In this study, we analyzed the effects of breed, diet energy source, and their interaction on adipose tissue transcriptome in growing Iberian and Duroc pigs. The study comprised 29 Iberian and 19 Duroc males, which were kept under identical management conditions except the nutritional treatment. Two isoenergetic diets were used with 6% high oleic sunflower oil (HO) or carbohydrates (CH) as energy sources. All animals were slaughtered after 47 days of treatment at an average live weight of 51.2 kg. Twelve animals from each breed (six fed each diet) were employed for ham subcutaneous adipose tissue RNA-Seq analysis. The data analysis was performed using two different bioinformatic pipelines. We detected 837 and 1456 differentially expressed genes (DEGs) according to breed, depending on the pipeline. Due to the strong effect of breed on transcriptome, the effect of the diet was separately evaluated in the two breeds. We identified 207 and 57 DEGs depending on diet in Iberian and Duroc pigs, respectively. A joint analysis of both effects allowed the detection of some breed–diet interactions on transcriptome, which were inferred from RNA-Seq and quantitative PCR data. The functional analysis showed the enrichment of functions related to growth and tissue development, inflammatory response, immune cell trafficking, and carbohydrate and lipid metabolism, and allowed the identification of potential regulators. The results indicate different effects of diet on adipose tissue gene expression between breeds, affecting relevant biological pathways. Most traits of interest for meat producers have a multifactorial background, with meat attributes being shaped by several genetic and environmental factors and their interactions. Within the environmental factors, feeding is the most important one in animal production. Nutrition influences animal body and tissue composition, and may be employed to improve carcass leanness, nutritional value of meat, as well as overall production efficiency .In general, nutritional and genetics approaches, both in applied and scientific fields, have followed separate paths, ignoring how genome–nutrition interactions affect physiological and metabolic processes with important phenotypic consequences. From a metabolic perspective, quantitative and qualitative properties of the diet components have important regulatory effects on muscle and lipid metabolism and influence gene expression . In thisPork, along with poultry, is one of the most consumed meat worldwide. Nowadays, modern pig breeding is essentially based on highly-selected genotypes from lean breeds, which are managed in the framework of intensive production systems focused on the production of fresh pork meat. However, in the Mediterranean area these selected breeds coexist with local breeds, with Iberian pig being the most representative one. The Iberian breed has important commercial value because of its use for the production of high quality dry-cured products  and is cIberian pig production is based on both purebred Iberian and crossbred Duroc × Iberian pigs. These two genotypes show important phenotypic differences in growth, fattening, tissue composition, muscle differentiation, and several metabolic processes ,10. SpecIn a previous work , in ordeThe present work employed the same experimental material as the quoted previous study , with thAll experiments were performed in accordance with the regulations of the Spanish Policy for Protection of Animals employed in Research and other scientific purposes RD53/2013, which meet the European Union Directive 2010/63/EU on the protection of animals used in experimentation. The project was approved on March 20, 2015, by the Comunidad de Madrid animal welfare and protection committee (reference number PROEX-007/15).2 pig−1), with a concrete floor and straw bedding. Temperature was controlled at a mean of 23.8 °C throughout the experiment. At 10 weeks of age (SD = 1.6 days), the animals were distributed in two experimental groups and fed two different isocaloric and isoproteic diets  provided for ad libitum consumption and differing in the energy source: HO diet contained 6% high oleic sunflower oil (17 Iberian and 10 Duroc pigs), and CH diet was formulated to contain carbohydrates as energy source (13 Iberian and 9 Duroc pigs). Feed composition is shown in The current study was carried out at the facilities of the Instituto Tecnológico Agrario de Castilla y Leon (ITACYL) Pig Test Center . The study comprised a total of 29 Iberian Torbiscal and 19 Duroc males born in 19 contemporary litters, which started the experiment at 19.9 kg (standard deviation (SD) = 3.8 kg) average live weight (LW). These animals were kept under identical management conditions, housed in batches of 4 pigs/pen . Total RNA was isolated from 50–100-mg samples of subcutaneous ham fat using the RiboPureTM RNA isolation kit , following the manufacturer’s recommendations. The obtained RNA was quantified using NanoDrop equipment , and the RNA quality was assessed with an Agilent 2100 bioanalyzer device  and submitted to the Centro Nacional de Análisis Genómico . Libraries were prepared using the TruSeq mRNA-Seq sample preparation kit  according to the manufacturer’s protocol. Each library was paired-end sequenced (2 × 75bp) by using TruSeq SBS Kit v3-HS in a HiSeq2000 platform .FastQC  was usedIn order to confirm that the mapping had been carried out correctly, a quality control was performed in all samples using two tools, Samstats  and QualFor the first employed pipeline, transcripts were assembled using Cufflinks (v2.2.1.), following the protocol proposed by the developer and including the Cufflinks, Cuffmerge, and Cuffdiff steps ; transcrq-value ≤ 0.05 for the breed effect and q-value ≤ 0.1 for the diet effect and FC ≥ 1.5. This software supports more complex experimental designs in addition to simple two-group setups. With DESeq2 software, RNA-Seq read counts were modeled by generalized linear models, including the breed and diet effects, the diet effect within each breed, and with a full model including breed, diet, and the breed–diet interaction effects.Differential expression analyses were also carried out in parallel with an R package, after raw counts for the genes and transcripts were obtained with HTSeq-counts . DESeq2 p-values < 0.05 were considered statistically significant.RNA obtained from the 24 animals used in the RNA-Seq assay was used to perform the technical validation of the differential expression of 11 genes that were either affected by the breed or the diet within each breed. This technical validation was performed by studying the Pearson correlation between the expression values obtained from RNA-Seq data (FPKM) and the normalized gene expression data obtained by RT-qPCR. To validate the global RNA-Seq results, the concordance correlation coefficient (CCC)  was calcPCK1, PLIN2, IGFBP3, JAZF1, PDLIM3, PYGM, RBP7, ASB2, EEF1A2, SERPINE1, and CYP1A1 was quantified employing the method previously described. Primer pairs were designed using Primer Select software  from the available GENBANK or ENSEMBL sequences. Primer pairs covered different exons to assure the amplification of the cDNA. Information on primer sequences, efficiency, and amplicon lengths are indicated in GAPDH, ACTB, TBP, 18S, PPIA, and B2M were selected for data normalization. The stability of the endogenous genes was tested with the Genorm and the Normfinder software [ACTB and PPIA genes were selected as endogenous genes.The expression of the genes software ,26. Thuswww.qiagen.com/ingenuity) software was used to identify and characterize biological functions, gene ontologies, canonical pathways, and regulatory networks affected by the DE genes. The IPA Canonical Pathways Analysis identified in its library the pathways that were most significant in our dataset. The significance of the association between the dataset and the canonical pathway was measured with Fischer’s exact test. IPA software transforms a set of genes into a number of relevant networks based on comprehensive records maintained in the Ingenuity Pathways Knowledge Base. Networks are presented as graphs depicting the biological relationships between genes. Complementarily, IPA software has a tool used to identify and characterize potential regulators . This tool identifies known regulators, including genes and other molecules that may affect the expression of DE genes. The analysis of upstream regulators considers every possible transcription factor and upstream regulator contained in the Ingenuity Knowledge Base repository, as well as their predicted effects on gene expression. Then, this tool analyzes whether the patterns of expression observed in the DEGs can be explained by the activation or inhibition of any of these regulators through the calculation of a z-score, a statistical measure of the match between the expected relationship direction between the regulator and its targets, and the observed gene expression [Ingenuity Pathway Analysis  transcriptome of 24 animals was characterized with RNA-Seq in order to study the effects of breed, diet, and breed–diet interaction. An average of approximately 50 million sequence reads was obtained for each individual sample and was assembled and mapped to the annotated Sscrofa11.1 genome. All samples passed the quality control and 91–93% of the reads were mapped to the porcine reference sequence. An average of 19,181 genes out of 22,452 annotated genes were expressed in the studied samples. Regarding mapping quality values (MAPQ), an average of 96% of the reads showed MAPQ ≥ 30 in the different samples .The differential expression analysis was performed using two pipelines, a standard protocol proposed in a previous study , involvin = 48). Genes for validation were selected from the lists of DEGs affected by the breed, diet, and interaction effects obtained in the different pipelines. For the technical validation we calculated the Pearson correlation values between RNA-Seq and qPCR data, which showed significant results in all cases . Moreover, the CCC coefficient, used to assess technical validation in high throughput transcriptomic studies, was calculated and a value of 0.81 was obtained, denoting a substantial general concordance between RNA-Seq and qPCR expression values [n = 48). Regarding the breed effects, six out of seven DEGs detected with RNA-Seq had confirmed significance in the qPCR analysis. For the diet effect, all four selected DEGs affected by diet in Iberian pigs according to RNA-Seq were also significant after qPCR analysis. Moreover, three additional genes were detected as significantly affected by diet in Iberian pigs by qPCR, which were not initially detected with RNA-Seq . For Duroc pigs, five