VDR) in the development of SpA. In this case–control study, a total of 244 patients with SpA and 197 individuals with no SpA were included. Among the patients, 174 had ankylosing spondylitis (AS) and 66 had psoriatic arthritis (PsA). Genotyping of FokI (rs2228570 C > T), BsmI (rs1544410 C > T), ApaI (rs7975232 A > C), and TaqI (rs731236 T > C) was performed using PCR-RFLP, while genotyping of HLA-B∗27 was performed using PCR-SSP. Serum levels for hydroxy (OH) vitamin D and the clinical activity index of the disease (BASDAI) were also evaluated. SNPStats and OpenEpi software were used for statistical analysis. The ApaI a allele and ApaI a/a genotype were less frequent in PsA compared with controls. The ApaI a/a genotype was associated with a protecting factor for PsA in females, and ApaI A/a was associated with a protecting factor for the disease in HLA-B∗27 positive patients. Notwithstanding, the ApaI a/a genotype was a risk factor for SpA and AS in males. The FokI f/f genotype was associated with a better clinical activity in PsA. When considering the covariates, vitamin D sufficiency, and gender, the FokI F/F genotype was associated with a risk factor in males with SpA and AS compared with females with this same genotype. In conclusion, the ApaI rs7975232 polymorphism was associated with PsA, and the FokI rs2228570 polymorphism was associated with better clinical PsA activity. ApaI and FokI were associated with SpA and AS when considering gender and vitamin D sufficiency.Spondyloarthritis (SpA) is an inflammatory rheumatic disease related to low bone mineral density. Because vitamin D plays an important role in bone metabolism and immune system modulation, the aim of this study was to evaluate the influence of polymorphisms in vitamin D receptor genes ( Spondyloarthritis (SpA) is a term used for inflammatory diseases that are clinically, epidemiologically, and genetically related. They comprise a group of chronic rheumatic diseases classified as ankylosing spondylitis (AS), psoriatic arthritis (PsA), and reactive arthritis (ReA) and SpA which is related to inflammatory bowel disease (IBD-SpA) and undifferentiated SpA (uSpA) , 2. The ERAP1, IL23R, and RUNX3 [The important hereditability of SpA comes from HLA-B27 codified in the major histocompatibility complex (MHC) and defined in 1973 –11 and m2D, an active form of vitamin D, are mediated by their receptor (vitamin D receptor (VDR)), a member of the nuclear hormone receptor superfamily expressed in almost all human cells including macrophages, lymphocytes, and dendritic cells. VDR complex influences the regulation of about 3% of the human genome [The gradual clinical worsening in the manifestations of SpA is linked to inflammation and low bone mineral density of the joints, due to a high production of inflammatory cytokines  and vitan genome . It is an genome , 18. Vitn genome , 19, 20.VDR gene is located on chromosome 12q13.11 and has 75 kb. The gene is composed of 11 exons, of which exons 2 and 3 encode the amino acids involved in DNA binding, and exons 7, 8 and 9 code amino acids involved in vitamin D binding [FokI C > T , ApaI A > C , TaqI C > T , and BsmI C > T . The FokI C > T change occurs in the first of the two potential translation initiation sites of exon 2, in the 5′ coding region of the VDR, and the ACG initiation codon results in a shorter peptide in the 3′ NH2 terminal (424 compared to 427), which has more efficient transcriptional activity. The BsmI and ApaI (both in intron 8) and TaqI (in exon 9) polymorphisms are located near the regulatory 3′ untranslated region of the mRNA and do not determine changes in the structure of the receptor. They are in linkage disequilibrium (LD), and specific haplotypes have been shown to affect VDR mRNA stability and rate of transcription [The  binding , 22. Thecription –33 and Acription , 34–36, VDR), FokI, BsmI, ApaI, and TaqI, with SpA and its clinical forms. These polymorphisms were also correlated with the serum level of vitamin D and with the clinical activity index of the disease, Bath Ankylosing Spondylitis Disease Activity Index (BASDAI).Because vitamin D is important in modulating the immune response and bone metabolism, the aim of this study was to evaluate a possible association between the polymorphisms in the vitamin D receptor gene (N = 141) and serum levels of hydroxyl (OH) vitamin D (N = 118) analyzed before starting treatment and none had vitamin D replacement at the time of diagnosis. The control group was selected by the same rheumatologists and was formed by 197 individuals with no SpA confirmed after clinical, laboratory, and radiological analyses and no vitamin D replacement (N = 23) at the time of the interview. The exclusion criteria for all samples were individuals with chronic diseases such as diabetes and other inflammatory and autoimmune diseases. Patients and controls were unrelated and matched by gender, age, and ethnicity. All participants were from the northwestern region of Paraná, in southern Brazil (22°29′30″-26°42′59″S and 48°02′24″-54°37′38″W), and were classified as mixed ethnicity, with predominantly European origin, based on the ethnic constitution in the state of Paraná as previously described [The study was approved by the Research and Ethics Committee of the State University of Maringá (UEM), number CAAE 27723114. All subjects agreed to participate in the study and signed a consent form. A total of 244 patients with spondyloarthritis (SpA) were included in this study, and among them, 174 had ankylosing spondylitis (AS) and 66 had psoriatic arthritis (PsA). The diagnosis for SpA was performed through clinical, laboratory, and radiological criteria according to the ASAS  2009/2011 criteria –39 by thescribed  and confDNA was obtained from peripheral blood collected in vacuum tubes with EDTA using the salting-out method . The conVDR: FokI C > T (rs2228570) [BsmI C > T (rs1544410), ApaI A > C (rs7975232), and TaqI T > C (rs731236) [The Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) technique was used for genotyping 2228570)  and BsmIs731236) . This me > T rs15410, ApaIHLA-B∗27 was performed using Polymerase Chain Reaction-Sequence Specific Primers (PCR-SSP) according to sequences previously described [Genotyping of escribed . This meescribed .Serum levels of hydroxy (OH) vitamin D were determined using the chemiluminescence technique , following manufacturer's recommendations. The serum levels of vitamin D were classified as sufficient (>30 ng/mL) and insufficient (<29.9 ng/mL) .The evaluation of the clinical disease activity index BASDAI was performed by the same rheumatologists according to criteria previously defined . The evat-test was used to calculate continuous data given as mean ± standard deviation . The possible association of VDR polymorphisms with the disease was carried out using Chi-square and logistic regression, and for these analyses and for estimating the distribution of genotype frequencies according to the Hardy-Weinberg (HW) equilibrium, we applied the OpenEpi Version 3.01 and SNPStats software (https://www.snpstats.net/start.htm) [HLA-B∗27, BASDAI, and vitamin D serum levels were included as covariates in the analysis. Odds ratios and their respective 95% confidence intervals (CI) were defined only for statistically significant values, considering P value to be less than 5%. The Bonferroni adjustment for multiple testing was applied and the corrected value (Pc) for a truly significant value was obtained after the multiplication of P value by the number of analyzed SNPs: two SNPs when considering linkage disequilibrium between BsmI, TaqI, and ApaI . The haplotype frequencies were estimated using the implementation of the expectation-maximization (EM) algorithm coded into the haplo.stats package. Bootstrapping by Haploview software version 4.2 was used to assess the robustness of the results [http://www.biostats.usc.edu/software) was used to calculate the sample size using the less frequent allele (0.16 for ApaI), population risk of 1.5%, and OR = 2.0 for statistical power of 80%.Student's art.htm) . The assart.htm) . Gender, results . QUANTO HLA-B∗27 was more frequent in SpA (35.66%), AS (44.47%), and PsA (15.15%) than in controls (6.59%), and these differences were statistically significant . The HLA-B∗27:05 allele was also more frequent in SpA (16.39%), AS (20.40%), and PsA (6.81%) than in controls (2.28%) . BASDAI >4.0, which corresponds to severe clinical forms of the disease, was 72.91% in SpA, 75.52% in AS, and 66.66% in PsA. The serum level of the hydroxy (OH) vitamin D < 29.9 ng/mL (classified as insufficient) was 52.54% in SpA, 52.56% in AS, and 65.00% in PsA.The characteristics of SpA, AS, and PsA patients and controls are presented in The correlation between serum levels of vitamin D, classified as sufficient (>30 ng/mL) and insufficient (<29.9 ng/mL), with the disease activity index (BASDAI <3.9 and >4.0), is shown in P > 0.05), which means they are good representatives of the control group and also ensure data quality. The genotype and allele frequency distributions of VDR were analyzed in patients and controls considering covariate genders . In the log-additive inheritance model, each copy of C modifies the risk in an additive form; in other words, the homozygous C/C, or ApaI a/a, have a double risk of T/C or ApaI A/a. In addition, the ApaI a allele frequency was lower in patients with PsA than in controls . The genotype and allele frequency distributions of FokI, BsmI, and TaqI in the SpA, AS, and PsA patients were similar to those found in controls, when values were adjusted for gender covariates. These results are shown in Significant differences were found in the VDR genotypes within and between genders, significant differences were also observed for ApaI . Otherwise, ApaI a/a genotype was associated with the risk of AS  and SpA in males when this same genotype was compared with females . These results are shown in After stratified analysis, and considering the distribution of for ApaI . The ApaHLA-B∗27 positive were separately analyzed, the ApaI A/a genotype was associated with protection for PsA, despite a small number of samples , the FokI f/f