and a protein flowering locus D-like (48.6 Mbp). Finally, we noticed that two genes are near to our marker-trait associations for the dichogamy (31.9 Mbp) and the male flowering dates (from 29.4 to 31.9 Mbp) on Chr11: a MADS-box transcription factor 27-like (29.9 Mbp) and a protein embryonic flower 1 (34.3 Mbp).Surprisingly, we did not find widely known genes to be involved in flowering process or dormancy in our candidate genes. In g others –52. For For the first time in walnut, a KASP marker related to phenology is released. By designing specific primers for the SNP AX-171179714, we found that the homozygous accessions A/A are significantly later leafing. Since this marker is dominant, it will greatly help breeders to accurately select individuals with delayed budbreak. However, due to the complex genetic basis of phenology-related traits in perennials , additio1 progeny is too small to pursue investigations but this study confirms that the INRAE walnut germplasm repository contains an array of plant material suitable for this type of work. New genome-wide analyses now are being initiated to further increase our knowledge concerning the genetic architecture of the main traits of interest in walnut.Due to the significant influence of environment on phenology-related traits in walnut, unravelling their genetic architecture is of upmost importance for the development of markers that could assist the selection of superior genotypes and, therefore, the release of new walnut cultivars adapted to different climatic conditions. Using GWAS with two different multi-locus models, we identified significant associations for budbreak date, and male and female flowering dates, confirmed by classical QTL mapping. In addition, we provide a list of candidate genes for these traits, that will be fundamental in future studies of functional genomics and understanding the metabolic pathways underlying phenology in walnut. We also developed and validated the first KASP marker for budbreak date in walnut, which will allow accurate selection of individuals with a delayed budbreak and, therefore, suitable for cultivation in France and other regions where late spring frosts are challenging. In parallel, the genetic bases for the expression of lateral bearing were confirmed. Since the future French walnut breeding program needs cultivars with high quality kernels, efforts are underway phenotype the entire collection regarding chemical content (e.g. fatty acids and tocopherols) and physical characteristics . Our FJ. regia grafted trees accessions originating worldwide and maintained at the Prunus and Juglans Genetic Resources Center located in the Fruit Experimental Unit of the INRAE, Toulenne, France (44°34′37.442″N – 0°16′51.48″W), near Bordeaux. This INRAE walnut germplasm collection is publicly available and is a result of important collecting work performed between 1988 and 2000 by Eric Germain  in 23 countries including the European, American, and Asian continents. Eric Germain initiated an international cooperation during his activity concerning the exchange of walnut plant materials  and obtained all permissions to bring them. Experimental research on this plant material complies with our INRAE institutional guidelines. The original source of each accession (institute/lab source or collecting source) is given . ‘UK 6–2’ has an early to intermediate budbreak date and lateral bearing. The progeny consists of 78 individuals, also located at the Fruit Experimental Unit of the INRAE in Toulenne, which were previously genotyped using the same SSR markers as those of the GWAS panel [The intraspecific FAS panel  to confi1 progeny, at a rate of two to three visits in orchards per week during the months of March, April and May: budbreak date, beginning, peak, end, and duration of female flowering, heterodichogamy, and beginning, peak, end, and duration of male flowering  for both the GWAS panel and the Fng Table . Heterodng Table  means th1 progeny, so only the GWAS panel was evaluated. Lateral bearing was found in recently released cultivars from France  and the USA , and in accessions coming from the Central Botanical Garden of Kiev, Ukraine  on three sites. These legacy data are available from the INRAE walnut germplasm collection database . Moreovene Table , suppose1 progeny, the means of genotypic effects were obtained for each accession by adjusting for known environmental factors using the BLUPs. When using two-year data, the means were predicted using a mixed linear model considering the year effect (a). When using phenological data from many years and three sites only available for the GWAS panel [For both the GWAS panel and the FAS panel , the meaPi(j)k refers to the observed phenotype of the kth accession in the ith year in the jth site; μ is the mean value of the trait; Yi is the fixed effect of the ith year, Sj is the fixed effect of the jth site, gk is the random effect of the k genotype; and ei(j)k is the residuals of the model. The BLUPs were performed using “R” package “lme4” [where e “lme4” .Based on the previous mixed linear models, broad-sense heritability of each trait was estimated using the variance components. When using two-year data, we accounted for the variance component of genotype × year interaction (a). When using phenological data from many years and three sites, we accounted for the variance components of genotype × year and genotype × site interactions (b):2G is the genotypic effect variance; σ2GxY is the genotype × year interaction variance; σ2GxS is the genotype × site interaction variance; σ2ε is the variance of residuals; ny is the number of years; and ns is the number of sites. We have always only one replication by genotype. For two-year data, ny = 2 (2018 and 2019). Nevertheless, the legacy data are very unbalanced for the years and sites available considering each genotype. We consider nyns = 5 because, in average, we have 5 observations by genotype.where σJ. regia accessions) and F1 progeny  were extracted from young leaves as described in Bernard et al. [J. regia 700 K SNP array containing 609,658 SNPs uniformly distributed over the 16 J. regia chromosomes [1 progeny were removed. After, Poly High Resolution (PHR) and No Minor Homozygotes (NMH) SNPs were filtered using stringent thresholds: SNP call rate (> 90%), minor allele frequency (MAF > 5%), and redundancy in the genome . Finally, 364,275 robust SNPs  were retained for the following genome-wide analysis.The genomic DNAs of both the GWAS panel . Single trait analysis was performed using a Multiple Interval Mapping (MIM) method [chr) was performed as follows:QTLs were determined using MultiQTL 2.6 software  method , in whicM is the total number of markers used for the QTL detection on each parental map, and m is the number of markers in the LG carrying the QTL [.where  the QTL .For the GWAS panel, the R packages “SNPRelate” and “gdsfmt”  were useGWAS was carried out using the R package “GAPIT” , accounthttps://www.ncbi.nlm.nih.gov/genome/annotation_euk/Juglans_regia/100/) and mapped onto the new chromosome-scale reference genome v2.0 [https://www.hardwoodgenomics.org/Genomeassembly/2539069).LD levels around the most associated loci were estimated using HaploView v4.2 software . Each phome v2.0   markers are based on the dual Fluorescence Resonance Energy Transfer (FRET) method, in which the sample DNA is amplified with allele specific primers conjugated to fluorometric dyes HEX and FAM at their 5′ end. Based on GWAS results regarding the budbreak date, the following KASP primers were developed  to target the most significantly associated SNP with this trait:Allele A: 5′-AGGACAGCAATAAACTCAATCACACA-3′.Allele C: 5′-GGACAGCAATAAACTCAATCACACC-3′.Allele A tail (FAM tail): 5′-GAAGGTGACCAAGTTCATGCT-3′.Allele C tail (HEX tail): 5′-GAAGGTCGGAGTCAACGGATT-3′.Common reverse: 5′-AGGTTCTGCCAAGCTACAGGGTATA-3′.https://www.biosearchtech.com/support/education/kasp-genotyping-reagents/how-does-kasp-work. The KASP reaction was prepared as follows: 1.95 μL of PCR mix PACE , 2 μL of genomic DNA (2 ng/μL), and 0.053 μL of the three primers , for a total volume reaction of 4.003 μL. Amplification was performed in a hydrocycler, starting with 15 min at 94 °C, a touchdown phase of 10 cycles at 94 °C for 20 s and at 61 °C for 60 s with a 0.6 °C decrease in temperature per cycle, followed by 35 cycles of 94 °C for 20 s and 55 °C for 60 s. Once the cycle was complete, Fluostar Omega reader  was used to read the fluorescence signal. The KASP method was tested using DNA from a set of 96 unreleased breeding line accessions from the Walnut Improvement Program of the University of