DEGs depending on diet were selected for validation, and in this case three of these DEGs were confirmed by qPCR, although one of them only showed suggestive significance value . The detection of additional DEGs in the qPCR step may be indicative of a higher precision of qPCR with respect to RNA-Seq, but mainly reflects the results of a larger sampling . The interaction effects breed–diet were confirmed for six genes, twice more than those selected from RNA-Seq data. Out of these, four showed quantitative interactions  and two genes (JAZF1 and SERPINE1) showed qualitative interactions, with opposite diet effects in both breeds. For JAZF1 gene we detected higher expression in CH group in Iberian pigs, but in HO group in Duroc pigs. For SERPINE1, in contrast, we observed higher gene expression in HO diet in Iberian pigs, but in CH diet in Duroc pigs.In order to validate the results obtained from the RNA-Seq analysis, the relative expression of 11 genes was assessed by qPCR in all available samples . DESeq2 detected as many as 1435 DEGs after the application of the same significance and magnitude thresholds . This soCLCA1  and PVALB . CLCA1 gene may act as an innate immune signaling molecule that activates macrophages, and thereby enhances pro-inflammatory cytokine release  and has an impact on the early innate immune response in mice [PVALB) is a member of the EF-hand superfamily  expressed in vertebrates in a tissue- and cell-specific manner, serving as a magnesium/calcium buffer. EF-hand proteins are involved in a variety of physiological processes, including cell-cycle regulation, second messenger production, muscle contraction, microtubule organization, and vision [Cufflinks detected 337 genes upregulated in Iberian pigs, with FC ranging from 1.5 to 45, and 500 DEGs upregulated in Duroc with FC ranging from 1.5 to 271. The genes showing the largest expression differences between breeds were  in mice . Parvalbd vision .LEP, PCK1, RXRG, IGBP3, RBP7, ME1, FADS2, or PLIN2. Higher expression of LEP (FC = 2.13) in Iberian AT is in agreement with previous findings [LEP gene is expected in animals with increased fat deposition, as has been observed in the fatty Iberian pig [PCK1) (FC = 1.81) is also an interesting candidate gene due to its involvement in lipid and carbohydrate metabolism. It is one of the main regulatory enzymes of gluconeogenesis and glyceroneogenesis [PCK1 c.2456A, associated with higher enzyme activity and better meat quality, is almost fixed in Iberian pigs [PCK1 activity due to the polymorphism, Iberians are characterized by higher PCK1 expression, supporting further increases in fat deposition in this breed, in agreement with the phenotype. Malic enzyme (ME1) gene is an essential gene directly involved in de novo lipogenesis that encodes a cytosolic enzyme that generates NADPH (Dihydronicotinamide-adenine dinucleotide phosphate) for fatty acid biosynthesis [ME1 gene (FC = 1.5) in Iberian versus Duroc pigs is in agreement with our previous results obtained by qPCR in ham subcutaneous fat samples [As expected, genes upregulated in Iberian pigs included several known biological candidate genes involved in lipid metabolism and energy homeostasis, such as P FC = 2. in Iberifindings . Leptin findings . Obesityfindings , thus, aP FC = 2. in Iberirian pig . Cytosologenesis , providiogenesis . Allele ian pigs  and is fynthesis . Higher P FC = 2. in Iberi samples .IGF2 (FC = 2.67) gene, which encodes for a member of the insulin family of polypeptide growth factors that involved in development and growth. It is a paternally imprinted gene, associated with fat deposition, muscle growth, and heart size. A causal mutation in IGF2 intron3 (g.3072G>A) has been detected in pigs [IGF2 g.3072G allele having strong adipogenic effects at the subcutaneous AT level. This mutation shows different alleles in the two analyzed breeds—a high frequency of the mutant IGF2 g.3072A allele is observed in Duroc breed, while it is almost absent in Iberian pigs [IGF2 expression [IGF2 in Duroc genotypes is in agreement with previous evidence and with the presence of alternative alleles in both analyzed breeds.Among the genes upregulated in Duroc we found the  in pigs ,44, whician pigs ,45. The p-value < 0.01) in the dataset of 837 DEGs   . Out of LXR/RXR activation, which is involved in the regulation of lipid metabolism, inflammation, and cholesterol metabolism [p53 signaling, which plays an important role in the coordination of the cellular response to different types of stress, including oxidative stress [For instance, pathways enriched in Iberian pigs included tabolism , glutathtabolism , or p53 e stress . In agreOn the other hand, calcium signaling and actin cytoskeleton signaling pathways were activated in Duroc pigs. Calcium signaling influences signal transduction in cells, thereby activating cellular growth and development , and actp-value < 0.05; The upstream analysis and regulator effects tools of the IPA package were employed to identify potential transcriptional regulators that may explain the differential patterns of expression observed between breeds, and allowed the identification of 739 regulators , mainly related to inflammatory response, such as KDM51, DNMT3A, AHR, NR1H3, NOS, or RBPJ, and lipid metabolism, such as AHR, NR1H3, or MED1. Especially NR1H3, also known as LXRA, is a transcription factor that modulates immune and inflammatory responses in macrophages and is a regulator of macrophage inflammatory signaling . Also, ip-value < 0.01, z-score > 2, In contrast, 23 upstream regulators related to cell proliferation, differentiation, and growth were activated in Duroc pigs , which is normal in the cereal-based concentrates commonly used for growing pigs.It is interesting to note that our results suggest metabolic alterations related to fattening, such as adipose tissue inflammation and insulin resistance, starting to develop in young growing Iberian animals. This early process may seem surprising, but there is evidence in humans demonstrating that the initiating events in obesity-induced inflammation can occur in all developmental stages, including early in infancy, childhood, and adolescence . In fact < 0.001 ). MoreovIGF2, MYCN, FMOD, FOSB, RGS7, AKT1, PHGBH, or FOS, and is in accordance with the usual phenotypic growth differences between Iberian and Duroc breeds, as explained in previous work [In contrast, in the Duroc breed we observed the enrichment of functional categories, pathways, and regulatory networks involved in the size of the animal or mass of the organism, both related to the GO category “organismal development”. This result is associated with the overexpression in Duroc of several genes and regulators with a known role in development, such as ous work ,30. InteCAMK2A and CAMK2B) are also implicated, regulating their co-localization at the plasma membrane of adipocytes [CAMK2A gene  and other genes implicated in calcium signaling pathways, such as CACNA1S, CACNG1, CANG6, and CACNB1, were also upregulated in Duroc pigs.In Duroc pigs, several biological functions, pathways, and regulators involved in the organization of the cytoskeleton and the extracellular matrix (ECM) were enriched. In AT, adipocytes are embedded in the ECM, which provides structural support and anchorage for cells and is composed of the same proteins found in other tissue types . The enripocytes . In agreThe ECM composition is related to development stage, viability, and subtype of the adipocytes . At earlCYP1A1, ADGRF2, and TMEM182) were common to both methods in Duroc pigs. Due to the scarce significant findings obtained with DESeq2 software, and taking into account that diet effects on gene expression detected in the Cufflinks pipeline were mostly successfully validated by qPCR, we decided to employ the Cufflinks output in order to be able to perform a functional interpretation of the diet effects on transcriptome. Thus, 207 DEGs affected by diet in Iberian pigs and 57 in Duroc pigs were employed for functional analysis. We have to consider that the performed intra-breed analysis of the diet effect implies that a lower biological replication was employed , meaning that the analysis may be considered less consistent than that of the breed effect and would benefit from additional analysis with a higher number of subjects, as employed in the validation step.Considering the strong effect of breed on transcriptome, and the fact that both breeds showed the quoted difference in feed intake, the effect of the diet was evaluated separately in the two breeds. DESeq2 and Cufflinks identified 49 and 207 DEGs according to diet in Iberian pigs, and 5 and 57 DEGs according to diet in Duroc pigs, respectively. Out of the DEGs affected by diet in Iberian pigs, 23 were common to both employed methods , while op-value < 0.01; p-value = 2 × 10−8), which is an important contributor to the low-grade chronic inflammatory status associated with AT metabolic alterations in obesity and metabolic disorders [p-values = 0.0002 and 0.003, respectively). Several other relevant enriched pathways were related to FA metabolism (acetyl-CoA biosynthesis I) or corticoid metabolism, such as mineralocorticoid biosynthesis, glucocorticoid biosynthesis, aldosterone signaling, androgen biosynthesis, or glucocorticoid receptor signaling .In Iberian pigs, 124 DEGs were upregulated in HO diet and 83 in CH diet . Functioisorders . This paCorticoid metabolism is related to adiposity and inflammation  and cortRegarding gene ontology enrichment, we detected that the enriched biological functions were involved in immune cell trafficking, inflammation, lipid, carbohydrate, and energy metabolism, and AT development . Many fuAlso, functional categories related to lipid and glucose metabolism and AT development were affected by the DEGs, including terms such as quantity of adipose tissue, mass of fat pad, insulin resistance, oxidation of long chain fatty acid, fatty acid metabolism, energy homeostasis, quantity of carbohydrate, quantity of glycogen, or synthesis of lipid . ResultsCCL14, CCL24, CCL26), and pro-inflammatory proteins , which were DE. For instance, SERPINE1 gene was 1.61-fold upregulated in HO. It is a pro-coagulant protein, expression of which is increased in AT of obese animals and is used as a marker of AT inflammation [SERPINE1 as one master regulator for the expression differences obtained, and a causal network was constructed that relates this gene to other 30 regulators and 62 DE genes  is the receptor for colony stimulating factor 1, a cytokine which controls the production, differentiation, and function of macrophages [Also, rophages . Severalrophages ,91. ThesROS biological function was also activated in HO diet.Also, some works