genotype frequency was higher in the PsA patients with better clinical activity ; however, significance was lost for PsA after Bonferroni correction. The large confidence interval is due to the small number of samples.When genotype and allele frequency distributions of  disease . The BsmBsmI_ApaI_TaqI bAt haplotype, which was a risk factor for PsA (haplotype frequency–hf = 0.078) compared with baT haplotype . Perhaps, this was due to the influence of the ApaI polymorphism.After the haplotype frequency distribution analysis, statistical significance was observed for the VDR polymorphisms and SpA, AS, and PsA was investigated. These associations were correlated with the covariates: gender, HLA-B∗27, BASDAI, and serum levels of vitamin D.Genetic polymorphisms have been studied in recent years in order to assist in the early diagnosis of spondyloarthritis and to understand the etiopathogenesis and improve treatment. Because vitamin D complex is important in the modulation of the immune response and in the homeostasis of calcium and phosphorus, the association between ApaI polymorphism was associated with psoriatic arthritis: ApaI a/a was a protective factor for PsA and for women with PsA, and ApaI A/a was a protective factor for PsA patients who were HLA-B∗27 positive. Furthermore, the FokI f/f genotype contributed to less clinical disease activity in PsA. For SpA and AS, the ApaI a/a and FokI F/F genotypes were risk factors for male patients. To our knowledge, this is the first study to address the association of VDR polymorphism with PsA.The main finding of this study was that the VDR polymorphisms and psoriasis, but few have included patients with PsA. In meta-analysis studies, ApaI, TaqI, BsmI, and FokI polymorphisms and susceptibility to psoriasis were evaluated in Caucasians and Asians [TaqI T/T genotype in Caucasians [ApaI polymorphism, only one study found that ApaI a/a, in a dominant inheritance model, was a risk factor for psoriasis in Caucasians [ApaI A allele was a risk factor for psoriasis in Chinese [ApaI variant was a protective factor for PsA, especially in women and HLA-B∗27 positive patients. Our findings may be explained by autoimmunity and by altered bone remodelling in psoriatic arthritis. In other studies, ApaI a/a was found to be a protector for idiopathic scoliosis [ApaI a allele correlated with higher levels of vitamin D in the serum [FokI f/f genotype was a protective factor for better clinical activity in PsA. Previously, FokI polymorphism was associated with lower bone mineral density [FokI F/F or F/f was a susceptibility factor for rheumatoid arthritis [Psoriatic arthritis is a chronic inflammatory musculoskeletal disease associated with psoriasis. Vitamin D is involved in calcium homeostasis, bone formation, osteoclastogenesis, and osteoclast activity and in the regulation of immune response. The action of vitamin D depends on vitamin D receptor (VDR). Many studies have been conducted to determine the association between d Asians , 31, 33.ucasians . Accordiucasians ; however Chinese . We founcoliosis , and it coliosis . In addihe serum . Althoug density  and FokIrthritis . Due to rthritis .VDR polymorphisms were not associated with AS. However, in a stratified analysis considering gender, the ApaI a/a was a susceptibility factor for AS and SpA in men, and FokI F/F was a risk factor for AS and SpA when compared with the same genotype in women. ApaI a/a was previously correlated with lower bone mineral density [ApaI a/a was also associated to be a susceptibility factor for rheumatoid arthritis [ApaI polymorphism in spondyloarthropathies, the correlation between different bone disorders and inflammatory processes in different clinical forms of the disease must be considered. Regarding the FokI polymorphism, studies have shown that FokI genotypes were associated with the lumbar but not the peripheral bone mineral density in male and Caucasian patients with AS, influencing inflammatory and pain indices [FokI F/F was a risk factor for AS after stratification with vitamin D. This result could be compatible with the lower osteocalcin scores previously associated with AS patients with the FokI F/F genotype [VDR polymorphisms associated with AS were found in two Chinese studies, and TaqI G allele and the TaqI haplotype for rs11168266 and rs11168267 were risk factors for the disease [Ankylosing spondylitis is a chronic inflammatory disease involving the axial skeleton, regions of enthesis, and peripheral joints and predominantly affects males . In this density –56 and w density . ApaI a/rthritis . To bett indices , 35. We genotype . Osteocagenotype . Other V disease , 59.BsmI b/b genotype was associated with the risk of SpA, though significance was lost after Bonferroni correction. Other studies conducted with rheumatoid arthritis have shown that the BsmI b/b genotype was a susceptibility risk factor for the disease [Vitamin D deficiency has been associated with symptoms of SpA, such as pain, inflammation, and structural damage due to altered bone activity , 60–62.  disease , 57.HLA-B∗27 is the main genetic factor associated with SpA and is present in about 90% of patients with AS and 20% with PsA in nonmixed populations [HLA-B∗27 has high polymorphisms named HLA-B∗27:01 to HLA-B∗27:226 . The most common subtypes associated with AS are HLA-B∗27:05, an ancestral allele from which other subtypes have derived (in Caucasians), HLA-B∗27:04 (Chinese), and HLA-B∗27:02 (Mediterranean populations). Two subtypes HLA-B27∗06 and HLA-B27∗09 were not associated with SpA [HLA-B∗27 and HLA-B∗27:05 were associated with SpA (with respective frequencies being 35.66% and 16.39%), AS (44.25% and 20.40%), and PsA (15.15% and 6.81%) when compared with controls. For controls, the HLA-B∗27 frequency was 6.59% and HLA-B∗27:05 was 2.28%, according to what is expected for the Brazilian population . The HLA-B∗27 frequency is lower in SpA in populations with a higher degree of heterogeneity, such as in Latin America, and for AS it was found in 50% to 70% of Brazilian and Ibero-American patients [ulations , 63. HLAwith SpA . In thispatients .VDR polymorphisms with serum levels of vitamin D before initiating treatment protocol in all patients. Studies conducted with patients before initiating treatment protocol and with follow-ups for their clinical evolution may contribute to a better understanding of the pathogenesis of the vitamin D complex in the SpA.The positive points of this study were the judicious selection of patients and controls, which had the diagnostics confirmed by an experienced rheumatologist. In addition, because SpA was primarily linked to males but has been recognized as an important cause of disability among females , the patVDR polymorphisms were associated with psoriatic arthritis: ApaI (7975232) polymorphism was a protective factor for disease and FokI (rs2228570) polymorphism was associated with better clinical disease activity. ApaI and FokI were associated with SpA and AS only when gender and vitamin D sufficiency was considered.In conclusion,


Charadrius nivosus) across its broad geographic range.Understanding the structure and variability of adaptive loci such as the major histocompatibility complex (MHC) genes is a primary research goal for evolutionary and conservation genetics. Typically, classical MHC genes show high polymorphism and are under strong balancing selection, as their products trigger the adaptive immune response in vertebrates. Here, we assess the allelic diversity and patterns of selection for MHC class I and class II loci in a threatened shorebird with highly flexible mating and parental care behaviour, the Snowy Plover (tenuirostris).We determined the allelic and nucleotide diversity for MHC class I and class II genes using samples of 250 individuals from eight breeding population of Snowy Plovers. We found 40 alleles at MHC class I and six alleles at MHC class II, with individuals carrying two to seven different alleles (mean 3.70) at MHC class I and up to two alleles (mean 1.45) at MHC class II. Diversity was higher in the peptide-binding region, which suggests balancing selection. The MHC class I locus showed stronger signatures of both positive and negative selection than the MHC class II locus. Most alleles were present in more than one population. If present, private alleles generally occurred at very low frequencies in each population, except for the private alleles of MHC class I in one island population (Puerto Rico, lineage Snowy Plovers exhibited an intermediate level of diversity at the MHC, similar to that reported in other Charadriiformes. The differences found in the patterns of selection between the class I and II loci are consistent with the hypothesis that different mechanisms shape the sequence evolution of MHC class I and class II genes. The rarity of private alleles across populations is consistent with high natal and breeding dispersal and the low genetic structure previously observed at neutral genetic markers in this species. The genes of the major histocompatibility complex (MHC) are crucial for the immune response in vertebrates , 2. TheiMHC genes display the highest degree of polymorphism within vertebrate genomes , 13. PatGallus gallus and some birds of prey, have an extraordinarily compact MHC region . Hoal” MHC, , 22. In al” MHC, , 23, 24.al” MHC, , 26. Theal” MHC, . Here, nal” MHC, , 27.Within the order Charadriiformes, study of the MHC have so far been restricted to three families: Alcidae, Laridae and Scolopacidae. These studies revealed considerable differences in the diversity and organization of the Charadriiform MHC , 28, 29.Charadrius nivosus, a member of the Charadriidae [Charadrius alexandrinus and considered to be a cosmopolitan species, but the analysis of genetic and phenotypic traits has shown that the two species are separate and that Snowy Plovers are characterized by low genetic diversity at neutral genetic markers [C. n. nivosus) in North America, Cuban Snowy Plovers (C. n. tenuirostris) in the Caribbean, and the Peruvian Snowy Plover  in South America [We investigated the diversity and organization of the MHC in the Snowy Plover adriidae . Until r markers –33. Thre America . Coastal America –37. HereWe discarded 32 samples because they did not pass the quality filters or because they did not have the minimum number of reads per amplicon. Among the 218 remaining samples from eight populations, we found a total of 40 alleles. Five alleles  showed a 3-bp insertion at position 178–180  in the α2 domain, retaining the correct reading frame. Meanwhile, the remaining 35 alleles  did not show this insertion. The alignment of exon 3 displayed five of the eight highly conserved amino acid (peptide main-chain) sites in birds  suggested stronger positive selection at PBR sites in comparison with non-PBR sites (Table C. n. tenuirostris (Puerto Rican population) showed the lowest level of positive selection. Using GARD (Genetic Algorithm for Recombination Detection) we found no evidence for recombination among the 40 alleles.The average nucleotide diversity (π) for the complete sequence was similar among the three lineages, ranging from 0.05 to 0.07. Populations at Nayarit and Puerto Rico showed the highest levels of nucleotide diversity, the nucleotides distance and average nucleotide diversity at PBR sites, whereas populations at Utah, San Quintin and Ceuta had the lowest levels of nucleotide diversity (both complete sequence and PBR sites) and nucleotide distance Table . Within es Table . Among gdN/dS proportion at PBR sites was higher for the Snowy Plover, indicating stronger positive selection at these sites in comparison with the other three charadriiform species.Average nucleotide diversity and number of segregation sites were lower than those reported from Red Knot, Icelandic Black-tailed Godwit and Red-billed Gull Table . NeverthAs in Red Knots and Red-billed Gulls, we found alleles with putatively non-classical functions among the Snowy Plover alleles. These non-classical alleles (Chni-UA*17 to UA*21) formed a well-defined cluster in the phylogenetic network Fig. a.Fig. 2We discarded 36 samples, as they did not pass the quality filters. In total, we found six alleles across 214 individuals of eight populations. We found no more than two alleles per individual  for subspecies es Table . The Bayes Table b. Sites es Table . All tenThe average nucleotide diversity of Snowy Plovers (π = 0.06) was well within the range of diversity observed in other Charadriiformes  but showed similar numbers of alleles . With the exception of the island population of Puerto Rico (lineage tenuirostris), private alleles generally occurred in low frequencies , and we found all six alleles in this population. The other populations had three to five alleles, except for Puerto Rico, where we detected only one allele  that shared a 3-bp insertion. The observed insertion kept the reading frame intact, presumably preserving their function. Contrary to the non-classical alleles, these five alleles with a 3-bp insertion show a higher nucleotide diversity, including several non-synonymous changes between these five alleles, suggesting these alleles have classical functions. In birds, insertions are less frequent than deletions in MHC genes, and it has been suggested that insertions have a reduced adaptive advantage . HoweverFor both MHC classes, we observed that the number of sites under negative (purifying) selection was higher than sites under positive (diversifying) selection when we considered the full sequence. As PBR sites are interacting directly with the antigens derived from pathogens, it is expected that these sites are subject to stronger positive diversifying selection than non-PBR sites. Consistent with this, we found that sites with signatures of purifying selection were predominantly non-PBR sites, whereas five of the six sites under diversifying selection in the MHC class I and in one site at MHC class II corresponded to PBR sites. Similar patterns were observed in another shorebird, the Icelandic Black-tailed Godwit , and shoPlasmodium and Haemoproteus spp.; [Mycobacterium; [Our finding that more sites showed signatures of positive selection at MHC class I than at MHC class II differs from a recent global analysis of selection at the avian MHC . Here, tus spp.; ) and baccterium; ) have alcterium; , 52. In nivosus and occidentalis), we found a similar number of alleles and nucleotide diversity. This result is consistent with the high gene flow observed among Snowy Plover populations [tenuirostris) showed two privates alleles at MHC class I at moderate frequencies .Brachyramphus marmoratus – 27 alleles, [Spheniscus humboldti – 0.03 to 0.04 – and Magellanic Penguin Spheniscus magellanicus – 0.05 to 0.06, [Fulica atra – 0.11, [Tetrao tetrix – 0.11, [Comparing allelic diversity across species, we found that diversity at MHC class I in Snowy Plovers  was similar to other Charadriiformes species . Despitto 0.06, ; or grouto 0.06, ). For MHto 0.06, ) and othto 0.06, ; Eurasia – 0.11, ; and Bla – 0.11, ).We developed novel MHC markers to amplify the PBR exon 3 of MHC class I and PBR exon two of MHC class II for the threatened Snowy Plover. These are the first markers for MHC in the family Charadriidae and we anticipate that they will be of high utility for studying MHC in other plover species. Overall, genetic diversity at MHC in Snowy Plovers was low to moderate and likely to be shaped by past demographic processes such as bottlenecks and island colonization. In line with population genetic studies, we find that there is limited genetic differentiation attributable to geographic variation, consistent with the high gene flow observed in this species. Contrasting differences in the allelic diversity between MHC class I and class II indicate stronger positive selection at MHC class I than at MHC class II. These differences may reflect variation in exposure to intracellular and extracellular pathogens , but furWe collected blood samples of 250 adult and juvenile Snowy Plovers from eight populations across North, Central and South America between 2006 and 2016 . We perWe determined the concentration of the PCR2 product using a fluorometer (FLUOstar OPTIMA) and 2 μl of the product. We pooled samples from eight individuals by taking 20 ng per sample and cleaning up the multiplexed PCR products with AMPure XP beads, as described above, with volumes adjusted to a 50 μl solution (concentration of 0.5 X). We used a TapeStation 4200 (Agilent Genomics) to confirm that there were no primer dimers present in the purified samples. We then quantified the PCR products using a qPCR with the KAPA library quantification kit (KAPA Biosystem) using 10 μl reaction volume (8 μl of SYBR Master Mix and 2 μl of template/standard or control), with the program: 95 °C for 5 min, followed by 35 cycles of 94 °C for 30 s and 60 °C for 45 s. We then pooled equimolar amounts per library, preparing six libraries in total, quantified the concentration of the pool with a Qubit  and submitted 4 nM per library for sequencing using 250 bp paired-end (500 cycles) Illumina sequencing on the MiSeq  in six separate runs at the Sheffield Diagnostic Genetics Service.For the raw MiSeq data processing, we used the Amplicon Sequencing Analysis Tool (AmpliSAT) web server . This toWe blasted all putative alleles from Illumina sequencing to the GenBank (NCBI) nucleotide database to examine their similarity to known MHC alleles from other species. The alleles were named Chni-UA*01 to UA*40 (MHC class I) and Chni-DAB*01 to DAB*06 (MHC class II), following the nomenclature suggested by Klein et al. .Saa), nucleotide diversity (π), evolutionary distance for nucleotide sequences (dnt) and evolutionary distance for amino acid (daa), for each of the eight populations included in the study. For the evolutionary distance analyses, we inferred the appropriate substitution models based on the best-fit model (using AICC) using JModeltest 2.1.10 [p-distance model with uniform rates to assess amino acid sequences distances. For MHC class II, we implemented the Jukes-Cantor + G model with a gamma distribution, setting the substitution rate α to 0.8 for nucleotide sequences, and we calculated amino acid sequence distances using a p-distance model with uniform rates.We used MEGA 7.0.18  for initt 2.1.10 . For MHCt 2.1.10  with a gdN/dS > 1). To assess the impact of positive selection on nucleotide diversity, we compared ω at PBR sites and non-PBR sites. We inferred PBR sites based on previously documented transcripts . We calclass I:  with Jukg/fubar, . As recog/fubar, , we testrg/gard, ).Calidris canutus [Limosa limosa islandica [Larus scopulinus [Uria alge [Alca torda [Fratercula artica [Rissa tridactyla [Gallinago media [Philomachus pugnax [Limosa limosa [Saa) and nucleotide (Snt) segregation sites, nucleotide diversity (π), as well the synonymous (dS) and non-synonymous (dN) substitution rates, using the same parameters described above. We visualized phylogenetic relationships between the MHC class I and class II alleles in Snowy Plovers through the Neighbor-net algorithm implemented in SplitsTree 4.14.6 [We compared nucleotide diversity at MHC loci across the Charadriiformes using data available at GenBank. For MHC class I, we obtained sequences from three other charadriiform species: Red Knot  canutus , Icelandslandica  and the opulinus . As onlyria alge , Razorbica torda , Atlantia artica , Black-lidactyla , Great Sgo media , Ruff Phs pugnax  and Blaca limosa . As we he 4.14.6 . This mee 4.14.6 , 25. FinAdditional file 1: Figure S1. Graphical representation of nucleotide diversity and dN/dS ratios for MHC class I and MHC class II. Silhouettes represent Charadriiform species, i.e. MHC class I  and MHC class II . Arrows represent strength of selection in both MHC classes for the Snowy Plover in comparison to the other species. Table S1. Results of generalized linear models testing the differences in the number of alleles per individual between populations for the MHC class I and class II in the Snowy Plover. Table S2. Results of generalized linear models testing the differences in the number of alleles between populations for the MHC class I and class II in the Snowy Plover. Table S3. Sequences ID list randomly drawn for the MHC class I and MHC class II species comparison.