California, Davis.These primers were developed by the BioGEVES laboratory  on the complementary strand. The KASP reaction and its components are explained at Additional file 1: Table S1. List of association panel accessions with their origin, breeding level, pedigree when known, and their membership to clusters based on fastSTRUCTURE results for K = 3.Additional file 2: Table S2. Number of principal components required for bearing habit trait using Bayesian Information Criterion.Additional file 3: Table S3. ‘Franquette’ and ‘UK 6–2’ parental maps for QTL detection.Additional file 4: Table S4. Genotyping results of SNP AX-171179714 by KASP.Additional file 5: Table S5. Walnut trait ontology.Additional file 6: Table S6. Phenotypic raw dataset of the GWAS panel.Additional file 7: Table S7. Phenotypic raw dataset of the F1 progeny.Additional file 8: Figure S1. Scatter plots showing the two-year data related to phenological traits in Julian days for the 170 accessions of the GWAS panel.Additional file 9: Figure S2. Density plots showing the two-year data related to phenological traits in Julian days for the 170 accessions of the GWAS panel.Additional file 10: Figure S3. Scatter plots showing the two-year data related to phenological traits in Julian days for the 78 accessions of the F1 progeny.Additional file 11: Figure S4. Density plots showing the two-year data related to phenological traits in Julian days for the 78 accessions of the F1 progeny.Additional file 12: Figure S5. Detection of the number of clusters using a) Prob(K), and b) deltaK method  in the GWAS panel.Additional file 13: Figure S6. Principal Component Analysis performed on the GWAS panel. The two first principal components show accessions colored according to fastSTRUCTURE results with EEAs for Eastern Europe and Asia, and WEAm for Western Europe and America.Additional file 14: Figure S7. Scatter plot showing the estimated kinship coefficients by the proportion of zero Identical-By-State (IBS0) in the F1 progeny.Additional file 15: Figure S8. Level plot showing the Identical-By-State (IBS) values for the 170 accessions of the GWAS panel.Additional file 16: Figure S9. Genetic maps and QTLs detected using two-year data. F(X) and U(X) are the linkage groups of ‘Franquette’ and ‘UK 6–2’ parental maps respectively. Legend of the QTLs: black for budbreak date, red for beginning female flowering date, deep green for full female flowering date, blue for end female flowering date, yellow for beginning male flowering date, pink for full female flowering date, and light green for end female flowering date. Solid bars indicate the 95% confidence interval of the QTL, and terminal bars indicate the 99.9% confidence interval of the QTL. The marker names were changed with the corresponding chromosome number and its physical position for a better visualization.


Misgurnus anguillicaudatus is a highly valued, aquaculture-relevant food fish in East Asian countries. In this study, the complete mitochondrial genome of Poyang loach was obtained by PCR. The genome is 16,646 bp in length, including 2 ribosomal RNA genes. 13 protein-coding genes, 22 transfer RNA genes, and a non-coding control region, the gene composition and order of the species were similar to most reported from other vertebrates. The phylogenetic tree showed that Misgurnus family got together for one branch, which includes Poyang M. anguillicaudatus, and the other loaches had their own branches.  Misgurnus anguillicaudatus , a small-sized freshwater fish species, is a highly valued, aquaculture-relevant food fish in East Asian countries. This loach species has also been given much attention as an important model organism to study developmental biology, polyploidy evolution and genetic breeding . All DNA was extracted using Phenol-Chloroform extraction method and stored at −80 °C. The mitogenome was amplified by primers, which were initially published  and eight tRNA genes  on the light strand (L strand). Eight PCGs  were terminated with a TAA stop codon and three PCGs  ended with TAG. On the other hand, remaining two PCGs (COIII and CYTB) ended with the incomplete stop codon represented as a single T. Frame overlapping occurred at three pairs of PCGs. ATP8 and ATP6 overlapped by 10 nucleotides (nt), ND4L and ND4 by 7 nt, and ND5 and ND6 (encoded on opposing stand) by 4 nt. Two ribosomal RNA genes, 12S rRNA (953 bp) and 16S rRNA (1679 bp) were located between PhetRNA and LeutRNA with separation by the ValtRNA as seen in other vertebrate mitogenomes.In this study, we sequenced the complete mitogenome of M. anguillicaudatus, we performed the phylogenetic relationship of Poyang M. anguillicaudatus stock with other natural populations in loach as inferred by entire mitogenome . The phylogenetic tree showed that Misgurnus family got together for one branch, which includes Poyang M. anguillicaudatus, and the other loaches had their own branches (M. anguillicaudatus was aligned by BLAST, compared with Cobitinae the sequence similarity could reach >95%, and the similarity to Misgurnus was >99%.To determine the taxonomic status of branches . Also th


Human DPSCs were isolated and cultured in osteogenic induction medium with or without RA or BMS 493, an inverse agonist of the pan-retinoic acid receptors (pan-RARs). Alkaline phosphatase (ALP) activity assays, alizarin red staining, quantitative calcium analysis, CCK8 assay, osteogenesis-related gene expression, and in vivo transplantation were conducted to determine the osteo/odontogenic differentiation potential and proliferation potential of DPSCs. We found that the expression of RARβ and CRABP2 decreased during crown calcification of DCs of miniature pigs. Activation of RA signal in vitro inhibited ALP activities and mineralization of human DPSCs and decreased the mRNA expression of ALP, osteocalcin, osteopontin, and a transcription factor, osterix. With BMS 493 treatment, the results were opposite. Interference in RA signal decreased the proliferation of DPSCs. In vivo transplantation experiments suggested that osteo/odontogenic differentiation potential of DPSCs was enhanced by inversing RA signal. Our results demonstrated that downregulation of RA signal promoted osteo/odontogenic differentiation of DPSCs and indicated a potential target pathway to improve tissue regeneration.Retinoic acid (RA) signal is involved in tooth development and osteogenic differentiation of mesenchymal stem cells (MSCs). Dental pulp stem cells (DPSCs) are one of the useful MSCs in tissue regeneration. However, the function of RA in osteo/odontogenic differentiation of DPSCs remains unclear. Here, we investigated the expression pattern of RA in miniature pig tooth germ and intervened in the RA signal during osteo/odontogenic differentiation of human DPSCs. Deciduous canine (DC) germs of miniature pigs were observed morphologically, and the expression patterns of RA were studied by  Retinoic acid (RA), the main active derivative of vitamin A, found in embryos and adult vertebrates , is esseAs mesenchymal stem cells (MSCs) have key roles in tissue engineering, their sources and the regulation of their differentiation mechanisms in tissue regeneration are active areas of research. Dental pulp stem cells (DPSCs) have been isolated from an adult dental pulp and are characterized by their high proliferation rate, self-renewal capability, and their potential to differentiate into osteoblasts, odontoblasts, adipocytes, etc. . CurrentIn this study, we used deciduous canines (DCs) of miniature pigs between late bell stage and calcification stage to study the expression pattern of RA in the dental papilla (DP) during crown calcification. Using human DPSCs, we investigated if RA had the presumed effects in osteo/odontogenic differentiation of human DPSCs. Our results revealed the negative effect of RA, both in crown calcification and in osteo/odontogenic differentiation of DPSCs, and we successfully improved the regeneration of bone-like tissue by inversing the RA signal, a novel method to promote the bone/dentin regeneration.Pregnant miniature pigs were obtained from the Animal Science Institute of Chinese Agriculture University. The gestation age was calculated from the day of insemination. Pregnancy was verified through B-type ultrasonography. All procedures acquired approvement from the Animal Care Use Committee of Capital Medical University  (Permit Number: AEEI-2016-063). Pregnant pigs were anesthetized and sacrificed as previously described . The DCsin situ hybridization were carried out as described previously [μg/mL proteinase K for 30 min at 37°C. After refixation with 4% paraformaldehyde- (PFA-) PBS, the