demonstrated that inflammasome activation depends on reactive oxygen species (ROS), another inflammation-associated group of compounds, which are produced extensively by the activated phagocytes . CirculaCYP1A1 or PDK4. CYP1A1 gene encodes a member of the cytochrome P450 superfamily of monooxygenases, which convert saturated and unsaturated FAs into small molecules [In relation to FA metabolism biological functions activated in HO diet, the up-regulated genes were mainly related to lipid and cholesterol metabolism, glycolysis, and gluconeogenesis, such as olecules . PDK4 prolecules . Thus, up-value < 0.05) upstream regulators in Iberian pigs depending on diet  in CH diet related to cell differentiation, proliferation, and development, such as MAPK1. In HO diet, 5 upstream regulators were significantly activated , some of them with roles in immune and inflammatory response, such as TGFB1 or IFNL1 [The upstream analysis allowed the identification of 687 : protein kinase A signaling, nNos signaling in neurons, and calcium signaling (p < 0.05). Other enriched functions were mainly related to immune cell trafficking , lipid metabolism, small molecule biochemistry, and vitamin and mineral metabolism  , with none of them being significantly activated or inhibited  . The upsnhibited . Thus, fSERPINE1 gene was up-regulated in HO diet in Iberian and in CH diet in Duroc pigs. These results are indicative of an interaction between the diet and the breed effects.Out of 207 DEGs affected by diet in Iberian pigs  and 57 iJAZF1 and two novel genes located in chromosomes 15 and 13, with strong homologies to human TTN and XIRP1 genes, respectively. Juxtaposed with another zinc finger, gene 1 (JAZF1) is a newly identified gene that is associated with various types of cancer and diabetes mellitus [JAZF1 gene reduced lipid synthesis and increased lipolysis by regulating the level of expression of genes related to fat metabolism in mice [In order to deepen the potential interaction effects unraveled from Cufflinks outputs, we used DESeq2 tool to perform a joint analysis using a full model fitting the breed–diet interaction. This analysis yielded three genes with significant interaction effects (FDR < 0.05): mellitus ,100. Rec in mice  and beca in mice . Therefo in mice . The RNASERPINE1), which is probably one of the most extreme cases of qualitative interaction, does not have a significant interaction q-value according to DESeq2 output. In fact, two genes showed significant nominal p-values in the interaction output  but did not reach the FDR threshold, namely SERPINE1 and CYP1A1, and were included in the qPCR validation step. Significant interaction effects were technically and biologically validated for these two genes with the more precise qPCR quantification and with a wider sampling . These findings suggest that available statistical tools for analyzing interaction effects are prone to false negative results, or the multiple testing correction is too demanding. Also, we have to consider that the magnitude of the expression changes which leads to biological consequences is unknown. Taking these considerations into account, we tried a less demanding threshold to employ a list of genes with suggestive interaction effects for functional interpretation. We considered the upper part of the list of genes obtained from DESeq2 output, ordered by significance, assuming as the threshold the nominal significance of CYP1A1 gene interaction effect . This list includes 49 transcripts corresponding to 44 known genes  and organismal survival or death , with half of the analyzed molecules being involved in these functions. Also, the network analysis showed that these genes were involved in metabolic disease, tissue abnormalities, and cellular assembly and organization. Although interaction effects are not clear and are difficult to interpret, joint results may agree with the differential fattening of the two breeds, leading to a clear inflammatory response being observed in Iberian pigs, which in turn is being developed with a different paucity in the two dietary groups. The between-breeds opposite regulation that diet exerts on the expression of some genes is intriguing and warrants further research with longitudinal time-series experiments and a more comprehensive study of tissue development and inflammation. Breed–diet interactions have been unveiled at the phenotypic level [Interestingly, the gene showing opposite diet effects in the two breeds (wn genes . Approxiic level ,105,106 ic level ,107,108.We found a deep effect of breed on adipose tissue transcriptome. Most differences in transcriptome between Iberian and Duroc breeds were related to growth, extracellular matrix formation, lipid and carbohydrate metabolism, and inflammatory and immune response. Results suggest low-grade inflammation and insulin resistance being developed in the obese Iberian animals in spite of the young age of the animals.On the other hand, diet and interaction effects indicate a more intense response to diet in Iberian pigs, which is essentially translated into changes in genes involved in inflammation, immune response, lipid metabolism, and fattening. The higher transcriptional response observed in Iberian pigs may be associated with structural variants increasing sensitivity of gene promoters to nutrient stimulation, for instance affecting glucose response elements located within regulatory regions. Future studies should address the structural variation in these genomic regions, as well as the expression of long non-coding RNAs, which could provide insight into other regulatory mechanisms supporting the breed and diet influences on transcriptome. More extensive research on the basis of breed and diet effects, as well as diet-breed interactions, may contribute to deepening the understanding of adipocyte cell biology, lipid, and glucose metabolism differences between this obese Iberian phenotype and lean genetic types.


Saturation mutagenesis was performed over six residues delimiting the substrate binding pocket of a fungal laccase previously engineered in the lab. Mutant libraries were screened using sinapic acid as a model substrate, and those mutants presenting increased activity were selected for exploring the oxidation of lignin-derived phenols. The latter comprised a battery of phenolic compounds of interest due to their use as redox mediators or precursors of added-value products and their biological activity. The new laccase variants were investigated in a multi-screening assay and the structural determinants, at both the substrate and the protein level, for the oxidation of the different phenols are discussed. Laccase activity greatly varied only by changing one or two residues of the enzyme pocket. Our results suggest that once the redox potential threshold is surpassed, the contribution of the residues of the enzymatic pocket for substrate recognition and binding strongly influence the overall rate of the catalytic reaction. Laccases participate in processes of polymerization, such as lignin formation in plants, cuticle sclerotization in insects, and sporulation and pigmentation in bacteria and fungi; and depolymerization, mainly lignin oxidation by fungi and bacteria [2O2-producing enzymes, cellobiose dehydrogenase, etc.) and oxidized species of low-molecular weight compounds . Although white-rot basidiomycetes produce the laccases with the highest redox potential in nature, with up to +0.8 V , this is still insufficient to oxidize certain components of lignin or other substrates of higher redox potential. However, laccases’ oxidative capabilities can be extended thanks to the presence of redox mediator compounds that, once oxidized by the enzyme, can in turn oxidize a recalcitrant substrate on which laccases cannot act on their own. In addition, mediators may act as electron shuttles, diffusing far away from the laccase active site and oxidizing complex compounds that cannot fit into the substrate binding pocket [Laccases are blue-multicopper oxidases that are widely distributed in nature. They primarily catalyze the oxidation of substituted phenols and aromatic amines, among other aromatic compounds, coupled to the reduction of Obacteria . Wood-roDue to their broad substrate range and low requirements, the use of laccases and laccase-mediator systems (LMS) is of great interest for different processes using lignocellulose as renewable raw material, including pretreatment and detoxification of straw materials for the production of second generation bioethanol, biobleaching of fibers in paper and textile industries, deinking of recycled paper or functionalization of lignocellulosic polymers ,5,6,7,8.p-hydroxycinnamic acids  [Although many LMS applications have been described, these typically rely on synthetic mediators such as ABTS , HBT (1-hydroxybenzotriazole) or violuric acid, which, apart from being expensive, may generate toxic species, hampering their utilization at an industrial scale. For this reason, the search for natural laccase mediators has become increasingly appealing. Indeed, we have described the ability of certain phenolic compounds derived from lignin to act as redox mediators in several works ,16,17, wc acids) ,19,20,21Pycnoporus cinnabarinus (PcL), by means of directed molecular evolution in Saccharomyces cerevisiae [etc. [Another bottleneck limiting the industrial use of laccases is the need of high amounts of enzyme, which also needs to be active and stable under the harsh operational conditions. This has encouraged numerous protein engineering efforts in order to heterologously express laccases in different hosts and to obtain tailor-made biocatalysts presenting the desired properties . In prevrevisiae ,24. The revisiae . One of ae [etc. ,26,27,28P. cinnabarinus and basidiomycete PM1 belong to (data not shown). Laccase mutants studied in this work come from the saturation mutagenesis of six residues of the substrate binding pocket of a high-redox potential laccase (3A4) previously engineered in the lab by recombination of PcL and PM1-L . In part14 in Three mutant libraries were constructed, each for the combinatorial mutagenesis of two positions: library A, for residues 162 and 164; library B, for residues 263 and 264; and library C, for residues 390 and 392. At least 3066 clones from each library were screened, in order to explore all possible codon-pair combinations with 95% library coverage. Mutant libraries were submitted to a high-throughput screening (HTS) assay based on the activity towards sinapic acid 14 in , a lignivs. 