Studies have shown that poor sleep could result in many unpleasant consequences and is prevalent in nurses. Considering the fact of high stress, overwhelming workload and many night shifts in the emergency department in China, this study aimed to evaluate the current status of emergency nurses’ sleep quality in public hospitals in Shandong, China and explored its influencing factors.A self-administered questionnaire incorporating the Job Content Questionnaire and Pittsburgh Sleep Quality Index (PSQI) was conducted among 4856 emergency nurses in five randomly selected city emergency command systems in Shandong, China. The association of potential influencing factors, including occupational, psychosocial and individual factors, with poor sleep (PSQI> 5) was quantified by multivariate logistic regression analysis.The average PSQI score of 4730 emergency nurses in public hospitals was 8.2 ± 3.9, including 3114 (65.8%) subjects with PSQI > 5 and 2905 (61.4%) > 8; these figures were found highest for 337 emergency nurses in 14 tertiary hospitals with 11.8 ± 4.3, 257 (76.3%) and 232 (68.8%), followed by 1044 emergency nurses in 43 secondary hospitals with 9.5 ± 3.9, 725 (69.4%) and 675 (64.7%) and 3349 emergency nurses in 167 primary hospitals with 7.4 ± 3.5, 2132 (63.7%) and 1998 (59.7%). The following factors were associated with poor sleep: hospital level , female sex, less of exercise, long work hours per week, many patients in the charge of at night, high monthly night shift frequency  and high occupational stress.The sleep quality of emergency nurses in public hospitals in China was poor, especially in tertiary hospitals. Many factors as listed above, especially occupational stress, night shift taking and workload at night, should be considered when improving emergency nurses’ sleep quality. With increasing modernization and accelerating life rhythms, sleep is increasingly becoming an important global public health issue influencing millions of people . Poor slNurses have been reported to suffer from poor sleep, especially in large-scale hospitals –24. In tStress, one’s response to environmental stressors, has been shown to be a hazard for many chronic diseases . In the In Mainland China, the emergency department is the first-line place to treat critical, serious and urgent patients and also the basic component of a whole city emergency network. But considering the shortage of emergency nurses, population aging, the large population base and patients’ inclination of going to large hospitals when face emergencies , 32, theA cross-sectional study was implemented in Shandong, China from June to December in 2019. As there were no more than 20,000 emergency nurses in Shandong, China and we set type 1 error as 0.05 and the allowable error as 0.02, given an estimated 50% prevalence rate of poor sleep based on previous studies , 24, theThe Ethics Committee of Shouguang People’s Hospital and Health Commission of Weifang City (wfwsjk_2019_188) approved the study. All the nurses participated in the study were voluntary and gave written informed consent.A paper-based self-administered questionnaire was developed, based on advice of epidemiologists and occupational health experts, field observation, interviews with emergency nurses and established questionnaires –39. The Section one dealt with individual information including sex, age, body mass index , educational level, marital status, children in household, smoking, alcohol/tea consumption, exercise in leisure time, income and professional rank .Section two dealt with work characteristics including hospital level , work years, number of patients in the charge of in the daytime, whether taking shift work (Yes/No), number of night shifts per month, number of patients in the charge of at night and employment status (permanent vs. temporary/contract).Sleep quality was assessed in section three, using the Pittsburgh Sleep Quality Index (PSQI) scoring system  which haThe forth section dealt with occupational stress evaluated by Job Content Questionnaire (JCQ) based on the job demand-control-support model, which has been translated into Chinese language and been validated in the Chinese people –39. In oP < 0.05 and P < 0.01, respectively. All tests were conducted at the 0.05 level of statistical significance.Statistical analysis was conducted on SPSS version 25.0. The differences were first tested by bivariate analysis such as one way analysis of variance (for continuous variables), chi-square test , or rank sum test . Every potential influencing factor for poor sleep was individually entered into a logistic regression analysis with poor sleep as dependent variable (PSQI> 5) and odds ratio (OR) with 95% confidence interval (C.I.) was calculated. Then multivariate logistic regression analysis was used to quantify the association of potential influencing factors, including workload, occupational, psychosocial and individual factors, with poor sleep. Variables selected at the level of 0.25 in bivariate logistic regression analysis were entered into the final multivariate logistic regression model with poor sleep (PSQI > 5) as dependent variable. The statistics for variable entry and removal were set at Of the 4730 emergency nurses, 3349 were from primary hospitals, 1044 from secondary hospitals and 337 from tertiary hospitals. The average age of the 4730 emergency nurses was 35.3 ± 6.8 years old, including 4185 female nurses and 545 male nurses. All nurses had at least 12 years of education. The average PSQI score was 8.2 ± 3.9, including 3114 (65.8%) nurses with a PSQI > 5 and 2905 (61.4%) nurses with a PSQI > 8.The emergency nurses in tertiary hospitals were younger than primary hospitals and secondary hospitals and had less years of service in the emergency department. The emergency nurses in tertiary hospital were in charge of more patients at night and in the daytime than those in primary hospitals and in secondary hospitals and experienced more work hours per week. The PSQI score of emergency nurses in tertiary hospitals was 11.8 ± 4.3, statistically higher than those in primary hospitals (9.5 ± 3.9) and in secondary hospitals (7.4 ± 3.5). The prevalence of poor sleep (PSQI > 5) and severe sleep problem (PSQI> 8) among emergency nurses was 76.3 and 68.8%, respectively, in tertiary hospitals, both statistically higher than in primary hospitals  and secondary hospitals . For more details, see to Table After bivariate logistic regression analysis between poor sleepers (PSQI> 5) and good sleepers (PSQI≤5), multivariate logistic regression analysis revealed that poor sleep quality was independently positively associated with hospital level , female sex, more work hours per week , higher number of patients in the charge of at night , higher nigh shift frequency per month , higher psychological job demand and higher job insecurity, and was negatively associated with more exercise in leisure time , higher workplace social support and higher job control. For more details, see to Table In the study, we found that the mean PSQI score of nurses in the emergency department in public hospitals in Shandong, China was 8.2 ± 3.9, most highly found in tertiary-hospital emergency nurses (11.8 ± 4.3), followed by secondary-hospital emergency nurses (9.5 ± 3.9) and primary-hospital emergency nurses(7.4 ± 3.5). The prevalence of poor sleep quality (PSQI> 5) and severe sleep problems (PSQI> 8) of emergency nurses was 76.3 and 68.8%, respectively, in tertiary-hospital emergency nurses, both statistically higher than secondary-hospital emergency nurses (69.4 and 64.7%) and primary-hospital emergency nurses (63.7 and 59.7%). The poor sleep quality among emergency nurses was associated with hospital level , workload , night shift work and work stressors , in addition to individual factors (sex and exercise in leisure time). More exercise in leisure time was found to be negatively associated with poor sleep among emergency nurses.To our knowledge, this may be the first large-scale multi-center study assessing sleep quality of emergency nurses working in public hospitals including primary, secondary and tertiary hospitals in China and its influencing factors, by applying standardized and well-validated instruments to diagnose sleep quality and measure occupational stress. The emergency nurses participated in the study were sampled from among all the 17 city emergency command systems in Shandong, China and the response rate of 86.6% was acceptable. The limitations of the study include the cross-sectional design and self-report measures, not use objective tests. Therefore, no solid causal directions could be made from the cross-sectional study. A cohort study might be needed in the future. As this study collected data on sleep among emergency nurses in public hospitals only, the results might not be generalized to other population, such as emergency nurses in private hospitals.In our study, the prevalence of poor sleep and severe sleep problem was 65.8 and 61.4%, respectively, in public-hospital emergency nurses, higher than other hospital workers , 40, 41.Studies , 45 haveEmergency nurses with more night shifts per month  suffered from poor sleep more easily, consistent with other studies , 51–54. In this study, high number of patients in the charge of at night and long work hours per week were associated with emergency nurses’ poor sleep. Number of patients in the charge of at night and work hours per week could be taken as signs of workload, and increased workload has been proved to be a hazard for fatigue or stress . Both deAttention should be given to the high prevalence of poor sleep of nurses in the emergency department in public hospitals in China. Factors of many night shift taking, high workload at night and long work hours per week were significantly associated with emergency nurses’ poor sleep, in addition to individual factors such as sex and exercise. Exercise and decreasing occupational stress might be considered when exploring preventive measures for poor sleep in emergency nurses.


Staphylococcus aureus (MRSA) is known for serious health problems. Testing new inexpensive natural products such as mango kernel  may provide alternative and economically viable anti-MRSA drugs. In the current study, we screened clinical isolates from Aseer Central Hospital, Saudi Arabia, during 2012–2017 for MRSA and tested an ethanolic extract of mango kernel for anti-MRSA activity. Brief confirmation of MRSA was performed by the Vitek 2 system, while antibiotic sensitivity of strains was tested for their clinical relevance. The In vitro disc diffusion method was used to test the anti-MRSA activity of the ethanolic mango kernel extract. The antimicrobial activity of mango kernel was compared to that of standard drugs (oxacillin and vancomycin). Of the identified 132 S. aureus strains, 42 (31.8%) were found to be MRSA and their prevalence showed a clear increase during the last two years . MRSA strains showed 100% sensitivity to vancomycin, teicoplanin, linezolid, tetracycline, daptomycin, tigecycline, and tobramycin and 100% resistance to ampicillin and 98% to penicillin. The ethanolic extracts of mango kernel were found active against both S. aureus and the MRSA strains. Inhibitory activities (mean ± SE) were achieved at concentrations of 50 mg/mL (20.77 ± 0.61), 5 mg/mL (16.18 ± 0.34), and 0.5 mg/mL (8.39 ± 0.33) exceeding that of vancomycin (p=0.0162). MRSA strains were sensitive to mango kernel extracts when compared to vancomycin. Therefore, ethanolic extracts of mango kernel can be escalated to animal model studies as a promising leading anti-MRSA drug candidate and can be an economic alternative to high-priced synthetic antibiotics.Methicillin-resistant  Staphylococcus aureus is a Gram-positive, ubiquitous bacterial pathogen that can perfectly adapt and is capable of living in different states. It has been reported that S. aureus can survive in an inanimate environment, existing as a colonizer or commensal, and may form biofilms [S. aureus is an important cause of a wide range of clinical infections including bacteraemia and infective endocarditis, osteoarticular, skin and soft tissue, pleuropulmonary, and device-related infections [S. aureus (MRSA), especially in nosocomial health settings, is a well-known health threat and economic burden worldwide [biofilms . S. aurefections . The sprorldwide .S. aureus related to various types of infection operations and a substantial contribution of MRSA. Preventive strategies aimed specifically at MRSA could reduce healthcare costs and improve patient outcomes [The introduction of newer potent systemic antibiotic combinations failed to control the endemic reservoir of multidrug-resistant bacteria and suggests that such policies have little impact . There aoutcomes , 8. The outcomes .Mangifera indica (mango kernel) is an example which was recorded to have antimicrobial activities. Application of such innovative results may help in controlling the infection and preventing their spread.It is necessary to search for new agents that are better, cheaper, and without side effect for treating infectious diseases especially in developing countries. It has been long known that an extensive variety of plant and plant-derived natural products are used in the treatment of infections. Phytoconstituents have been found to inhibit bacteria, fungi, viruses, and pests . The curEscherichia coli