sections were dehydrated in 25, 50, 75, and 100% ethanol successively before air-drying for 1 hour. Hybridization was performed overnight with diluted probes in an RNase-free incubator at 70°C. In the next day, the sections were rinsed for 3 to 4 hours and incubated with antibodies   overnight. Signal detection was performed with the NBT/BCIP substrate .RNA probe synthesis and nonradioactive eviously , 31. TheμM of ascorbic acid, 2 mM of β-glycerophosphate, 1.8 mM of KH2PO4, and 10 nM of dexamethasone. In the experimental groups, to activate the RA signal, RA  was used and, to inhibit RA signal, BMS 493 , an inverse agonist of pan-RARs, was used. For each experiment, RA and BMS 493 stock solutions were initially diluted in DMSO and then in culture medium for 10−7 M, 10−6 M, and 10−5 M according to a previous literature [Human tooth tissues were obtained from impacted third molars, under approved guidelines set by the Beijing Stomatological Hospital, Capital Medical University. All procedures were performed with informed consent from patients. The isolation and culture of DPSCs were performed as reported previously . DPSCs aterature , 19, 33.After osteogenic induction for 7 days, ALP activity assay was performed using an ALP activity kit following the instruction from the manufacturer's protocol (Sigma-Aldrich). Signal strength was normalized based on protein concentration. After 14 days of induction, mineralization was detected. The cultured DPSCs were fixed with 70% ethanol and stained in 2% Alizarin red (Sigma-Aldrich). To calculate their calcium contents, the above samples were destained with 10% cetylpyridinium chloride before measuring their absorbance at 562 nm on a multiplate reader. Their calcium contents were derived from a standard calcium curve, constructed using calcium dilutions of the same solution. In each group, the final calcium level was normalized to total protein concentration in duplicate plates.To analyze the effects of RA signal intervention on DPSC proliferation, cell proliferation assay was performed in 96-well plates using cell counting kit-8 . After osteogenic induction for 1, 2, 3, 4, and 5 days, 10% CCK8 reagent was added to each well and incubated at 37°C for 2 h before measuring the optical density (OD) values of the samples at 450 nm on a microplate reader. Cell proliferation capacities were represented by the OD values.μg of RNA, cDNA was synthesized with random hexamers or oligo (dT) and reverse transcriptase according to the manufacturer's protocol (Invitrogen). Real-time RT-PCR reactions were carried out using the QuantiTect SYBR Green PCR kit  and an iCycleriQ Multi-color Real-time RT-PCR Detection System. The primers used for the specific genes are shown in Total RNA was extracted from DPSCs using TRIzol reagent (Invitrogen). Using 2 The present study was performed in accordance with an approved protocol. Eight-week-old female BALB/c nude mice were maintained with free access to water and regular food. Human DPSCs were cultured at the presence or absence of BMS 493 for 3 days before combining with 40 mg of hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic particles. The mixture was then transplanted subcutaneously on the dorsal side of the nude mice. After 8 weeks, the transplants were harvested and fixed with 10% formalin for 48 h and decalcified in buffered 10% EDTA (pH 8.0) for a month prior to embedding in paraffin wax and sectioning.Sections were stained with hematoxylin and eosin (HE) to detect morphological changes in the DP of DC from miniature pigs and new bone formation in transplants. Masson's trichrome staining was applied to evaluate the collagen fibril deposits. Image-Pro Plus 6.0  was used for qualitative measurement of mineralization.2O2 for 10 min to quench the endogenous peroxidase. They were incubated with a primary antibody at 4°C overnight. The primary antibodies used here included those against dentin sialophosphoprotein (DSPP) , osteocalcin (OCN) , and collagen type I (COL-1) . In the next day, the sections were washed and incubated in secondary antibody at room temperature. DAB staining was performed with DAB Substrate Kit  and counterstaining with HE. Image-Pro Plus 6.0  was used for the qualitative measurement of mineralization.Immunohistochemistry staining was carried out as previously described . Brieflyt-test was performed to determine the statistical significance. P value ≤ 0.05 was considered to be significant.All statistical calculations were carried out using SPSS 13.0 statistical software. Student's RA receptors α (RARα) and β (RARβ) and cellular retinoic acid-binding proteins 1 and 2 (CRABP1 and CRABP2) in DCs from E50 to E60. We found that RARα was not expressed in the whole tooth germ of DCs from E50 to E60 and CRABP1 expression showed little difference between the two stages (data not shown), while the mRNA expressions of RARβ , osteopontin (OPN), and a related transcription factor osterix (OSX). Compared with the control group, there was significant reduction in the expression of the osteogenic markers  and OSX in RA-treated DPSCs on days 3, 7, 10, and 14 (Figures in vitro.DPSCs are dental stem cells isolated from the dental pulp, which is derived from the dental papilla, the mesenchymal compartment, during development. Based on our above finding, we raise a hypothesis that osteogenic potential of DPSCs could be controlled by RA signal, in addition to its involvement in mineralization of dentin during tooth development. To confirm this, DPSCs were cultured in osteogenic medium with or without RA supplement. First, ALP activity, an early marker for osteogenic differentiation, was analyzed. Experimental groups were treated with 10terature , 19. In re [−7 M  for furtre [−7 M  and quanre [−7 M  than in re [−7 M . Real-ti Figures . These r−7 M, 10−6 M, and 10−5 M of BMS 493, an inverse agonist of pan-RARs, were added in the culture medium to modulate the RA signal in DPSCs. With the addition of 10−7 M BMS 493, ALP activity increased after 7 days of culture (−7 M BMS (ALP, OCN, OPN, and OSX enhanced significantly in BMS 493-treated DPSCs, compared to the control group (Figures in vitro.To confirm further the role of RA signal in osteogenic differentiation of DPSCs, 10 culture , and mor culture  and high culture  were see−7 M BMS . Next, w Figures . Taken tin vivo, human DPSCs were treated with or without BMS 493 for 3 days before mixing them with HA/TCP ceramic particles and transplanting subcutaneously into nude mice. After eight weeks, the transplanted tissues were retrieved and decalcified. HE staining showed the formation of more bone-like tissue in BMS 493-treated DPSCs than in the control groups (Figures in vivo.To test whether the RA signal affects osteogenesis  Figures . In Mass Figures . Qualita Figures  and coll Figures  in BMS 4 Figures . Qualita Figures . Taken tβ expression is initiated during bell stage and is seen in odontoblasts [RARβ and CRABP2 in dental mesenchymal cells of miniature pigs during tooth development indicated the negative role of RA signal in odontogenic differentiation and dentin mineralization.Retinoic acid is an early signal in embryonic development and has a crucial role in the early stage of tooth development –12. Studtoblasts . The exptoblasts . CRABP2 toblasts . Miniatutoblasts . In the in vitro [in vitro and in vivo. The present results showed that BMS 493 enhanced the osteo/odontogenic differentiation potential in DPSCs in vitro and enhanced the regeneration of bone-like tissue in vivo. Although previous studies have shown the RA signal had a significant role in regulating the proliferation of different cell types [In recent years, DPSCs are proved to be a promising choice in dentin and bone regeneration. The ability to manipulate the stem cells accurately to a desired cell lineage remains a much pursued goal of research . Given tin vitro . Howeverll types , 43, intin vitro, we studied the expression of ALP, OPN, and OCN by real-time RT-PCR. Their synchronous decrease due to RA treatment indicated the negative effect of RA in osteogenesis of DPSCs in both early and late differentiation periods. OSX is a key transcription factor specifically expressed in developing bones [OSX was reported to be highly expressed in osteogenic mesenchyme and odontoblasts, and its overexpression in mouse odontoblast-like cells resulted in enhanced transcription of an odontogenic marker, DSPP [OSX due to RA and its enhancement due to BMS 493. These results suggested a role for OSX in regulating RA signal in osteogenic differentiation of DPSCs (in vivo [in vivo.Different stages of the osteo/odontogenic differentiation of MSCs are characterized by several markers. ALP participates in bone mineralization and is an early osteogenic differentiation marker , 45, whing bones  and enhang bones , 49. In er, DSPP . By realof DPSCs . Future (in vivo . The prein vitro induction time was mostly shorter than 7 days while osteogenesis is a long and dynamic process. In addition, a long-term in vitro culture of stem cells from periodontal ligaments and pulp of human exfoliated deciduous teeth showed improved osteogenic differentiation by RA [In contrast, some studies have suggested a positive role for the RA pathway in osteogenic differentiation of MSCs –56 and don by RA . Differein vitro and in vivo. The modulation of RA signaling has potential for improving the tissue regeneration and explains the mechanisms of osteogenesis and odontogenesis. The relationship between RA and its downstream signals needs to be studied in greater details to understand this process better.This study indicated that the RA signaling pathway was involved in dental crown calcification and demonstrated that activation of RA signal decreased the osteogenic differentiation potential of DPSCs and blocking RA signal enhanced it