48% for activity)  . Due to the mutagenesis strategy used for library construction, the mutations introduced were not expected to significantly affect laccase secretion levels by yeast  of 5 °C. For this reason, it was removed from the final evolved variant [Concerning the first point, one of the key principles of directed evolution is that “you get what you screen for” . For the variant ,32. In spara, including carboxyl (free or as methyl esters), ketone, aldehyde and propenoic groups. Besides these substitutions, they can hold different substituents in ortho respecting the hydroxyl: one or two hydroxyl groups, one or two methoxyl groups , or none (p-hydroxyphenyl or H-type). In addition, three phenolic compounds of renowned interest  were also evaluated. All these substrates are of plant origin and can be obtained as plant extracts or released during degradation of the lignin polymer either by physicochemical or enzymatic treatment of lignocellulose.We selected a battery of 28 compounds as substrates to study their oxidation by chosen laccase mutants produced in flask, following a systematic approach in order to evaluate the effect of the different chemical structures on their feasible oxidation by laccase . These cortho favor phenol oxidation by laccase, being the o-di-substituted phenols more easily oxidized than the mono- and non-substituted phenols . In fact, 4-hydroxybenzoic acid (6) was not oxidized at all in the assayed conditions. This is due to the fact that the hydroxyl and methoxyl substituents in ortho act as electron donating groups in the aromatic ring (making the π system more nucleophilic) and generally lower the substrate’s redox potential, thus allowing an easier oxidation by laccase. Furthermore, the parent laccase seemed to oxidize phenols with o-methoxyl substituents better than with hydroxyl substituents, even though hydroxyls are more activating substituents of the aromatic ring. The same has been described for laccases from Trametes villosa and Lentinula edodes [kcat/KM) for guaiacol (3) than for catechol (2). Similarly, laccase from Gaeumannomyces graminis var. tritici presented higher affinity (lower KM) for guaiacol over catechol and for 2,6-dimethoxyphenol (5) over pyrogallol (4) [Changes in the absorption spectra of the different phenols in the presence of parent 3A4 laccase along 24 h are shown in a edodes ,34, whicllol (4) . This hillol (4) , but alsllol (4) .para position, a general tendency  was found based on how rapidly the changes in the UV-Vis spectra were observed. The reactivity decreased in the following order: phenols without p-substituent > methyl p-hydroxycinnamates > p-hydroxycinnamic acids > p-hydroxybenzoic acids > methyl p-hydroxybenzoates > p-hydroxyketones > p-hydroxyaldehydes. This correlates well with the fact that all the p-substituents tested are moderately deactivating, except for the propenoic chain of cinnamic acids and cinnamic esthers due to the presence of the double bond. In fact, during fungal degradation of the lignin polymer, the oxidation of the side chains of the phenylpropane lignin units (Cα-Cβ cleavage) takes place in the initial stages, giving rise to vanillic and syringic acid as main degradation products from G- and S-type units, respectively [On the other hand, respecting the effect of the groups/side chains in ectively .Abs) observed for the parent type at the given wavelength was around 50% of the maximum ΔAbs. Relative activity for each clone respecting parent type was calculated according to the formula:In order to compare the activity of the different laccase mutants, an appropriate wavelength was chosen for each substrate. When changes in the absorption spectra were observed in more than one peak, increase in the visible wavelength range was selected, since these could be useful for developing future colorimetric high-throughput screening (HTS) assays. To detect differences between clones, time points were selected so that the change in absorbance  formed a distinct cluster, separate from the rest of substrates tested. The utmost enhancement of laccase activity on 14 and 25 is expected because laccase mutants were selected based on their activity towards sinapic acid, and methyl sinapate is the most structurally similar compound used in the screening. o-Monosubstituted phenols, on the other hand, were more evenly represented in the different clusters. It is worth noting that vanillin related compounds  clustered at the far left side of the map, with laccase mutants presenting even worse activity for these substrates than towards the theoretically more difficult to oxidize H-type phenols (except for 6). Interestingly, greater increases in activity were found for substrates with hydroxyl substitutions in ortho than for those with methoxyl substitutions (except for 7 and 8), even though parent laccase was more efficient oxidizing the latter. The mutations introduced in the enzyme binding pocket are not expected to significantly affect the laccases’ redox potential, since none of the target residues coordinate the T1 Cu . For this reason, the observed differences between mutants are most probably caused by different affinities. In fact, it is possible that not all substrates were used in saturating concentrations in the screening assay and, consequently, oxidation rates would be affected by the mutants’ affinity towards the different compounds.Results for relative activities were represented in a heat map and clustered according to substrate (columns) and laccase mutant (rows) . o-Di-suortho  and another group that did not . Finally, Cluster 3 was formed by four clones presenting the highest increases in activity towards several compounds (lower part of the map). These included three mutants from library C and one from library A. They showed enhanced oxidation over all o-di-substituted phenols tested and several o-monosubstituted phenols (including eugenol) and, interestingly, also increased activity on resveratrol.Respecting laccase mutants, roughly three main clusters were formed. Cluster 1 was formed by clones that performed worse than parent type laccase towards most substrates (upper part of the map). Except for the case of clone C9, mutants grouping in this cluster are those that presented lower activity towards ABTS in the culture supernatant a, meanino-di-substituted phenols but to a lesser extent, also maintained parent Pro390. The mutations found in these clones revealed that while position 392 is quite flexible, accommodating variable residues, position 390 is more restrictive. As for A2 mutant, substitutions A162K and T164I were found. Remarkably, clone A2 was significantly better at oxidizing o-monosubstituted phenols  than di-substituted phenols, in comparison to clones from library C. The same occurred for clones A1 (A162K T164V) and A4 (A162R). This suggests that the substitution of Ala162 for basic residues Lys or Arg could be beneficial for the oxidation of this type of compounds. Interestingly, several studies on laccase structure–function relationships hypothesize that a nonpolar residue in position 162 is important to establish correct hydrophobic interactions with the phenolic substrate [ortho. This highlights the often-difficult prediction of amino acid substitutions to enhance enzyme activity by rational design, and by contrast, the potential of directed evolution and random approaches to reveal unexpected mutations that might provide the solution to a problem.Analysis of DNA sequences revealed that all clones from library C mutated Phe392 for other residues. In particular, clones belonging to Cluster 3, namely C7, C1 and C6, mutated Phe392 for Asn, Thr and Gln, respectively, but maintained Pro390 from parent type. It is worth noting that the best mutants from cluster 2a, C8 (F392S) and C10 (F392V), which also presented better activity towards ubstrate . HoweverE. coli strain DH5α in order to obtain sufficient copies for DNA sequencing and for re-transformation in yeast cells.General considerations for DNA manipulation and library screening are described elsewhere ,24,25. M418 = 36000 M−1·cm−1) in 100 mM acetate buffer, pH 5.0, and protein concentration was determined with the Bio-Rad Protein Assay, using the microassay procedure for microtiter plates.Re-transformed yeast cells expressing laccase mutants selected in the re-screening were produced in duplicate in 100 mL flasks containing 30 mL of expression media ,24,25, g469 = 27500 M−1·cm−1). For each substrate, absorption peaks and appropriate time points were selected. Average ΔAbs per mg of protein was calculated for each mutant and normalized against parent type. Results were represented in a heat map and hierarchical clustering analysis was performed using the clustergram algorithm with Matlab software .All the assayed substrates were purchased from Sigma-Aldrich, except for methyl syringate, which was purchased from Alpha Aesar , and methyl coumarate, methyl ferulate and methyl sinapate, from Apin Chemicals . Stock solutions for the different phenols were routinely prepared at 10 mM in 20% ethanol, except for phenol, catechol, pyrogallol, guaiacol and 2,6-dimethoxyphenol, which were dissolved in water; and resveratrol and quercetin, which were dissolved in absolute ethanol. Reactions were carried out in duplicate for each mutant in UV-Star 96-well plates , by adding 10 μL of laccase crude extracts, 180 μL of 100 mM acetate buffer, pH 5.0, and 20 μL of stock substrate solution in each well. Control reaction without enzyme was included in order to subtract non-enzymatic substrate oxidation. UV-Vis absorption spectra  were recorded in a SpectraMax Plus 384 plate reader  at reaction times 0, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h. In the case of 2,6-dimethoxyphenol, reaction was too fast, so it was measured in kinetic mode at 469 nm (εIn this work, we have evaluated the oxidation of a battery of phenols with different chemical structures by a library of laccase mutants obtained by saturation mutagenesis of six residues of the substrate binding pocket. This has given us some insight into the structural factors, both from the substrate and the enzyme, that modulate laccase activity. Even though the substrates tested are structurally related to sinapic acid, high variability in laccase activity was found only by changing one or two residues of the enzyme pocket. This indicates that besides the difference in redox potential between laccase and substrate, which delimits the electron transfer, the efficient binding of the substrate to the enzymatic pocket determines the oxidative capabilities of laccases. These results also highlight once more the importance of the screening assay for directed evolution, which will define the fate of the protein subject of the study. The use of either a single or a multi-screening assay will determine whether we obtain a specialist or a generalist biocatalyst, respectively.