including members of Enterobacteriaceae family, while mangiferin, a bioactive component, has been reported to possess remarkable anti-influenza activity [Salmonella typhi at 250 mg/mL. Thai mango (M. indica L. cv. “Fahlun”) seed kernel extract was found to have potent antimicrobial properties [S. Aureus [Actinobacillus actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, Fusobacterium nucleatum, and Peptostreptococcus sp. It has been suggested to be used as an alternative remedial agent or an adjunctive treatment along with classic antibiotics [Previous studies have shown that extracts of mango kernel are promising candidates against numerous species of pathogenic bacteria –13. Leavactivity . Both acoperties . Aqueous. Aureus . Mangifeibiotics , 17.This study aimed to examine the clinical significance of MRSA and to determine their antimicrobial profile against routine antimicrobial agents and against ethanolic extracts of mango kernel.This study was conducted between 2012 and 2017 at definite time intervals and duration at the Department of Microbiology and Clinical Parasitology, College of Medicine, King Khalid University, and Aseer Central Hospital, Abha, Saudi Arabia. Ethical approval was obtained from the Ethics Committee of the College of Medicine, King Khalid University, vide reference letter, REC # 2017-02-17. Patient information, clinical data, and bacteriological and antimicrobial records of the isolates were collected and saved in a spreadsheet for analysis.S. aureus strains were included in the study for comparative reasons.All isolates were Gram-positive coccus and catalase- and coagulase-positive. Coagulase-negative staphylococci and duplicate isolates were excluded. Both MRSA and other coagulase-positive S. aureus strains (n = 132) were isolated from various clinical samples of patients attending Aseer Central Hospital, Abha, Saudi Arabia. Samples were processed for culture by standard conventional methods, and initial identification was performed by standard phenotypic tests [ic tests , 19. IsoAntimicrobial susceptibility testing was carried out using the Vitek 2 system, according to the manufacturer's information. The following antimicrobial agents have been tested for at least one or more strains: Amikacin; Amox/K Clav; Amp/Subactm; Ampicillin; Azithromycin; Cefepime; Cefotaxime; Cefoxitin; Cefuroxime; Cephalothin; Chloramphenicol; Ciprofloxacin; Clarithromycin; Clindamycin; Colistin; Cotrimox; Daptomycin; Erythromycin; Fosfomycin; Fusidc Acid; Gentamicin; Imipenem; Levofloxacin; Linezolid; Meropenem; Moxifloxacin; Mupirocin; Nalidixic; Neomycin; Nitrofurantoin; Norfloxacin; Ofloxacin; Oxacill/Methicill; Oxacillin; Penicillin; Pip/Tazo; Piperacillin; Rifampin; Sulfamethoxazole; Synercid; Teicoplanin; Tetracyclin; Tigecycline; Tobramycin; Trimeth/Sulfa; and Vancomycin.S. aureus according to the NCCLS methodology [μg/ml). Plates were inoculated with the 0.5 McFarland bacterial suspensions and incubated at 35°C for 24 h.Oxacillin screen agar testing was performed for hodology  using MuFresh mango kernel was collected from local markets in Abha, Saudi Arabia and was identified and authenticated at the Department of Biological Sciences, College of Science, King Khalid University. The collected seeds were stored in polythene bags at room temperature till further use.Mango kernel seeds were cleaned, air-dried, and coarsely powdered. 1000 grams of powder was extracted with ethanol in a Soxhlet apparatus. The extracts were filtered, and the filtrates were vaporized to dryness and weighed in order to determine the % yield of the extracts, following the formula: % yield =  × 100. The stock solutions of the crude ethanolic extracts were prepared by dilution of the dried extracts with 50% ethanol to obtain the desired final concentrations of 50 mg/mL, 5 mg/mL, 0.5 mg/mL, and 0.05 mg/mL. These concentrations were used to impregnate filter paper disks (5.5 mm diameters).Disks impregnated into 50% ethanol were used as control, while standard antimicrobial disks such as oxacillin and vancomycin (Difco) were used as positive control.n = 51) recovered from clinical samples isolated from patients at Aseer Central Hospital were used in the study. One to three loopful of 24 h old cultures from each test strains were used to prepare 0.5 McFarland standard suspensions.MRSA bacterial strains (Staphylococcus aureus and MRSA according to [Minimum inhibitory concentration (MIC) of ethanolic extraction of mango kernel was determined by using descending concentrations of the extract. The MIC of the extract was diluted using sterile saline and was tested for antibacterial activity against rding to  with somp value <0.05 was considered as the level of significance.Statistical analysis of data obtained from triplicate experiments was performed using SPSS software version 16.0. The nonparametric test for several independent samples was used to compare variation among means of different inhibition zones induced by extracts in comparison with positive controls. Linear regression was applied to assess the relationships among several independent samples. S. aureus strains isolated from various clinical materials of patients attending Aseer Central Hospital, Abha, Saudi Arabia in five years (2016-2017) is shown in S. aureus strains, 42 (31.8%) were found to be MRSA and the rest (68.2%) were S. aureus. The prevalence of MRSA demonstrated an obvious rise during 2016-2017 compared to the previous three years (p < 0.001).Prevalence of MRSA and other S. aureus strains originated from all age groups, but the majority was from 20- to 40-year-old patients. The strains were recovered predominantly from the respiratory specimens (77.3%) including endotracheal tube (53.8%), throat swab (10.6%), sputum (6.8%), and tracheal secretion (5.3%), followed by blood (10.6%) and wound specimens (4.5%).MRSA and other S. aureus strains to routine antimicrobials is shown in S. aureus and the MRSA strains. Inhibitory activities (mean ± SE) were achieved at concentrations of 50 mg/mL (20.77 ± 0.61), 5 mg/mL (16.18 ± 0.34), and 0.5 mg/mL (8.39 ± 0.33) but not at 0.05 mg/mL (0 ± 0.00). Inhibition zone by oxacillin was 9.45 mm while vancomycin was 20 ± 0.24 mm. The MIC was found (0.15625 mg/mL), whereas the MBC was (0.25 mg/mL). The antagonistic value of mango kernel at 50 mg/mL exceeded that of vancomycin (p=0.0162) but at 5 mg/mL was found to be less active than vancomycin (p < 0.001); nevertheless, at this concentration it exceeded oxacillin (p < 0.001).The susceptibility of MRSA and S. aureus strains to various concentrations of ethanolic extract of mango kernel is shown in The susceptibility of MRSA and S. aureus and the MRSA strains (The ethanolic extracts of mango kernel were found active against both  strains . InhibitS. aureus and MRSA is shown in A linear regression curve illustrating the activity of various concentrations of ethanolic extract of mango kernel against p=0.0162) but at 5 mg/mL, it was found to be less active than vancomycin (p < 0.001); nevertheless, at this concentration, it exceeded oxacillin (p < 0.001)  .The prevalence of MRSA is high and rapidly increasing worldwide but is a major public health worry in developing countries. MRSA is one of the important pathogens that result in heavy health and economic burden throughout the world . But theLack of surveys and collection of epidemiological data and absence of efficient health control programs are contributing to the spread of MRSA , 24. ThoMRSA is a major contributor and huge burden in the management of nosocomial disease. The overall global trend displays a disturbing rise of MRSA infections in addition to increased occurrence of multidrug resistance (MDR) strains . We had S. aureus, vancomycin-resistant enterococci, macrolide- and penicillin-resistant Streptococcus pneumoniae, extend-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae, carbapenem-resistant Enterobacteriaceae, and multidrug-resistant Pseudomonas aeruginosa and Acinetobacter spp., are becoming prevalent in many Asian countries including Saudi Arabia [Escherichia coli and staphylococci were the main etiological agents of infections in Aseer region, while purulent exudates of wounds and abscesses were the main source of S. aureus. Also, a higher rate of MRSA was detected [The antibiotic sensitivity pattern of the all the MRSA isolates were comparable thought the study period while antibiotic-resistant bacteria, including community-acquired and hospital-acquired MRSA, vancomycin-intermediate i Arabia . Our eardetected .S. aureus in the Kingdom with a growing prevalence of both nosocomial and community MRSA isolates [The antimicrobial resistance among these pathogens in Saudi Arabia in the last decades and their antimicrobial resistance has increased among isolates . The antisolates . Furtherisolates . High-rein vivo actions including toxicity, dose, and mechanism of anti-MRSA activity of mango kernel extracts.Ethanolic extracts of mango kernel showed a significant activity against MRSA isolates. We did not observe any variation in anti-MRSA activity over five years of time nor varied isolates. Mango kernel extracts could be the best and economic alternative to combat growing antibiotic resistance observed thoughout the Middle East and Saudi Arabia. The study could be escalated to small animal models addressing the


Breast cancer (BC) is the most common malignancy in women. We retrieved medical records from >2,000 Chilean BC patients over the 1997–2018 period. The objective was to assess changes in clinical presentation or prognosis of our patients throughout these 20 years of practice. Although most variables did not display significant variations, we observed a progressive increase in stage IV BC over this period. Our data showed that tumour stage III/IV or HER2-enriched subtype tumours were associated with poorer prognosis. In contrast, we found that patients diagnosed by mammography had better overall survival. We speculate that better screenings and more sensitive imaging could explain the unexpected rise in stage IV cases. Our results support mammography screenings as an effective measure to reduce BC-related mortality. Worldwide, breast cancer (BC) is a high incidence cancer among women. In Chile, it is women’s leading cause of cancer death . ReportsThe widespread use of mammography, better treatments and supportive care have allowed a progressive improvement in prognosis and a sustained 1%–2% increase in survival every year over the last 30 years in developed countries [Evidently, changes in diagnostic methods and staging of the disease  may be aRetrospective analysis of patients with invasive BC was performed, including all the women treated between January 1997 and August 2018 at Nuestra Señora de la Esperanza Cancer Centre in the Pontificia Universidad Católica de Chile or the Red de Salud UC-Christus Health Network, that were registered in our database. The Scientific and Ethics Committee approved this study. Assessed variables included: age at diagnosis, reason for attending the clinic (RAC) (this is the reason why patients consulted the clinic), dividing those who were motivated by symptoms or signs and those who were motivated by routine mammography findings, TNM  stage , histolop-value < 0.05 was considered statistically significant. Analyses were performed in SPSS v21 (IBM).According to their distribution, data were presented as average ± standard deviation or median (range). Categorical variables are presented as frequency or percentage. Continuous variables were compared by Student’s t-test, categorical variables were compared by chi-square or Fisher’s exact; p = 0.34). Tumour stage at diagnosis was obtained for 2,470 cases. As expected, most patients were stage I/II (74.4% combined). Tumour subtype was obtained for 2,286 patients (84%). Again as expected, the majority were luminal/hormone dependent BCs  were diagnosed by mammography, the remaining 63.1%  by symptoms. Especially, % of stage I was significantly higher among those diagnosed by mammography compared to symptoms .Medical records from 2,723 BC patients diagnosed between 1997 and 2018 were analysed . Clinicap = 0.0001). Solid line in n = 230), 40% GH2 (n = 514), and 42.1% HG3 (n = 540).Interestingly, we found a progressive and significant increase in stage IV cases over the assessed period . A Poissp = 0,621). As expected, survival rates were significantly lower on stage IV cases , with 451 deaths (16.6%). Five-year overall survival (OS) for the entire group was 88.6%. OS at 5 years was statistically similar between the first and second decade of the study  increase in early-stage BC incidence. This has been confirmed by demographic data from the Surveillance, Epidemiology and End Results (SEER) database in the United States (US), over the 1973–2008 period [d period  and 1B. Previous studies demonstrate that patients that come to the clinic after the appearance of signs/symptoms (versus mammography) almost double their risk of death. Conversely, breast neoplasms diagnosed by mammography display better prognosis, survival rates  and reduThe Scientific and Ethics committee at the Pontificia Universidad Catolica de Chile approved this research. Given the retrospective nature of the study no sensitive information was obtained from patients and all data were anonymised.The authors declare no conflicts of interest.FONDECYT-Iniciacion grant #11161103 (CS).