The aim of the present study was to evaluate the association between the histology of active and chronic lesions and urinary protein and serum creatinine (SCr) levels, as common clinical endpoints in clinical trials for lupus nephritis (LN).In total, 119 patients diagnosed with LN class III, IV, and V, as defined by the International Society of Nephrology/Renal Pathology Society, between 1990 and 2015, were enrolled in the present study. Multiple regression analysis was performed to explore semi-quantitative histological variables associated with urinary protein and SCr levels.The mean age of the enrolled patients was 45 years, and 79% were female. The mean SCr and mean urinary protein levels at the time of renal biopsy were 0.87 mg/dl and 3.00 g/gCr, respectively. Class IV (71%) was the most common type of LN followed by class III (17%), and class V (13%). Multicollinearity was confirmed between monocellular infiltration (variance inflation factor [VIF] = 10.22) and interstitial fibrosis (VIF = 10.29), and between karyorrhexis (VIF = 4.14) and fibrinoid necrosis (VIF = 4.29). Fibrinoid necrosis and monocellular infiltration were subsequently excluded, and multiple regression analysis revealed that only the urinary protein level was correlated with wire loop lesions , and that the SCr level was correlated with glomerular sclerosis (β: 1.08 and CI: 0.43 to 1.74).As urinary protein and SCr levels were not quantitatively associated with active lesions, they may not accurately reflect the response to remission induction therapy in patients with LN. While clinical trials for promising therapeutic agents for lupus nephritis (LN), such as B cell targeted therapy, cytokine-targeted therapy (IL-6 and IFN-a), and cytotoxic T lymphocyte-associated antigen 4, have been conducted, none have shown improved outcomes compared with controls . These fProteinuria and serum creatinine (SCr) have been considered as promising predictors for renal prognosis in patients with LN –8. ConseThe main objective of this study was to explore the histology of active and chronic lesions, as well as their association with proteinuria and SCr level, in patients with LN.We retrospectively reviewed patients with LN at Okayama University Hospital. Data from 1990 to 2006 and 2007–2015 were collected from paper- and electronic-based records, respectively. Data collection was completed in 2016–2017. The enrolled patients fulfilled the 1997 American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus (SLE) . PatientThe following information was collected at the time of the renal biopsy, prior to treatment: age, sex, SLE disease activity index 2000, daily maximum dose of prednisolone, use of immunosuppressants, SCr and eGFR levels, urinary protein levels (g/gCr), hematuria (dipstick test > 2+ and > 5 erythrocytes per high power field), and active urine sediments. The eGFR was evaluated by the equation developed by the Japanese Society of Nephrology .For all participants, the histology of the first renal biopsy sample was classified according to the ISN/RPS classification by experienced nephrologists and/or pathologists. Active glomerular lesions were defined by the presence of endocapillary hypercellularity, leukocyte infiltration, subendothelial hyaline deposits, interstitial inflammation, karyorrhexis, fibrinoid necrosis, and cellular crescent formation. Active interstitial lesions were defined by monocellular infiltration. Chronic glomerular lesions were defined by glomerular sclerosis, fibrosis adhesion, and fibrous crescent formation, whereas chronic interstitial lesions were defined by interstitial fibrosis. Arteriosclerosis was also defined by chronic lesions. Each renal biopsy sample was processed using light and immunofluorescence microscopy with standard methods of fixation and staining. For semi-quantitative analysis, the histological score was calculated as described in our previous study , where hThe primary outcome measures were the urinary protein and SCr levels. The secondary outcome measure was the cumulative 10-year renal survival rates from the date of the renal biopsy. The renal endpoint was defined as > 40% decline in the eGFR.p < 0 .05 was considered to indicate a statistically significant difference. Complete case analyses were performed, excluding patients with missing clinical data at the time of the first biopsy. The descriptive statistics are expressed as the mean and standard deviation (SD) for continuous variables, and as n (%) for categorical variables. The cumulative renal survival rates were calculated using Kaplan-Meier analysis. We censored patients that did not reach the renal endpoint when they completed the 10-year follow-up or at the date of the last recorded visit until December 31, 2015. We calculated the number of patients at risk for reaching the endpoint from the date of the renal biopsy. Patients were grouped according to urinary protein levels and the eGFR, and survival rates were assessed using log-rank tests.Statistical analyses were performed using JMP® 14 software  and STATA v15 . All statistical tests were 2-sided. Subsequently, multiple linear regression (ordinary least squares regression) analysis was performed to explore whether the histological findings contributed to urinary protein and SCr levels. The primary dependent variables were urinary protein and SCr levels at the time of renal biopsy, which were recorded as continuous variables, and the candidate variable was the renal histological score. Urinary protein levels were log-transformed to fulfill the assumption of a normal distribution of the residuals. To address multicollinearity, which was assessed using the variance inflation factor (VIF) , 19, we 2, respectively, at the time of the first renal biopsy. The mean urinary protein level was 3.00 g/gCr. Forty-six (39%) patients were treated with prednisolone alone and the others were treated with concomitant immunosuppressants for remission induction. Renal histology revealed that class IV (71%) was the most common type, followed by class III (17%) and class V (13%). The mean (SD) scores of each histological lesion were as follows: endocapillary proliferation, 0.26 (0.30); karyorrhexis, 0.06 (0.12); fibrinoid necrosis, 0.08 (0.14); rupture of the glomerular basement membranes, 0.01 (0.03); extracapillary proliferation, 0.05 (0.10); wire loop lesions, 0.14 (0.27); hyaline deposits, 0.02 (0.06); membranous lesions, 0.11 (0.28); glomerular sclerosis, 0.11 (0.16); fibrous adhesions, 0.04 (0.07); and fibrous crescents, 0.01 (0.04). The proportions of monocellular infiltration, interstitial fibrosis, and arteriosclerosis with histological grade ≥ 2 were 43 (36%), 41 (34%), and 23 (19%), respectively. The Pearson’s correlation coefficient for each histological lesion is presented in Supplementary Table From a total of 158 patients with LN, 119 patients with ISN/RPS class III, IV, and/or V were enrolled in the present study after eliminating 11 patients with class I, II, or VI; 20 patients with a lack of clinical data; and eight patients who underwent a re-biopsy. Patient characteristics are shown in Table p = 0.37). Similarly, the renal survival rate did not differ among patients stratified by SCr levels .During the mean observation period of 1931 days, 11 (9.2%) patients experienced a > 40% decrease in eGFR. There was no significant difference in the renal 10-year survival rate among the patients divided into four categories