Background and Purpose: There are recent reports of zolpidem being effective for the treatment of a variety of movement disorders, due to its action on the gamma-aminobutyric acid A receptors in the thalamus, subthalamic nucleus, and globus pallidus, hence facilitating inhibitory pathways in the basal ganglia motor loop. Its beneficial effects have been described for Parkinson's disease and other related disorders. The objective of this study was to assess the therapeutic effects of zolpidem for various types of dystonia.Methods: We conducted a literature search using MEDLINE via PubMed, Cochrane Library, EMBASE, Scopus, and Google Scholar.Results: There were no randomized controlled trials. The literature included 6 case reports, 4 case series, and 1 non-randomized, non-controlled interventional trial. Overall, 49 adult participants (range 1–34 participants) with a mean age of 49.5 years were treated. Regardless of the dystonia subtype, a single dose of zolpidem at 10 mg causes improvement of symptoms for a mean duration of 3.4 h until patient returns to baseline. The main adverse effect noted was drowsiness, which was dose-dependent.Conclusion: While the current available literature suggests that zolpidem may be an effective pharmacologic option for treating dystonia, however the quality of evidence remains limited. Larger sample size, methodological consistency, and randomized controlled trials with long-term patient follow-ups are necessary to come up with definitive conclusion. Dystonia is a hyperkinetic movement disorder characterized by sustained or intermittent muscle contractions causing abnormal, often repetitive, movements, postures, or both. Movements are typically patterned, twisting, and may be tremulous. It is often initiated or worsened by voluntary action and associated with overflow muscle activation . EstimatThere are several treatment options for dystonia, including pharmacological therapies such as the use of anticholinergics (most commonly trihexyphenydil), botulinum toxin injections, and surgical treatment such as deep brain stimulation (DBS) of the globus pallidus interna –6. HowevN,N,6-trimethyl-2-(4-methylphenyl)-imidazo-pyridine-3-acetamide hemitartrate (1 subunits of gamma-aminobutyric acid (GABA)A receptors in the central nervous system, facilitating inhibitory neurotransmission . Since very few of these studies have been done, no limits were placed on study design, patient demographic variables, or outcome measures.clinicaltrials.gov. All searches were run on or before September 30, 2018.Searches were run in MEDLINE via PubMed, the Cochrane Library, EMBASE, Scopus, and Google Scholar. Currently, there are no zolpidem trials registered with The studies were screened by the primary investigator (SPB). Any study that used zolpidem to treat dystonia was included in the next stage of the review. A second round of screening was performed by the authors based on abstract and/or full text and studies that met the eligibility criteria above were included. Reference lists of all studies identified during the first round of screening were cross-checked for additional studies that would meet the eligibility criteria. The Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) statement guidelines were followed .Data items included study design, diagnosis, country of first author's institution, sample size, total number of participants, mean age and sex of study participants, mean duration of diagnosis, outcome measures, dose of zolpidem, number improved, duration of effect, and adverse effects. No assessment for risk of bias was performed since the accepted tools for this type of assessment are designed primarily for randomized controlled trials (RCTs). This review includes all study designs and hence these tools would not be appropriate. Instead, data was collected on randomization, controls and masking (if available), as well as informal notes on each study's limitations.n = 8/11, 73%) described outcomes using clinical observation only , MEDLINE (n = 241), Cochrane (n = 9), Scopus (n = 121), and PUBMED (n = 33). Thirty studies were included after initial screening based on the title only. A second round of screening was done by applying eligibility criteria to study abstracts and/or full texts. Systematic reviews, commentaries, experimental studies using animal subjects and other studies that used zolpidem for the treatment of other movement disorders not including dystonia  were excluded from this review  produced 2,294 studies after duplicates were removed. Individual database results included Google Scholar  having only one participant. The ethnicity of the subjects was often not reported; however, studies were conducted in 5 countries: Korea (n = 4), USA (n = 4), Japan (n = 2), and Argentina (n = 1). The total number of adult participants was 49, with 30 males (61.2%). One study did not mention the patient's gender . The numOut of a total of 49 adult patients included in this review, 25 patients (51%) showed almost complete resolution of symptoms immediately after intake of zolpidem 10 mg, with an onset of effects being observed in 15–45 min and peak effects in 1–2 h , 11–19. Because of the short duration of action of zolpidem, dosing varied from once daily to four times daily, with a mean total daily dose of 30 mg, usually given at 10 mg three times daily. Doses of 20 mg daily or higher were divided into three to four times per day to avoid the side effect of somnolence , 15. TheIn the three studies that used the BFMDRS scores as an outcome measure, a significant improvement was seen after zolpidem therapy in various types of dystonias , 6, 19. There was also noted improvement in dystonias unresponsive to other medications. A case report of a patient with Meige syndrome, initially unresponsive to botulinum toxin A, baclofen, and trihexyphenidyl, showed complete resolution of symptoms within 1 h of intake of zolpidem 12.5 mg, with symptom free periods lasting for 4 h . A patieZolpidem is a sedative-hypnotic initially approved for the treatment of sleep disorders. It was first marketed in Europe in 1987. In April 1992, it was approved by the United States Food and Drug Administration as a hypnotic for patients with sleep disorders, with a recommended dose of 5–10 mg to be taken at bedtime . Since tmax) of 0.75–2.6 h postdose, an oral absolute bioavailability of 70%, and a short mean half life of 1.5–3.2 h  . The binding sites for zolpidem and for benzodiazepines is in the α subunit. Benzodiazepines can bind to multiple types of GABAA receptors that contain α1, α2, α3, or α5. However, zolpidem binds selectively to GABAA receptor isoforms that contain α1 subunits  . GABAA rs system .A receptors was found in the basal ganglia and thalamus , an extended release tablet (6.25 mg and 12.5 mg), an oral spray (5 mg/spray), and a sublingual tablet  . Most stThe main adverse effect of zolpidem as described in the literature was drowsiness (when it is not being prescribed as a sedative). Daytime somnolence was observed among patients taking a single dose of zolpidem 20 mg or more in the morning , 6, 19. There were no studies that described the adverse effects of long term use of zolpidem among patients with dystonia. However, these have been extensively described in studies among patients with sleep disorders. Zolpidem is not known to cause hepatic, respiratory, cardiovascular, or renal dysfunction . No effeWhile the available literature suggests that zolpidem may be an effective pharmacologic option for treating dystonia, the quality of evidence remains limited since the studies available were case reports and small observational studies only. Regardless, the potential of zolpidem to improve quality of life and functional outcome especially among patients with medically refractory and chronic dystonias merits further investigation with additional, larger-scale, standardized controlled trials.All datasets generated for this study are included in the manuscript.SB: study concept and design, acquisition of data, analysis and interpretation and writing of the initial draft. RJ: study concept and design, acquisition of data, analysis and interpretation, critical revision of the manuscript for intellectual content, and study supervision.RJ serves on the advisory board of Lundbeck Phils and Torrent Phils. He has received honoraria and CME grants from the Philippine offices of Allergan, Innogen, Lundbeck, Medichem, Natrapharm, Sandoz, Sun, Torrent, and Zydus. He has research grants from the Collaborative Center for X-linked Dystonia Parkinsonism and the Philippine Neurological Association. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Supplemental Digital Content is available in the text Vitamin D deficiency (VDD) has been implicated in the pathogenesis of tuberculosis (TB), but most studies have not reported a significant association. We conducted a meta-analysis to explore the association between vitamin D status and TB in children.Web of Science, Ovid Medline, and EMBASE were searched for studies in English that discussed vitamin D status and TB in children before January 22, 2018.P < .05) in children. Vitamin D levels were significantly lower in TB patients than in controls, with a mean difference d = −5.49 nmol/L , indicating that VDD was significantly associated with TB  in children.From the 585 initially identified studies, we selected those that addressed an association between vitamin D status and TB according to our preselected inclusion criteria. Our meta-analysis included 10 studies. According to the random effects model, TB was significantly associated with VDD (ORs, 1.70; 95% CI, 1.20–2.42; This study suggests that vitamin D levels are significantly lower in children with TB/latent TB infection than in controls. TB may contribute to VDD in children. Therefore, VDD may be associated with TB in children. VDD is highly prevalent worldwide and leads to immune dysregulation and systemic inflammation.The definition of vitamin D deficiency (VDD) is inconsistent. Some studies define VDD as serum 25-hydroxyvitamin D3 (25(OH)D) levels less than 25 nmol/L (<10 ng/mL),5,6 Vitamin D regulates the immune system by modulating both innate and adaptive immunity. Various cytokines are expressed after the treatment of macrophages with 25(OH)D. Additionally, 2 antimicrobial peptides, cathelicidin and β-defensin, which stimulate autophagy in Mycobacterium tuberculosis, are expressed after exposure to vitamin D. Matrix metalloproteinases (MMPs) are inactive in the pathogenesis of pulmonary cavitation, and the process is induced by reactive nitrogen and oxygen via vitamin D. VDD may affect immunity, leading to an increased incidence of TB and sepsis. Williams et al found that VDD was common in children with active or latent TB, and sex, ethnicity, and TB status had no effect on vitamin D levels.The mechanisms underlying the association between VDD and tuberculosis (TB) may be as follows. Vitamin D is known to be essential for health and controls cell functions via vitamin D receptors (VDRs), which are present on myocytes, endothelial cells, and major immune cell types such as T and B lymphocytes, macrophages, dendritic cells, and neutrophils.M tuberculosis, is a leading health problem worldwide. One-third of the world's population is considered to have latent TB infection (LTBI). Several reports have shown an association between TB and malnutrition, demonstrating that multiple factors contribute to TB.