Postoperative pulmonary complications (PPCs) and hypoxaemia are associated with morbidity and mortality. We aimed to evaluate the feasibility and efficacy of lung ultrasound (LUS) to diagnose PPCs in patients suffering from hypoxaemia after general anaesthesia and compare the results to those of thoracic computed tomography (CT).2) less than 92% for more than 30 s under ambient air conditions. LUS was performed by two trained anaesthesiologists once hypoxaemia occurred. After LUS examination, each patient was transported to the radiology department for thoracic CT scan within 1 h before returning to the ward.Adult patients who received general anaesthesia and suffered from hypoxaemia in the postanaesthesia care unit (PACU) were analysed. Hypoxaemia was defined as an oxygen saturation measured by pulse oximetry  undergoing abdominal surgery , video-assisted thoracic surgery , major orthopaedic surgery , neurosurgery  or other surgery  were included. CT diagnosed 327 of 1356 lung zones as atelectasis, while LUS revealed atelectasis in 311 of the CT-confirmed zones. Pneumothorax was detected by CT scan in 75 quadrants, 72 of which were detected by LUS. Pleural effusion was diagnosed in 144 zones on CT scan, and LUS detected 131 of these zones. LUS was reliable in diagnosing atelectasis , pneumothorax  and pleural effusion .Lung ultrasound is feasible, efficient and accurate in diagnosing different aetiologies of postoperative hypoxia in healthy-weight patients in the PACU.NCT03802175, 2018/12/05, www.ClinicalTrials.govCurrent Controlled Trials  Postoperative pulmonary complications (PPCs), including atelectasis, pulmonary oedema, pneumonia, etc., are common, persistent and related to poor patient outcomes, medical costs, hospital readmissions and even significant mortality . HypoxaeRapid diagnosis and appropriate management must be made by the anaesthesiologist once hypoxia occurs postoperatively. The use of chest X-rays (CXR) is limited due to the disadvantage of poor quality . AlthougThe aim of this study was to evaluate the feasibility and efficacy of LUS to diagnose PPCs in patients suffering from hypoxaemia after general anaesthesia in the postanaesthesia care unit (PACU) and compare LUS results with those of thoracic CT.ClinicalTrials.gov (NCT03802175) before patient enrolment. Informed consent was obtained from all patients. Adult patients who received general anaesthesia and suffered hypoxaemia in the PACU were included in this study. Postoperative hypoxaemia was defined as decreased oxygen saturation measured by pulse oximetry (SPO2) less than 92% for greater than 30 s while under ambient air conditions 20 min after extubation [2; lack of cooperation due to cognitive dysfunction; residual muscle relaxants resulting in incomplete recovery of muscle strength ; respiratory forgetfulness from residual opioid; haemodynamic instability; anaemia; and significant bleeding, fever or hypothermia. In addition, patients were withdrawn if their SPO2 declined to 85% or less or if admission to the intensive care unit (ICU) occurred.The study was approved by the review committee of the Second Affiliated Hospital of Zhejiang University  and registered at tubation . The exc2) of 1.0 for 3 min. Anaesthesia was induced with midazolam 0.05–0.1 mg/kg, sufentanil 25–35 μg, etomidate 0.2–0.4 mg/kg and rocuronium 0.5–0.7 mg/kg. A proper double-lumen endotracheal tube was intubated to perform one-lung ventilation (OLV) during video-assisted thoracoscopic surgery (VATS), whereas a common tracheal tube was inserted for two-lung ventilation (TLV) during non-VATS. Continuous intravenous propofol, remifentanil and inhalational sevoflurane were utilized for anaesthesia maintenance after intubation. Supplemental cisatracurium was provided for adequate muscle relaxation when needed. Volume-controlled ventilation with tidal volume of 5–8 mL/kg (5–6 mL/kg for OLV and 6–8 mL/kg for TLV), respiratory rate (RR) of 12–15 breaths/min, FiO2 of 0.5–0.6 and positive end-expiratory pressure (PEEP) of 5 cm H2O was utilized to maintain an end-tidal carbon dioxide pressure (PETCO2) between 35 and 45 mmHg and a peak airway pressure of less than 30 cmH2O (specific parameter was adjusted according to the type of surgery and patient’s condition). Depth of anaesthesia monitoring was completed by bispectral index (BIS) with an appropriate value of 40–60. Before closing the chest, each patient undergoing VATS received a recruitment manoeuvre (RM) by forcing sustained inspiration at the level of 30–40 cm H2O airway pressure for 10–20 s, and then OLV was converted to TLV until extubation. In addition, a chest tube was connected to a water-sealed bottle to provide drainage of any leaked air or fluid. Those undergoing non-VATS did not receive RM. All patients were transported to the PACU after the operation. Before extubation, the mechanical ventilations in the PACU was the same as that in the operating room. Extubation was performed when the following criteria were met: VT > 5 mL/kg, minimal RR of 11 breaths/min, haemodynamic stability , normothermia, and TOF ≥0.9. Neostigmine (0.02 mg/kg) was used for the reversal of neuromuscular blocking before extubation. After extubation, the patient inhaled oxygen through a face mask at 3–6 L/min for approximately 15 min, and then the face masks were removed. Patients were supplemented with oxygen again through masks as temporary treatment if the SPO2 declined to less than 92%.Before the induction of anaesthesia, all patients were preoxygenated with an inspiration oxygen fraction , LUS imaging was performed immediately in the PACU by two trained anaesthesiologists  once hypoxaemia occurred. The anterior and posterior axillary lines divided each hemithorax into three regions , and each region was further divided into two quadrants (superior and inferior)  are used to assess aeration changes, and a higher grade represents more serious aeration loss but is inapplicable for pneumothorax . With a window width of 1500 Hounsfield units and a section thickness of 0.5 mm, all CT sections were stored for reconstruction and computerized analysis. A trained radiologist blinded to the study reported the CT findings by assessing the absence or presence of consolidation, effusion or pneumothorax as negative (−) or positive (+) in the same anatomic quadrant.2), and arterial partial pressure of carbon dioxide (PaCO2), were also recorded.Demographic data, including sex, age, height, weight, American Society of Anesthetist (ASA) score, BMI, vital signs and smoking habit, were recorded. Medical history, pulmonary function test and physical examinations were extracted from the Electronic Medical Record. At the bedside, we collected surgical information, duration of mechanical ventilation and PACU stay, time needed for LUS examination and time needed for CT scan . Cumulative opioid dose , volume of fluid administration , blood products and arterial blood gas at the end of the operation, including haemoglobin, arterial partial pressure of oxygen (PaOPASS software (version 16.0) was used to calculate the sample size. Assuming the allowable error was 10% and α was 0.05 , on the basis of a previous study, the estimated sensitivity and specificity of LUS were 87.7 and 92.1%, respectively . The calFrom January to May 2019, 138 adult patients were evaluated for eligibility. Twenty-five patients  were excluded, and 113 patients were ultimately enrolled  and VATS , followed by major orthopaedic surgery , neurosurgery  and other types of surgery . Eighty-two patients (72.6%) were diagnosed with atelectasis by both CT and LUS. CT scan diagnosed 327 of 1356 quadrants as atelectasis, while LUS revealed the same diagnosis in 311 of the 327 CT-diagnosed quadrants. In patients undergoing nonthoracic surgery, atelectasis was found to be in the posterior zones of both lungs, while the remaining atelectasis was discovered only in the operative lungs of VATS patients. Among the 82 patients with postoperative atelectasis, 19 patients showed signs of atelectasis on preoperative routine chest imaging examinations, while the remaining atelectasis was diagnosed only after surgery.Twenty-eight patients (24.8%) (75 quadrants) were diagnosed with pneumothorax by CT scan, whereas 72 quadrants of these 75 quadrants were also diagnosed with pneumothorax with LUS. The majority of these pneumothorax patients (26 patients) were in the VATS group, and the pneumothorax was mainly distributed in anterior and lateral quadrants. In VATS patients, 11 cases of pneumothorax were small, while fifteen were medium in size. The other two patients underwent partial hepatectomy surgery. Regarding the last 2 patients, one was diagnosed with small pneumothorax both by LUS and CT scan, while another was diagnosed with medium pneumothorax, and CT reported an approximate 50% prevalence of tension pneumothorax.Pleural effusion was found in 144 quadrants on CT scan in 39 patients (34.5%), which was primarily exhibited in posterior quadrants. LUS examination detected 131 quadrants with effusion among these CT-diagnosed zones. Nineteen patients (48.7%) received a diagnosis of pleural effusion on preoperative chest radiographs. The other 20 patients were newly diagnosed in the VATS group, all on the operative sides. One patient was diagnosed with massive pleural effusion on the left side, with visible anechoic effusion in the six quadrants.One patient was diagnosed with diffuse interstitial syndrome due to multiple B-lines in all 12 lung quadrants, and a CT scan led to the same diagnosis. Both LUS examination and CT scan showed no abnormalities in 12 patients.P < 0.001). Kappa values for the agreement between the first two observers of atelectasis, pneumothorax and pleural effusion were 0.951 (P < 0.001), 0.858 (P < 0.001) and 0.964 (P < 0.001), respectively. To resolve this disagreement, the third reviewer was mainly devoted to evaluating the diagnosis of pneumothorax. Table The time needed for the LUS examination was significantly shorter than that needed for the CT scan  were excluded. Considering the safety risk of transferring patients to CT scans, those with haemodynamic instability were also excluded. Hypoxaemia might also frequently occur in these patients, but the whole study only excluded 3 relevant patients (Figure 2, higher RRRM and PEEP have been reported to significantly decrease postoperative respiratory complications [2O in our study has been reported in a previous study, a higher PEEP may be much more beneficial for reducing atelectasis formation, as has been recommended by some researchers [PPCs are common after general anaesthesia, while hypoxaemia is primary triggered by atelectasis from compression, gas absorption and loss of surfactant . Postopeications –37 and wications –40. Althearchers . RM combearchers , but it earchers . Under pearchers , 44, 45.Similar to Xirouchaki et al.’s findings, our study showed that LUS was effective in the diagnosis of pneumothorax . The absAlthough LUS is an operator-dependent skill and adequate training is needed for effective clinical usage, it can be readily learned with a very simple device. See et al.  confirme2 were excluded.There are several limitations of our study. First, the 1-h time interval between LUS examination and CT scan and the suction impact of the water seal bottle may create false-positive results, while the obstruction of ultrasound views by the scapula and ribs could introduce false-negative results. Second, the presence of consolidation on LUS alone was insufficient for diagnosing pneumonia. In a recent study by Zhou et al. , the comIn conclusion, we showed that the application of LUS to diagnose the aetiologies of hypoxaemia in healthy-weight PACU patients is feasible and quick. LUS was sensitive and specific to diagnose PPCs when compared to the sensitivity and specificity of thoracic CT scans.