of urinary protein levels (Fig. Multiple regression analysis was used to explore the explanatory variables related to urinary protein levels Table .Table 2Similarly to the analysis for urinary protein level, multiple regression analysis using all the histological variables was performed to explore the explanatory variables related to the SCr level Table .Table 3In this study, we explored the associations between histological findings and urinary protein and SCr levels at renal biopsy. The urinary protein level was reflected only in wire loop lesions, whereas the SCr level was only correlated with glomerular sclerosis.We could not confirm urinary protein and SCr levels as predictive factors for the renal outcome of patients with LN. As several previous reports showed that urinary protein and SCr levels are the main predictors for renal outcome –8, thereUrinary protein level mainly reflected wire loop, but not endocapillary or extracapillary, proliferative lesions in our study. Our previous report showed that only extracapillary proliferation was associated with poor renal predictors in active lesion . To the The SCr level was the only factor indicative of glomerular sclerosis in our study. This finding is supported by previous studies showing that the SCr level mainly reflects chronicity in LN , 17, 22,The present study had certain limitations. Firstly, this is a cross-sectional study performed at renal biopsy; therefore, we were unable to evaluate whether urinary protein and SCr levels directly reflect the treatment response of active lesions. However, we concluded that SCr was unlikely to recover to a normal range if glomerular sclerosis was present. Secondly, we did not evaluate treatment effects related to outcomes. Treatment may be adjusted according to not only histological findings, but also urinary protein and renal function. As our patients exhibited a better renal outcome than previous reports , we may The present study revealed that urinary protein and SCr levels did not quantitatively reflect active lesions in LN. Therefore, they may not be adequate biomarkers for measuring the response to remission induction therapy in patients with LN. Comparing the changes in candidate biomarkers and histological findings before and after treatment may aid the identification of potential biomarkers for monitoring the treatment response.Additional file 1 Supplementary Table 1. Correlation matrix of the histological findings. * p < 0.05. Supplementary Table 2. Multiple regression analysis of log-transformed urinary protein levels at renal biopsy and histological variables. Proteinuria was log-transformed to obtain a closer approximation to normal distribution. Covariates; Model 1: All independent explanatory variables; Model 2: Independent explanatory variables excluding fibrinoid necrosis and monocellular infiltration; Model 3: Independent explanatory variables excluding fibrinoid necrosis and interstitial fibrosis. 95% confidence intervals in brackets. * p < 0.05, ** p < 0.01, *** p < 0.001. Supplementary Table 3. Multiple regression analysis of serum creatinine levels at renal biopsy and histological scores. Covariates; Model 1: All independent explanatory variables; Model 2: Independent explanatory variables excluding fibrinoid necrosis and monocellular infiltration; Model 3: Independent explanatory variables excluding fibrinoid necrosis and interstitial fibrosis. 95% confidence intervals in brackets. * p < 0.05, ** p < 0.01, *** p < 0.001.


Actinobacillus pleuropneumonia (App), Mycoplasma hyopneumoniae (Mhyo) and swine influenza virus (SIV). A second objective was to estimate differences in antibody levels in pigs presenting pluck lesions. This was an observational study whereby pigs were managed as per routine farm practice. Data on sow parity, number of born alive (NBA) pigs per litter, cross-fostering status, birth and weaning body weight were recorded from 1016 pigs born from one farrowing batch. At slaughter, blood samples were collected for serological analysis and pigs were inspected for presence of enzootic pneumonia (EP)-like lesions, pleurisy, pericarditis and heart condemnations. Pigs were retrospectively classified into three production flows, depending on time spent in each production stage: flow 1 ; flow 2 ; and flow 3 . A nested case-control design was applied by matching pigs from each flow by sow parity, birth weight and NBA.Serological screening is a common method to monitor antibody response to pathogen exposure, but results could vary due to several factors. This study aimed to quantify animal and management related factors associated with variation in antibody levels in finisher pigs at slaughter, in an Irish farrow-to-finish farm endemically infected with P < 0.05) and there was no association between any of the pathogens investigated and other early life indicators (P > 0.05). Pigs in F1 had lower antibody levels for App but higher antibody levels for SIV than F2 and F3 pigs (P < 0.05). There was no association between pluck lesions and respiratory pathogens (P > 0.05), except for increased antibody levels for Mhyo when EP-like lesions were present (P = 0.006).Pigs born from primiparous sows had higher antibody levels for App than those born to parity ≥5 sows (Results indicate that offspring from primiparous sows develop higher antibody levels for App IV toxin when exposed to this disease and that enforcement of a strict all-in/all-out production system would reduce on-farm disease circulation. A high percentage of pigs were affected with EP-like lesions which were associated with higher antibody levels for Mhyo. Actinobacillus pleuropneumoniae (App), Mycoplasma hyopneumoniae (Mhyo) and swine influenza virus (SIV) are among the most important respiratory pathogens in pigs; they are agents involved in the porcine respiratory disease complex ; grower (18.1% CP and 10.0 MJ/DE per kg of feed) and finisher diets (16.9% CP and 9.9 MJ/DE per kg of feed). Pigs had ad libitum access to water via a nipple drinker for each 10–15 pigs.In brief, a total of 1016 pigs, born within one weekly farrowing batch, were followed through the different production stages. All pigs were individually tagged at birth and information on sow parity, number of piglets born alive per litter (NBA), sex and cross-fostering status (i.e. cross-fostered or not cross-fostered) was recorded. All pigs were weaned at approximately 28 days of age. At weaning, entire litters were transferred to the first nursery stage and housed in groups of 55 pigs, , 24 pigs died during the nursery stages (12.5% out of 192 pigs), 3 pigs died during the grower stage (1.5% out of 192 pigs) and 14 pigs died during the finisher stage (7.3% out of 192 pigs). The remaining 47 pigs (24.5% out of 192 pigs) were selected for euthanasia due to the presence of abnormalities such as external lesions, hernias, tail loss, severe lameness, external abscesses and emaciation. Eight-hundred-and-twenty-four pigs reached slaughter age and they were slaughtered within 1 week, regardless of body weight at 24 weeks of age, for the purpose of the experiment. Pigs were retrospectively classified into three production flows, depending on the time spent in each production stage: flow 1 = pigs that advanced through the normal production stages ‘in a timely manner’; Mycoplasma hyopneumoniae Antibody Test ; SIV – Influenza A Ab Test  which detects antibodies to nucleoprotein of SIV for serotypes H1N1, H1N2 and H3N2 for swine sera. Manufacturer’s instructions were strictly adhered to during analysis, with positive in-house and also positive and negative test-kit controls incorporated during the serodiagnostic testing.At slaughter, individual blood samples were obtained at exsanguination using labelled red-stopper sterile BD Vacutainer® blood collection tubes . All blood samples were individually labelled on collection, with corresponding sample delivery documents. Samples were transported to the Irish Department of Agriculture, Food and the Marine’s, Blood Testing Laboratory, Cork, for analysis. Blood samples were processed following clot formation, with serum aliquoted into individually labelled, anonymised cryovials  and stored at -80 °C until required for analysis. All serum samples were analysed by ELISA using commercial pathogen-specific ELISA kits  for the three respiratory pathogens of interest [App - ApxIV  Ab Test ; Mhyo – HerdChek® For each sample, the serostatus was determined for each pathogen of interest using the immunodiagnostic assay. Quantification of the antibody response was determined by colorimetric detection by spectrophotometry using TECAN Sunrise™ microplate reader  in conjunction with TECAN Magellan™ data analysis software v7.1 . Sample-to-positive (S/P) ratio values for App and Mhyo and sample-to-negative (S/N) ratio values for SIV were extrapolated from the optical density values obtained as per manufacturer’s instructions. Samples with sample-to-positive values ≥0.40 for Mhyo, ≥ 0.50 for APP and samples with sample-to-negative values SIV ≤0.60 were considered as positive as per the criteria given in the manufacturer’s instructions.At slaughter, lesions of the lungs and heart were visually scored by a single trained observer. The macroscopic enzootic pneumonia (EP) like lesions were scored according to severity using BPEX Pig Health Scheme  on a scaAll data were analysed in R v3.5.2 . Initialstats package. A Gamma distribution was fitted to each predicted variable. Univariable linear models were used with S/P or S/N ratio values as predicted variables with early-life indicators , production flow and pluck lesions  as predictor variables. For all analyses, alpha level for determination of significance and trends was 0.05 and 0.10, respectively. Results for fixed effects are reported as least square means (LSM) ± standard error (SE), and results for continuous variables are reported as the regression coefficient ± SE.Residuals of predicted variables were tested for normality using the Shapiro-Wilk test and by examining the normal test plot. Predicted variables were not normally distributed and thus, were analysed using generalised linear mixed models using the P values) for the univariable models between the different predictor variables and App and Mhyo S/P and SIV S/N ratio values are presented in Table P < 0.05) and there was no difference in App S/P ratio values for pigs born from other sow parity groups . Seroprevalence for App and SIV decreased as sow parity increased and 100% of samples were Mhyo positive.Mean S/P ratio values were 0.87 ± 0.361 range 0.02 to 1.63) for App, 2.04 ± 0.382 (range 0.53 to 2.8) for Mhyo and mean S/N ratio values were 0.36 ± 0.242 (range 0.06 to 1.09) for SIV. Associations (i.e.  to 1.63 P < 0.001) and SIV S/N (P = 0.002) ratio values compared with pigs in flow 2 and flow 3 . Calculated Mhyo S/P ratio values did not differ between production flows (P > 0.05). Seroprevalence for App increased with each subsequent production flow; in contrast, SIV seroprevalence decreased with each subsequent production flow. The overall calculated APPI score for the studied batch of pigs was 0.50 and it increased from 0.28 for flow 1, 0.45 in flow 2 to 0.98 in flow 3.Pigs in flow 1 had lower App S/P ( P = 0.00 ratio vaP = 0.006). There were no differences in App and Mhyo S/P or SIV S/N ratio values between pigs presenting pericarditis, heart condemnations and pleurisy compared with pigs not having such lesions at slaughter (P > 0.05).Pigs presenting EP-like lesions had higher Mhyo S/P ratio values compared with pigs not presenting EP-like lesions  for disease classifications instead of antibody levels, our results are similar to those reported by Sibila et al.  and Arseo et al.  reportedo et al.  reportedo et al. , consequo et al. ).Actinobacillus pleuropneumonia circulates in the herd for long periods of time [Production flow was associated with antibody levels for App and SIV, confirming the relationship between management practices and pig health. Pigs in flow 2 and flow 3 shared air space (i.e. flow 2) or were re-mixed (i.e. flow 3) with pigs that had returned from the hospital facilities having recovered from illness and/or injury . During  of time , with an of time . Therefo of time  that cou of time . Similar of time , the low of time . This hi of time . Finally of time , or rein of time .Although the result of higher antibody levels for Mhyo in pigs with presence of EP-lesions was expected, there was a high percentage of pigs (42.1%) presenting lesions despite the farm vaccinating piglets against Mhyo. However, vaccinating for Mhyo is reported to reduce clinical signs, and decrease infection levels , 45; andWe hypothesised that mean antibody levels would be higher for pigs presenting pluck lesions at slaughter; this was only observed between EP-like lesions and Mhyo antibody levels. The lack of the expected association between other pluck lesions and antibody levels in this study may be explained by the amount of time required for the development of a macroscopic lung lesion, resulting in the absence of the lesions at slaughter. Other possibilities include the ability of the lesion to resolve, or to the fact that pluck lesions are multifactorial and multiple pathogens can cause them .Finally, when interpreting the results, it is worth noting/considering that results represent finding from only one farrow-to-finish farm and a small sample size. Management and vaccination practices, sow parity distribution and on-farm prevalence of respiratory pathogen vary between farms and thus, results would likely differ if we were to include more farms and/or sample more pigs. However, the merit of this study is the fact that there is scarce information available in the scientific literature about animal and herd management factors, serological response to respiratory pathogens and pluck lesions at an individual animal level; most studies report such relationships on a farm level. Also, it seems that although a high proportion of Irish farmers claimed to practice strict AIAO during the grower-finisher period, they mix older pigs younger pigs in the different production stages . Also, aSow parity and production flow were associated with variation in antibody levels for the investigated respiratory pathogens. Offspring from first parity sows had more elevated App antibody levels, indicating the importance of adequate gilt management programs due to the high seropositivity detected. The inclusion of an App vaccine would assist with improved passive immunity and increased immune development of their progeny. The differences observed between the production flows in the detected antibody levels for App and SIV reflect moving pigs in a timely manner is beneficial to reduce on-farm disease circulation. Enforcement of a meticulous AIAO production system would minimise adverse effects of respiratory disease on pig performance and health. Under the conditions of this study, only an association between Mhyo and presence of EP-like lesions was observed. Furthermore, although the farm vaccinated piglets against Mhyo, extension of the programme to include the sows and gilts may be beneficial to controlling the infection spread within the herd.


Pain-related fear-avoidance (FA) is a common problem affecting many patients with painful medical conditions. As there is great interest in the clinical importance of the relationship between FA and disability, several questionnaires have been developed to measure FA. The Fear-Avoidance Components Scale (FACS) is a recently developed patient-reported instrument that addresses critical issues not previously considered in previous FA-related questionnaires. The original English version of the FACS demonstrated good reliability, internal consistency, and construct, criterion, and predictive validity. Two factors were determined: General Fear Avoidance and Types of Activities That are Avoided. The aim of this study was to to translate the FACS into European-style Spanish (FACS-Sp), and validate its psychometric properties.This two-stage psychometric study included 330 subjects with various chronic musculoskeletal pain disorders. An initial translation and cross-cultural adaptation of the FACS, from English to Spanish, was performed. Then, critical psychometric properties were analysed, including internal consistency by Cronbach’s α coefficients, structural validity from the Maximum Likelihood Extraction (MLE), and convergent validity by Pearson correlation with the Central Sensitization Inventory (CSI).This study reports for the first time the psychometric properties of the Spanish version of the FACS. Total scores ranged from 0 to 88 points, with a mean of 30.49 (±17.18). The FACS-Sp showed a high internal consistency for factor 1 (α = 0.902) and factor 2 (α = 0.88). Factor structure was two-dimensional and supported structural validity, accounting for 48.75% of the total variance. Convergent validity analysis found a significant Pearson correlation r = 0.414.This study reports for the first time the psychometric properties of the Spanish version of the FACS-Sp. Psychometric properties supported the validation of FACS-Sp and ensured the conceptual equivalence with the original English version. In primary care and chronic pain