–10 Additionally, malnutrition affects the immune system and thus increases the risk of developing TB. In recent years, some studies–13 have performed meta-analyses of the association between TB and VDD. Zeng et al showed that only 2/15 studies included in their meta-analysis included children. Most recent meta-analyses including patients of all ages did not present results for the subgroup of children.,14 The studies presumed that the association between VDD and TB in children is limited. In addition, the sample sizes of the studies were small, and many of the studies lacked sufficient power to evaluate the relationship between vitamin D level, vitamin D status, and TB. Therefore, we conducted this meta-analysis to overcome these limitations.TB, caused by 22.1This meta-analysis followed the Meta-Analysis of Observational Studies in Epidemiology (MOOSE) and the Preferred Reporting Items for systematic Reviews and Meta-Analyses (PRISMA) guidelines. The Ovid Medline, EMBASE, and Web of Science databases were searched through January 22, 2018. The search consisted of the following 3 terms: child, TB, and vitamin D. We used the medical subject heading (MeSH) terms and key words to search for the first and second terms, and we used “AND” to connect child with TB. We used vitamin D and the relevant key words to search for the third term. In addition, we used “AND” to connect the third term and the combination of the first 2 terms. For MeSH terms and search strategy, see supplementary data. We restricted the search to human studies published in English. The retrieved studies were screened by reading the titles and abstracts, and 2 authors (XG and LP) independently read the full texts of the remaining publications and then discussed disagreements to reach a consensus.2.2The study inclusion criteria were as follows: the participants were children <18 years old whose vitamin D levels had been measured so that the association between their vitamin D status and any of the following 3 outcomes could be investigated: the difference in the concentration of 25(OH)D (reported by the mean and standard deviation [SD]) between patients with active TB/LTBI and controls, the incidence, prevalence, relative risk (RR) or odds ratio (OR) of VDD in children with active TB/LTBI and controls, and the incidence, prevalence, RR, or ORs of TB/LTBI in children with VDD and controls; the study defined VDD; the study described the assessment of exposure and outcomes and reported unadjusted and/or adjusted RR and corresponding 95% confidence intervals (CIs) or unadjusted and/or adjusted ORs and 95% CIs; and the study was published in English, and the study design was case–control, cohort, or cross-sectional.The exclusion criteria were as follows: the participants of the study were adults; the study was a meta-analysis, review, or case report; the study was not published in English; the article described an animal study; a study included data that overlapped with the data of another study; and the study did not have usable data D was not reported as the mean and SD but as the median and interquartile range [IQR], the controls were not truly free from TB infection, and the second or third outcomes with ORs or RR were not directly or indirectly available from the study).2.3The data were independently extracted from the studies by 2 reviewers (XG and LP) and included the following standardized forms: author, publication year, country, study population, study design, sample size, and outcomes.2.4 to examine all included studies for the methodological quality. The reviewers evaluated the quality score (with a maximum score of 9) in the following 3 domains: the study population selection, comparability, and evaluation of exposure and outcomes. The reviewers resolved disagreements as previously described.The 2 reviewers (XG and LP) independently used the Newcastle-Ottawa Scale2.5 In addition, the original included studies used ORs and/or RRs and 95% CIs to assess the association between VDD and TB among the subjects. Both ORs and RRs were considered to be ORs. All data of the included studies were converted into log(ORs) and standard error (SE). We pooled the log(ORs) and SEs of each study separately using the DerSimonian–Laird formula (random effects model). Statistical heterogeneity between studies was assessed using the Q and I2 statistics. Values of I2 > 50% and P < .1 indicated a high degree of heterogeneity.The original included studies used the mean difference (MD) in 25(OH)D levels to evaluate the difference between the active TB/LTBI and control groups. We pooled the means and SDs of each study separately using the DerSimonian–Laird formula (random effects model). of SEs versus ORs to assess the publication bias and used Review Manager 5.3 software to perform the statistical tests.We used funnel plots33.1We identified 585 potential studies including 103 from Ovid Medline, 204 from EMBASE, and 278 from Web of Science. After careful screening, 10 studies that reported the 25(OH)D levels or vitamin D status and presence of TB in children were included D levels in children with TB and controls; two,23 of these studies calculated 25(OH)D levels in nmol/L, and two,22 calculated 25(OH)D levels in ng/mL. Four studies–27 evaluated the association between the presence of any TB and VDD. One study showed a significant association between the presence of any TB and VDD, while 2 studies,25 showed a significant association between the presence of active TB and VDD; however, 3 studies,26,27 showed a nonsignificant association between the presence of any TB and VDD. Four studies,23,28,29 showed that VDD contributes to TB.The included studies were published between 2012 and 2017. Four studies4P < .05, I2 = 87%), and the level of 25(OH)D was lower in the active TB/LTBI group than in the control group. The MD in 25(OH)D levels (nmol/L) between children with active TB or LTBI and controls was −6.98  and −4.20 , respectively. The pooled data demonstrated that active TB was significantly associated with VDD, with a pooled OR of 2.09    .5To the best of our knowledge, although several recent meta-analyses on TB have included children, none has reported the association between vitamin D status and TB/LTBI in children. This meta-analysis, which included 10 studies, provides evidence that vitamin D levels are significantly lower in children with TB/LTBI than in controls. TB/LTBI significantly contributed to VDD in children. Therefore, VDD was significantly associated with TB in children.,30 suggest that VDD affects immunity; it is possible that a low level of vitamin D may contribute to M tuberculosis infection by increasing chemokine production, activating dendritic cells, and altering T cell activation. Children with VDD in developing countries have a greater risk for developing TB. A recent meta-analysis showed that VDD in adults and children was associated with TB, with a pooled OR of 2.57 . However, the results of the studies focusing on the relationship between TB and VDD in children were inconsistent. Ludmir et al evaluated the relationship between active TB and VDD and identified an OR of 1.25 ; however, Venturini et al demonstrated an association between TB and VDD, with an OR of 1.61 . Differences between studies may also be explained by interindividual differences due to VDR polymorphisms. The results of our meta-analysis showed that lower vitamin D levels were observed in children with TB than in controls. The definition of VDD varied from a 25(OH)D level <50 nmol/L (20 ng/mL) to <75 nmol/L (30 ng/mL) or <10 ng/mL, which may have contributed to the heterogeneity in the meta-analysis results. A consensus definition of VDD in children is thus required in future studies.VDD might contribute to TB in children through multiple pathways. A growing body of evidenceTo better understand the relationship between TB and VDD in children, some studies have evaluated the association between active TB/LTBI and VDD. This meta-analysis demonstrated that the risk of developing VDD was prevalent in children with active TB and LTBI, with an OR of 1.70 . Furthermore, the results of this meta-analysis suggest that active TB increases the risk of VDD in children. Nevertheless, the analyzed studies did not include strict criteria for active TB, which may have led to an overdiagnosis of active TB and contributed to the heterogeneity among the studies.,28 Furthermore, the definition of VDD varied among studies, which may have contributed to the observed heterogeneity. Therefore, a consistent definition of VDD is required.Additionally, we found a significant association between VDD and active TB/LTBI. We found that VDD increased the risk of TB and that TB increased the risk of VDD. The lower level of 25(OH)D in children with active TB than in controls may be explained by poor nutrition or VDR polymorphisms. TB increases the risk of VDD via malnutrition, limited vitamin D absorption, limited sun exposure, immune dysregulation, and VDR polymorphism.,28 due to immune dysregulation. Jubulis et al and Gupta et al found that HIV status was a significant predictor of TB.Other factors may interact with TB via pathogenetic pathways leading to VDD in children. Although the included studies controlled for some confounding variables, many other factors that contribute to VDD in children could not be excluded. For example, HIV-infected subjects and malnourished subjects are more susceptible to active TB/LTBISimilarly, some factors may interact with VDD via pathogenetic pathways to cause TB in children. Although the included studies controlled for some confounding variables, many other factors that contribute to TB in children could not be excluded.Our meta-analysis has some limitations. First, we included only articles published in English, which may have contributed to publication bias. Second, the results of this meta-analysis should be interpreted with caution because of the limited number of subjects. Third, the bias inherent in observational studies was not eliminated in a quantitative synthesis. Additionally, the differences in design among studies may have affected the pooled results of the studies. Finally, the 25(OH)D level can be affected by many factors, and the measurement of 25(OH)D may not directly reflect vitamin D status.Our meta-analysis also has several merits. First, we evaluated the association between vitamin D status and TB in children. Second, we demonstrated that the lack of a uniform definition of VDD contributes to heterogeneity among studies and that a consistent definition of VDD is required for future studies.–34The results of our study indicate that supplementation with vitamin D may be beneficial for active TB/LTBI treatment and prevention, although the few relevant trials that have been conducted have had disappointing results.6In conclusion, our pooled analyses provide strong evidence that vitamin D levels are significantly lower in children with TB/LTBI than in controls. TB may contribute to VDD in children, and thus, VDD may be associated with TB in children. This knowledge may support the design and evaluation of randomized case–control clinical trials on the role of vitamin D supplementation in preventing TB in children.www.aje.com) for editing the English language of the manuscript.XG and LP contributed to the conception and design of the study, as well as to the drafting of this article. FT and HG contributed to the collection and analysis of the data. DX contributed to the conception and design of the study and approved the final version of the manuscript for publication. In addition, we would like to thank American Journal Experts (Conceptualization: Xiaoyun Gou, Lingli Pan, Dongqiong Xiao.Data curation: Xiaoyun Gou, Lingli Pan, Dongqiong Xiao.Formal analysis: Xiaoyun Gou, Lingli Pan, Fajuan Tang, Hu Gao, Dongqiong Xiao.Investigation: Xiaoyun Gou, Fajuan Tang, Hu Gao.Methodology: Xiaoyun Gou, Hu Gao, Dongqiong Xiao.Project administration: Xiaoyun Gou, Lingli Pan, Dongqiong Xiao.Resources: Xiaoyun Gou, Fajuan Tang.Software: Xiaoyun Gou, Lingli Pan, Fajuan Tang, Hu Gao.Supervision: Xiaoyun Gou, Lingli Pan, Fajuan Tang, Hu Gao, Dongqiong Xiao.Visualization: Xiaoyun Gou, Fajuan Tang.Writing – original draft: Xiaoyun Gou, Lingli Pan.Writing – review & editing: Dongqiong Xiao.