Are serum folate levels associated with cardiovascular mortality risk among adults with rheumatoid arthritis (RA)?In this cohort study including 683 patients with RA, those with serum folate levels between 4.3 and 8.2 ng/mL and greater than 8.2 ng/mL had 48% and 56% lower cardiovascular mortality risk, respectively, compared with patients who had serum folate levels less than 4.3 ng/mL after adjusting for demographic characteristics, body mass index, C-reactive protein level, smoking, RA medication use, and comorbid conditions.In this study, serum folate levels of at least 4.3 ng/mL were associated with lower cardiovascular mortality risk among patients with RA. This cohort study examines the association of serum folate concentration with cardiovascular mortality risk among patients with rheumatoid arthritis. Patients with rheumatoid arthritis (RA) are at high risk for cardiovascular (CV) mortality, attributed to chronic inflammation coupled with elevated circulatory homocysteine levels. Increasing the serum folate level reduces homocysteine, but the association of serum folate concentration with CV mortality in patients with RA has not been previously examined.To examine the association of serum folate concentration and CV mortality risk among patients with RA.A cohort study of the third National Health and Nutrition Examination Survey (1988-1994) and 2011 Linked Mortality File was performed. Adults aged 18 years or older with self-reported physician-diagnosed RA were included. Data analysis was performed between April 2019 and June 2019.Serum folate level.All-cause and CV mortality risk estimated using Cox proportional hazards models, adjusted for the complex survey design and patient characteristics, including demographic characteristics, body mass index, C-reactive protein level, smoking, RA medication use, and comorbid conditions.P for trend = .01). Findings for CV mortality were consistent in a sensitivity analysis that estimated 10-year risk; patients in tertile 2  and tertile 3  had lower CV mortality risk compared with those in tertile 1 (P for trend = .04).A total of 683 patients with RA  were classified into tertiles based on serum folate levels, as follows: tertile 1, folate levels less than 4.3 ng/mL (n = 239); tertile 2, folate levels 4.3 ng/mL to 8.2 ng/mL (n = 234); and tertile 3, folate levels greater than 8.2 ng/mL (n = 210). During a median (interquartile range) follow-up of 17.4 (10.0-19.4) years, a total of 392 all-cause deaths and 258 CV deaths occurred. Compared with tertile 1, patients in tertile 2 had lower all-cause mortality risk . The risk of CV mortality was lower among patients in tertile 2  and tertile 3  compared with those in tertile 1 (Among patients with RA, a serum folate level of at least 4.3 ng/mL was associated with lower CV mortality risk. Further research is needed to examine whether a causal relationship exists between serum folate and CV risk among patients with RA. Folate deficiency is common among patients with autoimmune diseases, including RA,11 and it is also a well-documented adverse effect of the disease-modifying antirheumatic drug methotrexate.12 To counteract reduced folate levels, folate supplements are recommended to patients with RA.16 Among patients with RA, an inverse association between serum folate level and homocysteine has been reported.17 However, to our knowledge, no published study has examined the association of serum folate levels with long-term CV outcomes. The objective of this study was to determine the association of serum folate levels with CV mortality among patients with RA. Using the third National Health and Nutrition Examination Survey, 1988 to 1994 (NHANES III) and linked mortality data, we examined whether optimal serum folate levels were associated with lower CV mortality risk in adults with RA.The risk of cardiovascular (CV) mortality among individuals with rheumatoid arthritis (RA) is 60% greater than among the general population.18 For this study, we merged the NHANES III with the 2011 National Center for Health Statistics Linked Mortality Files. The institutional review board of the University of Texas Health Science Centre at Houston deemed this study exempt from review and informed consent because it uses publicly available deidentified data. This report followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline.This is a cohort study of the NHANES III, which contains information from 33 994 US residents aged 2 months or older. The NHANES is a complex, stratified, multistage probability sample of noninstitutionalized US civilians. The survey collects information on demographic characteristics, socioeconomic status, and health conditions and behaviors, which are administered through a personal or telephone interview. Physiologic, dental, and laboratory examinations are included in the survey; these are conducted by trained medical professionals in mobile examination centers (MECs). A detailed description of the NHANES is available elsewhere.We identified adults aged 18 years or older with a self-reported diagnosis of RA. Self-reported data on medical conditions were collected at the MEC through a standardized questionnaire. Participants were asked, “Has a doctor ever told you that you have arthritis?” Those who responded yes were further asked, “Which type of arthritis was it?” Our cohort was restricted to participants who responded that they were diagnosed with rheumatoid arthritis. We excluded participants who were pregnant during the interview and those with missing data.20 Serum folate levels were measured using the Bio-Rad Laboratories Quantaphase Folate radioassay kit. The assay consists of the preparation of labeled 12 × 75 mm tubes in duplicate for each blank, standard, control, and patient sample. Standard manual procedures, as outlined by the manufacturer for the instrument, were followed. Reportable values for serum folate ranged from 0.4 to 199.0 ng/mL .Blood specimens of participants were collected in the MEC by trained laboratory technicians, and frozen specimens were shipped overnight per protocol. The NHANES III examined both serum and red blood cell folate values. We used the serum folate value because it is influenced by fewer analytical variables and it shows a higher correlation with homocysteine compared with red blood cell folate.We also evaluated C-reactive protein (CRP) levels as a biomarker for inflammation. The CRP assay was performed on a Behring nephelometer analyzer system. Samples were centrifuged at 3000 rpm for 15 minutes and transferred to a fully automated nephelometer analyzer. Results were automatically printed and calculated by the nephelometer analyzer when the tests were completed. Reportable values for CRP in the NHANES III were between 0.03 mg/L to 2.52 mg/L . Participants with undetectable values were labeled accordingly. The NHANES III defined normal CRP values as 0 mg/dL to 0.1 mg/L in normal healthy adults based on in-house testing of a sample of 300 patients. For this study, we classified CRP level into the 3 following categories: undetectable (<0.03 mg/L), 0.03 mg/L to 0.1 mg/L, and greater than 0.1 mg/L.Serum homocysteine (HCYS) levels were analyzed using residual serum samples. The samples were reduced using tributylphosphine to disassociate HCYS from nonrelevant proteins and other disulfides, followed by acid precipitation and the addition of 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonic acid, a fluorescent reagent. Subsequently, reverse-phase high-performance liquid chromatography and fluorescence detection were used to quantify HCYS. Values for HCYS in the NHANES III ranged from 0.27 mg/L to 17.8 mg/L .21Demographic data, including age at the time of the interview, sex, and race/ethnicity, were collected through a standard questionnaire administered in-home by trained interviewers using a computer-assisted personal interviewing system. Body mass index  was calculated for each participant based on body measurements collected at the MEC. A detailed questionnaire regarding tobacco use was also administered. We identified participants as current smokers if they responded yes to the question, “Do you smoke cigarettes now?” Hypertension, diabetes, and cardiovascular diseases (CVDs) were also self-reported. For the purpose of this study, we identified participants with CVD as those who responded yes to the questions, “Has a doctor ever told you that you had congestive heart failure?” or “Has a doctor ever told you that you had stroke?” or “Has a doctor ever told you that you had heart attack?” A similar case definition for CVD was used in a previously published report.During the survey, participants were asked if they had taken any prescription drugs and dietary supplements during the past 30 days. Participants who answered yes provided containers of medications and dietary supplements to a trained professional who then matched the product using a prescription drug database  and a dietary supplement database . Information regarding the strength, dosage form, and duration of use was recorded for each medication, provided by the participant. Similarly, information on the dietary blend, strength, and duration of use was recorded for all dietary supplements. We used prescription drug use data to identify patients who reported that they were using disease-modifying antirheumatic drugs, steroids, or nonsteroidal antiinflammatory drugs. Similarly, we used dietary supplement data to identify patients who consumed a dietary supplement containing folic acid.International Classification of Diseases, Ninth Revision (ICD-9) (through 1999) or ICD-10 (after 1999) code reported on the participant’s death certificate.22 We defined all-cause mortality as death because of any reason, while CV mortality included deaths reportedly due to a CVD .The 2011 National Center for Health Statistics Linked Mortality File consists of mortality information  of NHANES III participants from the date of survey participation until death or December 31, 2011. The Linked Mortality File consists of a leading cause of death variable, which includes 9 cause-specific death categories based on the 2 test for categorical variables. The distribution of numbers of patients in the 3 CRP categories was determined and compared using a χ2 test. Cox proportional hazards models were used to estimate the risk of all-cause and cause-specific  mortality.23 The models were adjusted for several potential confounding variables including sex, body mass index, CRP value, and use of steroid or nonsteroid anti-inflammatory drug, based on prior studies.29 We calculated tests of trend with the median folate concentration in each tertile as an ordinal variable. The analyses were repeated for the subgroups of patients without CVD and diabetes. Statistical significance was tested at P < .05, and all tests were 2-tailed. All analyses were conducted per the NHANES III analytical guidelines30 and were performed with SAS version 9.4 (SAS Institute). We used the survey procedures in SAS, which included weight, cluster, and strata statements, to incorporate sampling weights and to account for the complex survey design. Data analysis was performed between April 2019 and June 2019.We used descriptive statistics to summarize the characteristics of the entire RA cohort and by tertiles of serum folate level. Frequencies and means were estimated for categorical and continuous variables, respectively. Differences in patient characteristics by serum folate tertiles