rehabilitation, FA assessment is crucial for clinical decision-making and treatment guidance. The FACS-Sp offers a new measure of FA in Spanish speaking populations. Future research on the FACS-Sp should evaluate test-retest reliability, treatment responsiveness and psychometric comparisons with other translated versions. Primary care has traditionally functioned within a biomedical model, which only considers the physical components of illness, and has tended to disregard psychosocial components . One of As there is great interest in the clinical importance of the relationship between pain-related FA and disability, several questionnaires have been developed to measure FA . Patientr = .90–.94) and internal consistency (Cronbach α = .92). The original English version, and other translated versions of the FACS, are available at https://www.pridedallas.com/questionnaires/.Recently, the Fear-Avoidance Components Scale (FACS) was developed, which incorporates important components of previous FA-related measures, and includes components of the FA model not previously considered in the earlier-developed questionnaires, within a framework of the most current FA model of Vlaeyen , 16. TheIt is estimated that 14% of Europeans speak Spanish, which is about 50 million people . SpanishThis cross-sectional study was conducted in two stages. First, an initial translation and cross-cultural adaptation of the FACS, from English to Spanish, was performed. Secondly, for evaluation of the FACS-Sp’s critical psychometric properties, patient volunteers from a physical therapy outpatient clinic were used.Psychometric properties were assessed according to the “COnsensus-based Standards for the Selection of health status Measurement INstruments” COSMIN guidelines . ReliabiAn English–to-Spanish translation was carried out to ensure conceptual equivalence of all of the test items, while maintaining appropriate Spanish cultural linguistic qualities. For this purpose, a direct- and reverse-translation methodology was utilized, with the help of a specialist in the field, as recommended in the literature . For thiA total of 330 volunteers with Chronic Musculoskeletal Pain Disorders (CMPDs) were recruited consecutively from the community-based Physiotherapy Program at the Malaga University. Demographic variables of the subject sample can be found in Table The FACS contains 20 separate items that are scored from 0 (“completely disagree”) to 5 (“completely agree”), with a total possible score of 100. Five severity levels are available for clinical interpretation: subclinical (0–20), mild (21–40), moderate (41–60), severe (61–80), and extreme (81–100) .The CSI-Sp assesses 25 health-related symptoms common to Central Sensitization Syndromes (CSS). It contains 25 items that are scored from 0 (“never”) to 4 , with a total possible score of 100. The CSI-Sp has high internal consistency (α = 0.872) and test-retest reliability (r = 0.91) .Normality of the distribution of data was determined by one-sample Kolmogorov-Smirnov test (significance < 0.05) for descriptive variables. Means and standard deviations of anthropometric variables were extracted for descriptive analyses.Factor structure was calculated from the Maximum Likelihood Extraction (MLE) by varimax rotation. Requirements for factor extraction as proposed in the literature , includeConvergent validity was calculated with Pearson correlation coefficients by P values  with the CSI-Sp. Along with FA, Central Sensitization is often present in individuals with CMPDs –32. TherInternal consistency was obtained by Cronbach’s α coefficients at an anticipated value range of 0.80–0.95 , 34 for All statistical analyses were conducted using the Statistical Package for Social Science version 21.0 (SPSS 21.0) for Windows. Ethical clearance was approved by the Tribunal of Review of Human Subjects at the University of Malaga.The final Spanish version of FACS is shown in Additional file No missing responses were found in the data collection. FACS-Sp scores ranged from 0 to 88 points, with a mean of 30.49 ±17.18). FACS severity level subgroups are displayed in Table 7.18. FACp < 0.001). Maximum Likelihood Extraction detected four components with Eigenvalues above 1, explaining 39.68, 11.57, 7.25, and 5.12% of the variance, respectively. The last two factors, however, accounted for less than 10% of the total variance, so they did not completed requirements for factor extraction, according to established methodology. Hence, a two factor solution was extracted according to the established criteria, explaining 48.75% of total variance. Table p < 0.000) after analysis.It was determined that the correlation matrix was adequate for the Maximum Likelihood Extraction from the results observed in Kaiser–Meyer–Oklin values (0.900) and the Bartlett’s test of sphericity  in the total sample of 315 participants.The Pearson correlation for the FACS and CSI questionnaires was r = 0.414  in factor #1 and α = 0.808 (ICC = 0.775–0.837) in factor #2.The present study completed a cultural adaptation and validation of the FACS questionnaire to Spanish, resulting in a FACS-Sp version. In a first step, the translation process followed established guidelines explained in the literature, following a recommended direct- and reverse-translation- and back-translation methodology . This prThe two-factor solution that emerged in the factor analysis accounted for 48.75% of the total variance. The English  and SerbActivity Avoidance and Harm, as maintained in a 11-items Spanish version [Activity Avoidance and Pathological Somatic Focus) in 17-items English version [Other FA-related questionnaires have shown several factors. FABQ contains two dimensions: While FA beliefs about work explain a 43.7% of the total variance, FA beliefs related to physical activity explain 16.5% . The Spa version . However version , 4 facto version , and 2 d version , which pRegarding items loading from the present version items related to cognitive and affective pain tended to load higher on factor 1, while items related to reasons of avoidance loaded higher on factor 2. In the English version of FACS, factor 1 was composed of items 1–14, and factor 2 was composed by items 15–20; while item 6 was cross-loaded in both factors but finally included in factor 1 . In the As expected, the FACS was positively correlated with the CSI, although this correlation was not strong r = 0.414). This finding was expected as both questionnaires measure different construct. While the FACS measure FA, the CSI measures symptoms present in Central Sensitization Syndromes. However, the CSI was likely to converge (positive correlation) because both symptom dimensions often appear in CMPDs –32 and o4. This fr = .464; p < 0.001) in patients with chronic spinal pain [r = .518; p < 0.001), but lower and not significant with the TSK questionnaire  [The convergent validity of CSI has previously been studied with questionnaires measuring FA, such as the PCS, showing similar results  . HoweverThe FACS-Sp showed a high internal consistency illustrated by α = 0.902 for factor 1 and 0.808 for factor #2. Similar results were found in the Serbian version, with higher internal consistency in factor 1 (α =0.904) than 2 (α = 0.880) The original version of FACS showed similar results for the entire questionnaire (α = 0.92), but separate results for each of the two factors were not reported .. This hAccording to the biopsychosocial model of chronic pain, psychological factors, like FA, contribute to the onset and progression of both pain and disability  and playAlthough avoidance behaviors may be adaptive in the context of acute pain, long-term avoidance can impair daily functioning and lead to physical disability . As it iApproximately 55% of our sample was suffering from LBP. Evidence suggests that fear avoidance beliefs are prognostic for poor outcomes in subacute LBP, so early treatment, including interventions to reduce fear avoidance beliefs, may avoid delayed recovery and chronicity . A recenOne of the strengths of the present study was the good psychometric results of the FACS-Sp, supporting previous research on the original English  and SerbAs a limitation, there was a lack of longitudinal data, so it did not include test-retest reliability, responsiveness and error scores or provide information on minimal clinically important difference values.This study reports for the first time the psychometric properties of the Spanish version of the FACS (FACS-Sp). The reliability, in terms of internal consistency, and structural validity were comparable to the original English version , ensurinAdditional file 1.