Patients suffering from chronic kidney disease (CKD) are at a 20-fold higher risk of dying due to cardiovascular diseases (CVDs), primarily thrombosis following vascular injury. CKD is connected with retention of uremic toxins, especially indoxyl sulfate (IS), which are currently considered as a non-classical CKD-specific risk factor for CVDs. The present study aimed to examine the effect of chronic exposure to IS on the hemostatic system and arterial thrombosis in a model without greater interferences from the uremic milieu consisting of additional uremic toxins. Forty-eight male Wistar Crl:WI (cmdb) rats were divided into three groups: one control group and two experimental groups, which were exposed to 100 or 200 mg/kg of b.w./day of IS in drinking water for a period of 28 days. The control group received water without IS. At the end of the experiment, the induction of arterial thrombosis was performed. We investigated the impact of IS on thrombosis incidence, kinetics and strength of clot formation, platelet activity, aortic contents of sirtuin (SIRT) 1 and sirtuin 3 (SIRT3), hemostatic system, cardiorespiratory parameters, biochemistry of plasma and urine as well as histology of the thrombus, kidney, and liver. Obtained data revealed that chronic exposure to IS promotes arterial thrombosis via increased levels of complex tissue factor/factor VII, plasminogen activator inhibitor-1 (PAI-1), platelet activation, as well as decreased aortic levels of SIRT1 and SIRT3. Therefore, we hypothesize that IS enhances primary hemostasis leading to augmented formation of platelet plug with increased amounts of fibrin and affects secondary hemostasis through the influence on plasma coagulation and fibrinolysis factors, which results in the increased kinetics and strength of clot formation. The findings described may contribute to a better understanding of the mechanisms leading to increased thrombotic events in patients with CKD with elevated levels of IS. Chronic kidney disease is a growing global health problem that affects almost 15% of the world population . PatientChronic kidney disease is associated with the retention of uremic toxins that are currently considered as a non-classical CKD-specific risk factor for CVD . One of in vitro model. However, there are no literature data currently available regarding the influence of IS on Sirt1 and Sirt3 and its involvement in CKD thrombosis in in vivo studies.Sirtuins – Sir2 proteins – are members of the class III type nicotinamide adenine dinucleotide-dependent histone deacetylases. Their roles are commonly linked to aging, metabolism, and antioxidant defense . FurtherThe present study is a continuation of our previous work  and it aad libitum access to sterilized tap water and standard chow in specific pathogen-free conditions. The number of animals was estimated after the calculation of the sample size. All procedures involving animals were approved by a Local Ethics Committee (Agreement Nos. 124/2015 and 31/2017/WNP) and conducted in accordance with institutional guidelines and the principles of the Basel Declaration.Male Wistar rats obtained from the Center of Experimental Medicine in the Medical University of Bialystok were housed in a temperature- and humidity-controlled room according to Good Laboratory Practice rules. Rats were allowed to have n = 16) and two experimental groups, which were exposed to 100 mg/kg of b.w./day  or 200 mg/kg of b.w./day  of IS  in drinking water for a period of 28 days  rats weighing 180–210 g were divided into three groups: one control group : PF4, vWF, FV, FXa, TF, TF/VII complex, TAFI, PAI-1, PAP, and d-dimers.Activated partial thromboplastin time, PT, TT, concentration of fibrinogen, and AT III activity were measured in citrated plasma using coagulometer Coag-Chrom 3003  and Bio-Ksel plasma kits.The evaluation of the effect of chronic exposure to IS on the dynamics of clot formation was performed using ROTEM technology as described previously . AnalysiThe following blood morphology parameters were evaluated in whole blood samples using Animal Blood Counter  according to the manufacturer’s directions: WBCs, RBCs, HGB, HCT, MCV, MCH, MCHC, and PLTs.Plasma biochemical parameters such as ALAT, ASAT, total cholesterol, TGs, LDL-d, HDL, creatinine, UA, urea, and total proteins, as well as urinary biochemical parameters such as creatinine and urine proteins were measured using an automated biochemical analyzer .Collected tissues were fixed in formalin and processed routinely for embedding in paraffin. Sections of thrombi, kidneys, and livers were cut on a microtome at a thickness of 4 μm and stained by H&E and additionally by PAS in the case of kidneys. The analysis of renal microscopical findings was performed using the semiquantitative method for the following histopathological indicators of tubular injury: interstitial fibrosis, tubular atrophy, and interstitial inflammation . Additio2 were monitored during 50 min after electrical stimulation of the common carotid artery using a rodent vital sign monitor . Body temperature was monitored via a rectal probe .The cardiorespiratory parameters: heart rate, tissue perfusion of rat’s paw, and SpOAortic levels of SIRT1 and SIRT3 in homogenates (1:25) were measured using rat ELISA kits supplied by Cloud-Clone, Corp., Houston, TX, United States according to the manufacturer’s instructions.t-test or non-parametric Mann–Whitney U-test. The correlations were calculated by Spearman’s rank correlation analysis. A two-tailed p-value < 0.05 was considered statistically significant. Statistical analyses were performed with GraphPad 6 Prism .Shapiro–Wilk’s test of normality was used for data distribution analysis. The normally distributed data were presented as mean ± SD, while the non-Gaussian data were presented as median (full-range). Comparison between data was made by Student’s p < 0.05). The 100 IS and 200 IS groups presented higher plasma concentrations of ALAT, total cholesterol, HDL, and creatinine (p < 0.05). Only the 200 IS group had increased concentration of UA in plasma (p < 0.05) and urine protein excretion (p < 0.01). Creatinine clearance was decreased in both experimental groups (p < 0.05).The general characteristics of rats are shown in Table p < 0.001) . However, the concentration of IS in the urine in the 200 IS group was also increased when compared to the 100 IS group (p < 0.001)  Figure .p < 0.05, 100 IS; p < 0.001, 200 IS) . In addition, the frequency of thrombosis incidence was also higher in both experimental groups than in the control group  and decreased CFT (p < 0.05) when compared with control. Other parameters were not affected. Furthermore, we did not observe any changes in the 100 IS group.As shown in Figure p < 0.05)  Table .p < 0.05). In turn, concentrations of TF/VII complexes were significantly higher in the IS groups . Moreover, the 200 IS group showed augmented concentrations of PAI-1 (p < 0.05 compared to the control group) and PF4 – a marker of platelet activation of which clearance is not dependent on the functioning kidney tissue (p < 0.05). There were no differences in concentrations of vWF, TF, TAFI, and d-dimers  as well as SIRT3  when compared to control group . Additionally, the 200 IS group presented higher WBC when compared to control (p < 0.05). Moreover, the highest dose of IS decreased RBC  whereas it increased MCH and MCV (both p < 0.001 compared to control and 100 IS). The HCT, HGB, and MCHC did not differ between all studied groups  were administered chronically to rats. The doses used were enough to obtain significant elevations of IS in rats’ plasma and reflect level of IS observed in patients with CKD . A definOur previous study demonstrated an acceleration of thrombotic response following vascular injury after acute exposure to IS . SimilarIn addition to the earlier observations, a histological evaluation revealed that thrombi of IS-treated rats were richer in fibrin with PLT when compared with control rats. This fact suggests that IS not only contributes to an increase in PLT, but also to increases the amount of fibrin. The above-mentioned hypothesis is also supported by data collected from our previous study, which showed that IS significantly enhances fibrin generation . MoreoveGenerally, the platelet plug formed during primary hemostasis is not stable. In turn, ROTEM analysis showed that IS induces an increase in MCF, a parameter reflecting the strength of the clot formed. Augmented stability of clots after IS administration can also be one of the causes of increased thrombosis incidence, which is observed in our model. Therefore, we hypothesize that IS not only enhances primary hemostasis, but also affects secondary hemostasis through influence on plasma coagulation and fibrinolysis factors. In addition, an IS-induced increase in clot firmness seems to be linked to slight impairment of fibrinolysis that was reflected by a decreased level of PAP and increased concentration of PAI-1. Moreover, the level of PAI-1 positively correlated with the concentration of IS. Obtained data are in line with work of Motojima et al., in which they observed that IS is able to upregulate gene expression of PAI-1 in human renal proximal tubular cells . A furthin vitro formation of the DNA-histone complex, which is a marker for the formation of NETs. In the current work, we show for the first time that chronic exposure to IS leads to reduced aortic contents of Sirt1 and Sirt3 in rats, which, in turn, can contribute to a prothrombotic state. Thus, enhancing the activity of Sirt1 and Sirt3 may provide new therapeutic options for the treatment of thrombosis in patients with CKD with an elevated level of IS. However, data obtained in our study showed that both IS doses reduced the level of SIRT1 and SIRT3 at a similar magnitude, whereas changes in hemostatic parameters differed depending on the IS dose. Therefore, IS-mediated changes in Sirt1 and Sirt3 definitely need to be assessed at transcriptional and post-transcriptional levels, and the molecular mediators of these regulations should be determined to better understand the contribution of Sirt to the thrombotic process. In addition, due to the fact that uremic milieu consists of toxins other than IS, further studies are required to confirm changes in sirtuins in well-established models of CKD.Recently, particular attention is being paid to Sir2 proteins — sirtuins. Hitherto, the role of sirtuins described is mainly associated with protection against senescence, oxidative stress, or inflammation . HoweverThis study demonstrates that IS promotes arterial thrombosis and prothrombotic state that is connected with an increased level of complex TF/VII, PAI-1, and platelet activation, as well as decreased aortic contents of SIRT1 and SIRT3. Obtained data suggest that IS enhances primary hemostasis leading to an augmented formation of platelet plug with an increased amount of fibrin and affects secondary hemostasis through influence of plasma coagulation and fibrinolysis factors that results in increased kinetics and strength of clot formation. Our findings may help to better understand mechanisms leading to increased thrombotic events in patients with CKD with elevated level of IS. However, the model from the current work described solely the IS impact. Considering the above, further studies are necessary in order to reveal the influence of complex interactions between other uremic toxins on thrombotic events in patients with CKD.MK, TK, and DP conceived and designed the experiments and wrote the paper. MK, TK, BZ, TD, TM, TR, AP, KP, and DP performed the experiments. MK, TK, TM, AP, KG-U, KP, and DP analyzed the data. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Psoriatic arthritis (PsA) is a chronic, inflammatory disease. The effects of PsA real-world treatment patterns on patient-reported outcomes in the US and 5 European countries  were evaluated. Respondents from the 2016 National Health and Wellness Survey received advanced therapies , other therapies, , or no treatment. Assessments included demographics, disease severity (patient-reported), comorbidities (Charlson Comorbidity Index), health status , depression , work productivity (Work Productivity and Activity Index), and treatment adherence . Overall, 1037 respondents from the US and 947 respondents from the EU5 were included. Of these, 21.7% US and 7.3% EU5 respondents received advanced therapies; 16.6% and 28.5%, other therapies; and 61.7% and 64.2%, no treatment, respectively. During treatment with advanced or other therapies, 40.8–54.7% US and 57.7–58.9% EU5 respondents self-reported moderate or severe PsA. Respondents receiving advanced therapies had the highest Charlson Comorbidity Index score ; the lowest scores were with no treatment . Employment was lowest with other therapies . Overall work impairment was reported by 57.9% US and 62.6% EU5 respondents receiving advanced therapies. Medication adherence was generally low in the US and medium in the EU5 . Advanced and other therapies reduced PsA severity; however, > 40% of respondents reported moderate or severe PsA during treatment. Better management and adherence may reduce unmet need and disease burden. Further work is required to improve PsA diagnosis and time to treatment initiation.The online version of this article (10.1007/s00296-018-4195-x) contains supplementary material, which is available to authorized users. Psoriatic arthritis (PsA) is a chronic, debilitating, inflammatory disease. The domains of PsA have been defined by the Group for Research and Assessment of Psoriasis and Psoriatic Arthritis (GRAPPA) as peripheral arthritis, axial disease, enthesitis, dactylitis, psoriasis (PsO), and nail disease . More reDue to the complexity and range of symptoms involved, PsA may be diagnosed and managed by both dermatologists and rheumatologists. However, as PsA is often misdiagnosed or undiagnosed , 6, estiPsA is associated with considerable disease burden, increased healthcare costs, and impairments in HRQoL and work productivity , 18–20, PsO and rheumatoid arthritis (RA), unique conditions that share symptoms with PsA, are associated with a number of comorbidities including increased risk of cardiovascular events and disease, mortality, infections, and malignancies compared with the general population –29. PatiTreatment guidelines from GRAPPA , the AmeEULAR guidelines recommend that patients with active PsA receive non-steroidal anti-inflammatory drugs (NSAIDs), then conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) if they do not respond to NSAIDs, followed by a biologic DMARD  or targeted synthetic DMARD  if needed . AAD guiDespite the development of treatment guidelines by GRAPPA, AAD, and EULAR, along with a greater understanding of the disease burden of PsA, the effects of real-world treatment patterns on PROs and health status have not been fully evaluated to date. Here, we report a descriptive, exploratory analysis of data from the cross-sectional, 2016 National Health and Wellness Survey (NHWS). The objectives of this analysis were to characterize patients with self-reported PsA and to describe the effect of treatment patterns  on PROs, with the aim of improving understanding of how treatment affects health status in the real world in the US and EU5.Data were taken from the 2016 NHWS , a self-NHWS respondents were asked ‘Which of the following conditions have you ever experienced?’ and were provided with a list of conditions, with PsA listed under ‘chronic pain conditions’. Respondents who reported having PsA were directed to complete a section called the “arthritis module”. Respondents who did not self-report having PsA or who self-reported having PsA, but did not complete the arthritis module were excluded from the analysis. Respondents reporting they had PsA who completed the arthritis module were grouped by mutually exclusive categories based on self-reports of treatments currently being used to treat their disease. Three pharmacologic treatment categories were defined: advanced therapies -12/23 and IL-17 antagonists, and PDE-4i; patients were included in this group regardless of any non-advanced therapies currently being used), treatment with other therapies , and no treatment.PRO data were collected as components within the NHWS questionnaire and summarized descriptively. PRO instruments included the Short Form-36 Health Survey (SF-36) to measure health status, including physical health  and mental health  ; the WorPatients were asked to self-report the current and pre-treatment severity of their PsA as mild, moderate, or severe, based on their perception of their current health state and recollection of their health state prior to receiving treatment; no specific definitions or criteria were provided to patients for self-rating of PsA severity. Sociodemographics and health history details were also collected.t tests (for continuous data) were used to assess unadjusted associations. All analyses were performed using SAS version 14.1. No missing value imputation was performed. No multiplicity correction adjustments were made.Descriptive statistics were calculated for personal characteristics and included means (± standard deviation) and relative frequencies (%) as applicable. Basic inferential statistical tests such as Chi-square  and In total, 97,503 US and 80,600 EU5 adults completed the 2016 NHWS; 1140 (1.17%) respondents in the US and 1085 (1.35%) in the EU5 self-reported having PsA.Among US respondents, 1037 who reported having PsA completed the arthritis module and provided information on treatment. Of these, 225 (21.7%) reported receiving advanced therapies, 172 (16.6%) other therapies, and 640 (61.7%) no treatment. In the EU5, 947 respondents who reported having PsA completed the arthritis module and provided information on treatment, 69 (7.3%) reported receiving advanced therapies, 270 (28.5%) other therapies, and 608 (64.2%) no treatment.p < 0.01) and EU5 (64.1%) compared with the no treatment group .Age distribution was similar across US and EU5 patients Table . SignifiIn the US and EU5, 57.6% and 52.9% of patients, respectively, were employed and the majority of these  were employed full time. In both the US and EU5, the treatment group receiving other therapies had the lowest proportion of employed patients ; Medicare and coverage through a current or former employer were the most common sources of insurance (Online Resource 2). In the EU, 92.0% of patients had health insurance, with public insurance alone being the most common (Online Resource 3). A similar proportion of patients had health insurance across the treatment groups in EU5, with significant differences between advanced therapies and other therapies (p < 0.01) and between other therapies and no treatment (p < 0.05) in the UK only.In the US, the no treatment group had the lowest proportion of patients with some form of health insurance (86.4%) vs. advanced (93.3%) and other therapies groups  or other therapies (3.1%) compared with pre-treatment , although 54.7% of patients receiving advanced therapies and 40.8% of patients receiving other therapies still rated their PsA as moderate or severe , and the lowest proportion of patients self-reported severe disease  and EU5 patients (p < 0.001 for both).SF-36 MCS and PCS scores, and PHQ-9 scores, were broadly similar in the US and EU5 Table  39]. Di. Di39]. p < 0.05 vs. other therapies]; EU5, 57.2% ), followed by those with no treatment  and those receiving other therapies  in EU5 patients.As measured by their completion of the WPAI, patients in both the US and the EU5 reported missing work. The range of absenteeism was similar for both US  and EU5  across all treatment groups . In the EU5, patients in both the advanced and other therapies groups were more likely to self-report medium adherence . For patients receiving treatment with either advanced or other therapies, fewer patients self-reported severe disease , and a higher proportion reported mild disease , compared with self-reported pre-treatment severity, suggesting that patients responded to treatment. Despite this improvement, 54.7 and 40.8% of patients in the US and 57.6 and 58.9% of patients in the EU5 still rated their PsA as moderate or severe for advanced and other therapies, respectively.This was a population-based survey relying on self-reported PsA rather than a study conducted in rheumatology or dermatology clinics. This likely impacted the patient population identified, resulting in a larger number of patients with lower disease activity, and therefore, a lower proportion of patients receiving advanced therapies and a higher proportion of patients receiving no treatment than might be expected. Despite this, both age distribution and the proportions of female/male patients were similar across the US and EU5 treatment groups. The largest proportion of patients in the US with a high BMI received other therapies, whereas in the EU5, the largest proportion of patients with a high BMI received advanced therapies. In addition, in both the US and EU5, patients receiving advanced therapies had the highest age-adjusted Charlson Comorbidity Index score compared with other treatment groups. These data indicate that high BMI and comorbidities are common in PsA, with the highest disease burden being observed in patients receiving advanced therapies.The findings of these analyses confirmed the impact of PsA on HRQoL, with SF-36 PCS and MCS scores below the normative values seen in the general population . SF-36 PThe majority of US patients were in full-time employment and a greater proportion of these patients received advanced therapies compared with other treatment groups. In EU5, full-time employment levels were similar across treatment groups. Similar to the previous investigations in PsA , 47, proSelf-reported medication adherence was generally poor in the current study. This may be a reflection of access to advanced therapies in the EU5, with public insurance coverage being more prominent compared with the US healthcare systems. A recent systematic review of medication adherence in patients with a range of conditions, including PsA, identified varying levels of non-adherence, which can impact negatively on health outcomes and have associated economic costs . In the A number of limitations of this analysis must be acknowledged. Patients were included if they selected PsA in response to the question ‘Which of the following conditions have you ever experienced?’, where PsA was listed under ‘chronic pain conditions’. The diagnosis of PsA was not further validated by physician or chart review. Grouping of patients by treatment was based on self-reports of treatments used to treat arthritis; it is, therefore, possible that some patients might have been receiving treatment for PsO that might also be effective for PsA, but would not necessarily have been captured. Disease severity  was not defined and was determined based on a patient’s response to the question ‘What is the level of severity of your condition?’, with no criteria provided to guide self-rating of severity of PsA. Results may, therefore, differ from those that would have been obtained if respondents had been required to receive a formal physician-reported PsA diagnosis and severity rating. It is worth noting, however, that participants receiving advanced therapies and other therapies reported seeing a rheumatologist or dermatologist more frequently than those participants receiving no treatment, lending support to the self-reported PsA in these treatment groups. Patients were not required to be under the care of a clinician for their PsA, which might have resulted in different treatment patterns, with fewer patients being treated, particularly with advanced therapies, compared with patients under the care of a rheumatologist or dermatologist. Pre-treatment severity levels were based on patients’ recollection of their health state before they started treatment. Information regarding the delay between a patient starting their current treatment and responding to the survey was not collected in the survey. In addition, given the design of the survey questionnaire, it was not possible to analyze the influence of the various symptoms and comorbidities of PsA on the treatment received or the patients’ perception of disease severity. Selection bias may have affected these analyses; patients who are satisfied with their treatment are less likely to respond to these types of questionnaires, so this type of survey may select for patients who are unhappy with their treatment. Only patients with PsA who completed the arthritis module of the NHWS and provided information on treatment were included in the analysis; 9% and 13% of US and EU5 respondents, respectively, with self-reported PsA were excluded from the analysis because of missing data. Data were limited to those obtained from patients who volunteered to participate, and therefore, results may not be applicable to the entire PsA population. Although this study included patients from the US and five European countries, it may not reflect clinical experience in all countries. Finally, although adherence was recorded, reasons for lack of adherence were not recorded, and it is possible that adherence was lower in patients who felt their treatments did not work or were too expensive. In addition, results here were not adjusted by age or gender, which may limit their interpretation.Finally, although disease severity in patients receiving advanced and other therapies was reported to have changed after initiating treatment, 40–60% of patients reported moderate-to-severe PsA while being treated. The findings underscore the need for overall better management and identification of PsA, which, as demonstrated in this study, has a substantial impact on patients’ mental and physical health, employment, and healthcare resource utilization. Key areas for improvement include the early recognition of the disease and utilization of the guideline recommendation of a shared decision-making process between patient and physician on a treatment strategy to achieve remission or, alternatively, low disease activity as a treatment goal.Below is the link to the electronic supplementary material.Supplementary material 1 (DOCX 53 KB)Supplementary material 2 (DOCX 45 KB)Supplementary material 3 (DOCX 45 KB)Supplementary material 4 (DOCX 136 KB)