Considering the inconsistent available findings regarding the cardioprotective effect of dietary fatty acid composition, we prospectively examined the feasible association between the dietary fatty acids and the cardiovascular disease (CVD) incidence in framework of the population-based Tehran Lipid and Glucose Study.A total of 2369 participants  without CVD at baseline (2006–2008) were included and followed-up for 6.7 years. Fatty acids’ dietary intake was estimated using a 168-item semi-quantitative food frequency questionnaire. The CVD incidence risk across tertiles of dietary fatty acids was predicted via Cox proportional hazards regression models.2 at baseline. Over 6.7 years of follow-up, 79 cases of CVD were detected. The risk of CVD was lower in upper tertile of monounsaturated fatty acids, oleic acid, and docosahexaenoic acid + eicosapentaenoic acid among the tertiles. No significant associations were found between total fat, saturated and polyunsaturated fatty acids’ intake, and CVD.The average age and body mass index of the included population were 38.5 ± 13.3 years and 26.6 ± 4.8 kg/mOur results suggest that the dietary fatty acid composition might affect the incidence risk of CVD within the Iranian population. Cardiovascular diseases (CVD), which carry high psychological and medical impact as well as economic burden, has emerged as the leading factor of global morbidity and mortality, claiming 17.3 million lives each year . It’s beSeveral studies have previously investigated the association between dietary fatty acids and CVDs, most of which reported controversial results. In this regard, polyunsaturated (PUFA) and monounsaturated (MUFA) fatty acids-enriched dietary patterns were associated with lower CVD mortality and morbidity , 7. SomeThe observed inconsistency can be attributed to participants’ characteristics, which has resulted in unclear conclusions in population-based studies. The importance of addressing the CVD dietary-related risk factors calls for a full clarification and firm conclusion on this matter. Also, the global dietary guidelines developed by the international health institutions require reconsideration in light of these findings; meanwhile, there should be clear recommendations in these guidelines. Therefore, the current study aimed to assess whether dietary fatty acid composition can predict future CVD events.This study discussed the fatty acid composition in association with the CVD incidence risk in the prospective Tehran Lipid and Glucose Study (TLGS) framework , 12. Then = 626), with suspected energy consumption  (n = 579), CVD history at baseline  (n = 90), and those with missing data or loss to follow-up (n = 14), a total of 2369 adults (1030 men and 1339 women) were recruited.Figure P = 0.001).During the follow-up, the response rates for in-person visits and annual telephone call follow-ups were ranging from 55 to 75% and 80 to 90%, respectively. The participants were 19 to 70 years, with a mean age of 58.4 ± 9.7 and 37.4 ± 12.8 for those with CVD and no CVD, respectively  at baseline and were not following any specific diets. Participants who have completed the FFQ shared similar baseline characteristics with the total population of phase three of the TLGS.Demographic information was collected through interviews with participants via standardized questionnaires. The interviews were conducted by a group of trained interviewers. Variable measurements of the TLGS were described in detail elsewhere . The antThe required demographic, dietary, biochemical, and anthropometric data were evaluated at the baseline. The typical dietary habit of the participants was evaluated by a validated 168-item semi-quantitative FFQ in the primary examination . The relThe consumption frequency of certain food items was designated from the participants on a daily, weekly, or monthly basis by highly experienced dietitians . The IraBlood samples were collected at baseline and follow-up after an overnight fasting. The levels of triglyceride (TG) and fasting serum glucose (FSG) were measured by enzymatic colorimetric using phosphate oxidase and glucose oxidase, respectively. Similarly, the post-precipitated apolipoprotein B-containing lipoproteins along with phosphotungstic acid, were used to designate the HDL-c. The analyses were conducted by a Selectra 2 auto-analyzer  and the Pars Azmoon kits .The risk score of CVD was calculated by the “general CVD” algorithms specified for each gender. This algorithm took the TC, HDL-C, age, SBP, hypertension medication, smoking, and diabetes into account . The defAnnual telephone call follow-ups were used to collect information regarding possible medical events by a physician or a trained nurse. Additional data were retrieved from medical records. An adjudication committee consisting of a physician, an internist, an epidemiologist, a cardiologist, and an endocrinologist would review the obtained data, and the final diagnosis was confirmed by a predefined coding protocol. Other experts were invited to the committee as needed .CVD was primarily described as any history of coronary heart disease (CHD) or definite fatal CHD, definite fatal and non- fatal stroke, or CVD mortality . CHD terTaken together, in the present study, CVD definition was based on any history of CHD or definite fatal CHD, definite fatal and non- fatal stroke, or CVD mortality. Since the main purpose of the current study was to address the risk of CVD outcomes related to the dietary fat composition at the end of the follow-up period, it worth mentioning that none of the participants had any history of CVD-related outcomes at the baseline.p-value < 0.05 were considered as statistically significant. To compare the frequency and mean of the baseline characteristics across tertiles of total fat intake, univariate analysis, and chi-square tests were used. The significant differences in the frequency (%) of qualitative variables were detected by the chi-square test. Correspondingly, the CVD incidence score, total dietary fiber, and energy intake were considered as the cofounding variables of this one-way analysis (PE < 0.2). Due to the highly significant value of the physical activity factor (PE > 0.2), it was not ultimately selected for the final multivariable model.Data analyses were performed by IBM SPSS version 20.0  and To compute the 95% confidence intervals (CIs), and the Hazard ratios (HRs), the Cox proportional hazards regression model was used. The potentially confounding variables were adjusted in three models to estimate the HRs of CVD incidence risk across the tertiles of total fat, SFA, MUFA, PUFA, oleic acid, omega 6/omega 3 (ω-6/ω-3) ratio, and eicosapentaenoic acid + docosahexaenoic acid (EPA + DHA). In the first model, no adjustment was applied for the variables. For the CVD risk score, the second model was adjusted for TC, type 2 diabetes, smoking, age, HDL-c status, and hypertension treatment, and the calculations relied on the sex-specific “general CVD” algorithms . Subsequ2, respectively. Within a follow-up period of 6.7 ± 1.4 years, 79 cases of CVD events were detected, and angiographic proven CVD (40.4%), definite MI (24.6%), unstable angina (12.2%), and stroke (8.8%) were the most common. Approximately 30.1, 10.6, and 9.6% of the total energy consumption were contributed to dietary fat (76.2 g), MUFA (26.2 g), and oleic acid (23.6 g), respectively. Almost 90% of dietary MUFA was driven from oleic acid resources.In the current study, 2369 participants (1030 men and 1384 women) were recruited. At baseline, the mean age and BMI of the participants were 38.5 ± 12.7 years and 26.6 ± 4.8 kg/mP = 0.001) and hypertension  tended to be more prevalent among those diagnosed with CVD-related outcomes. Also, at baseline, the CVD patients appeared older and were more likely to have higher BMI, WC, FPG, TG, and blood pressure levels as well as CVD risk score (P < 0.05). Also, no significant difference was observed concerning the physical activity and the proportion of the inactive individuals between the two groups.The distribution of CVD common risk factors among patients diagnosed with CVD and those free of CVD at baseline (2006–2008) is described in Table Baseline characteristics and dietary intake of TLGS studied population after the follow-up and across tertiles of total fat intake are described in Table P = 0.030) and SFA  were inversely associated with the incidence of CVD events in the crude models; nevertheless, after adjusting for potential confounders, no association was observed. In contrast, the consumption of dietary MUFA resources was negatively associated with the incidence of CVD .The HR of the CVD incidence (95% CIs) across the tertiles of dietary fat constitutions is described in Table P = 0.016) was higher against the lowest tertiles. Despite so, the association between greater load of EPA + DHA and decreased risk of CVD incidence was insignificant . Nevertheless, no statistically significant association was detected between the dietary PUFA and omega 6/ omega 3 intakes and the risk of CVD.The risk of developing CVD events in the highest adjusted model of dietary MUFA and oleic acid  , 35, 47.The potential lipid-heart mechanisms can be attributed to the reduced production of inflammatory and reactive oxygen species by PUFA , inactivThe current study had some limitations and strengths. The major strengths included having a population-based and prospective design, high follow-up rate and duration, obtaining detailed information on the common risk factors and potential confounders, and using a validated FFQ to collect dietary intake data. The main Limitations was the limited control of the potential residual confounders, unavailability of MUFA components other than oleic acid, and unexplained and unmeasured confounders (i.e. genetic and serum-free fatty acids levels). Also, due to the potential modifications in the routine dietary habits and CVD risk factors of the participants over the study duration, certain misclassifications were unavoidable, which could have led to biased HRs towards the null. However, this issue is not far from expectations in the typical prospective studies. It worth noting that the present study was performed on a population with a relatively low incidence of CVD outcomes to make a definitive and firm conclusion. Therefore, further investigations on populations with higher rates of CVD events are required to obtain more accurate and profound results. Although the low number of CVD cases probably has affected the ultimate results, it is yet comparable to other Iranian populations .Dietary interventions are the primary strategies to prevent CVDs. In conclusion, this prospective study demonstrated that the fatty acid composition of different food sources influences CVDs incidence rate. A diet rich in MUFA, oleic acid, and EPA + DHA significantly reduced the risk of CVDs in the current study, whereas such association was not found for total fat, SFA, and PUFA. Caution should be taken when generalizing the results to populations with higher rates of CVDs, yet the findings can be generalized to other populations with identical dietary patterns. Since the incidence of CVDs is on rise worldwide, these investigations reveal the need for studies with larger samples and more extended follow-up periods.