were tested for statistical significance using an analysis of variance for continuous variables and a χAfter applying study criteria, the final cohort included a total of 683 patients with RA ; 239 (35.0%) belonged in tertile 1 , 234 (34.3%) in tertile 2 , and 210 (30.7%) in tertile 3 . The total follow-up duration was 23 years . The study flow is illustrated in eFigure 1 in the P = .005; current smokers: 142 [68.0%] in tertile 1 vs 113 [53.2%] in tertile 2 vs 102 [53.4%] in tertile 3; P = .04; disease-modifying antirheumatic drug use: 7 [3.5%] in tertile 1 vs 9 [1.9%] in tertile 2 vs 11 [8.0%] in tertile 3; P = .03). There was no significant difference in the mean CRP level for 313 patients in detectable range (P = .23)  .P for trend = .01). The cumulative incidence curves are presented in A total of 392 all-cause and 258 CV deaths occurred during the follow-up period. The unadjusted and adjusted results for all-cause and CV mortality risk are presented in P for trend = .20). Patients with RA in tertile 2  and tertile 3  had significantly lower CV mortality risk than those in tertile 1 (P for trend = .04) , but the test for trend was not significant. Similarly, the risk of CV mortality was lower for patients with RA in tertile 2  and tertile 3  compared with those in tertile 1, but the test for trend was not significant (P for trend = .051). Results for the subgroup of RA patients without diabetes at baseline are also presented in P for trend = .17). The risk of CV mortality was lower among patients in tertile 3 vs tertile 1  (P for trend = .04). Comparisons of characteristics by serum folate tertile for the RA subgroups without CVD and diabetes are presented in eTable 3 and e Table 4 in the The main analyses were repeated in the subgroup of patients with RA but no CVD at baseline . Compare32 In the study by Morrison et al,31 participants with serum folate levels less than 3.0 ng/mL had higher risks of coronary heart disease compared with those with serum folate levels greater than 6.0 ng/mL . Similarly, Loria et al32 reported an increased CV mortality risk among participants with serum folate levels less than 4.2 ng/mL compared with those with serum folate levels greater than 7.4 ng/mL . Furthermore, in a previously published study, patients with RA using supplements containing folate had lower odds of CVD compared with those not receiving folate supplementation .33 The diagnosis of CVD is an intermediate end point, unlike mortality. Nonetheless, the lower odds of CVD corroborates the findings of the current study, ie, higher serum folate levels were associated with lower CV mortality risk.To our knowledge, our study was the first to examine the association of serum folate level with CV mortality risk among adults with RA. We found that patients with RA and serum folate levels less than 4.3 ng/mL had nearly 50% higher CV mortality risk compared with those with serum folate levels of at least 4.3 ng/mL. Data specific to the RA population are unavailable; however, 2 previous studies of the general, non-RA population have reported increased CV risk with low serum folate levels.34 reported no CVD or all-cause mortality benefits of folate supplementation in patients with history of vascular disease, a 2016 meta-analysis35 reported a 4% lower risk of CVD among patients with preexisting CV or renal diseases. The heterogeneity of cohorts may have led to the discrepancies in CV outcomes observed in these studies. We conducted a subgroup analysis restricted to participants without CVD and diabetes to examine effect modification in our study. Regardless of preexisting CVD or diabetes, patients with RA and serum folate levels less than 4.3 ng/mL in our study had a high risk of CV mortality. Our findings suggest that serum folate levels are associated with CV risk among patients with RA regardless of existing diabetes and CVD.Several studies have found an association between folate supplementation, circulating folate concentration, and CVD. The literature on serum folate supplementation provides indirect evidence of the association of serum folate level with CV mortality risk. While a 2006 meta-analysis37 Empirical studies have reported a successful reduction in HCYS levels among patients with RA supplemented with folic acid. In a multicenter study of patients with RA, plasma HCYS was significantly lower among patients receiving folate supplementation compared with placebo .15 Two other studies reported an inverse association of serum folate with plasma HCYS in patients with RA.39 Consistent with these findings, HCYS concentration decreased with increasing folate level in our study. However, it is important to note that we did not observe a dose-response association for serum folate and CV mortality risk. In general, HCYS levels above 1.4 mg/L are considered elevated, and CV risk has been shown to increase in a linear fashion for HCYS levels greater than this threshold.41 Because patients in both tertile 2 (HCYS reduction of 1.3 mg/L) and tertile 3 (HCYS reduction of 1.1 mg/L) had achieved HCYS reductions of less than 1.4 mg/L, CV risk estimates for these serum tertiles were not statistically significantly different. The lack of dose-response association for serum folate and CV mortality is consistent with findings in the general population.32It is unclear why higher folic acid concentrations would be associated with lower CV mortality risk among patients with RA. A possible explanation is that serum folate lowers CV risk through HCYS reduction. Serum folate levels and plasma homocysteine have an inverse relationship.43 It is possible that limitations of survey methodology  may have led to lower number of treated patients with RA in NHANES III; however, incorporation of drug information is recommended, and previous studies also have reported poor treatment rates in RA.45 The folate status of patients was based on a single serum folate concentration measurement that may not accurately represent long-term folate levels; however, pathological studies have shown positive correlation (R = 0.53) between serum folate and liver folate (a laboratory biomarker for tissue stores of folate).46 Next, consistent with findings in the general population,32 estimates for all-cause mortality were not significant. All-cause mortality, by definition, includes deaths due to any non-CV cause  and presumably has no association with serum folate. Although most confounders were taken into consideration, we did not adjust our analyses for factors that might affect serum folate levels, including health conditions in which folate absorption lowers  or use of drugs such as phenytoin. Furthermore, the observational nature of the data precludes causal inference, and the timeframe of NHANES III data collection limits the generalizability of our findings to current clinical practices.This study has limitations. The diagnosis of RA was self-reported in our study. The case definition for arthritis  used in our study is recommended by the US Centers for Disease Control and Prevention and has been validated.Our findings suggest that serum folate level is associated with CV mortality risk among patients with RA and might be a useful indicator for assessing patient risk in clinical practice. Additionally, if a causal link is validated in future clinical studies, folate supplementation can be an inexpensive strategy for reducing CV mortality risk in patients with RA.


Case Presentation. A 62-year-old female patient had a history of CLL. A lesion with the appearance of an atheroma was removed from the right upper arm. Histology confirmed the diagnosis of MCC. She was admitted to the neurology department with her first GM seizure. The cranial MRI/MRA showed bone metastases in the right parietal and both frontal areas, compressing the brain. Flow cytometry of CSF did not reveal metastasis of MCC.  Merkel cell carcinoma (MCC) is a rare primary neuroendocrine cutaneous tumor, rarely metastasizing to the brain. Chronic lymphoid leukemia (CLL) is a disease predisposing to MCC. According to previous reports, headache and focal neurological deficits suggest disease progression to the brain. We present a patient with MCC whose seizure was not elicited by a cerebral metastasis, but by bone metastases compressing the brain.  The case history of the patient was unique even among the rare cases of MCC with neurological involvement. The seizure was not elicited by a cerebral metastasis, but by bone metastases compressing the brain. In addition to patient history, clinical presentation and radiological findings enabled a suspected diagnosis of skull metastasis of MCC compressing the brain, causing symptomatic epileptic seizures. Merkel cell carcinoma (MCC) is a rare and extremely aggressive neuroendocrine malignancy. Both clinically and histologically, it is difficult to differentiate it from neuroendocrine carcinomas, skin metastasis of small cell lung cancer, small cell malignant melanoma, malignant lymphoma, atypical keratoacanthoma, and leiomyosarcoma. In addition to patient history, clinical and radiological findings as well as immunohistochemistry contribute to a definitive diagnosis. Due to the above reasons, it is a significant challenge for the neurologist to diagnose this very rare and malignant disease in a timely manner which needs rapid treatment to provide a chance of recovery.The 62-year-old female patient's chronic lymphoid leukemia (CLL) was diagnosed 10 years ago and was nonprogressive following chemotherapy. The only other past medical history of note was laparoscopic cholecystectomy in 2006.A lesion with the appearance of an atheroma was removed from the right upper arm 2 years ago. Histology with immunohistochemistry confirmed the diagnosis of MCC, and the patient received combined chemotherapy based on National Comprehensive Cancer Network (NCCN) guidelines. This was discontinued due to bone marrow depression. The lesion recurred 7 months later and was reoperated. At this time, the patient also had right axillary block dissection due to the local recurrence of MCC and its metastasis to the axillary lymph nodes, confirmed by aspiration cytology, showing metastases from both CLL and MCC in the lymph nodes. Postoperative radiotherapy was performed, but the patient's condition deteriorated. Progressively, a right axillary soft tissue conglomerate mass infiltrated the right lateral thoracic wall, and pathologic upper mediastinal and parasternal lymph nodes appeared. These all showed metastases from MCC. Chemotherapy following the ECO regimen (epirubicin (E) given weekly in combination with cyclophosphamide (C) and vincristin (O) applied during a 3-week interval) was reinitiated due to clinical progression, and the last chemotherapy session (vincristin) was administered 3 days before the patient was admitted to our Department of Neurology due to her first epileptic seizure (GM). The patient complained about headache during the week before admission.BP: 115/75 mmHg and HR: 75/min; no bruit over carotid or subclavian arteries; an oblique surgical scar with primary healing on the right upper arm; liver edge at the costal margin. No other abnormality was found during physical examination.No abnormality was found.No abnormality was found.∗9/L; neutrophils (%): 5.9, ratio of stab cells: 1%; lymphocytes (%): 86.4%; monocytes (%): 1.0%; eosinophiles (%): 0.1%; basophiles (%): 0.4%; RBC: 3.90 10∗12/L; Hgb: 112 g/L; Hct: 0.335 L/L; platelets: 85 10∗9/L; blood smear: Gumprecth's shadows.WBC: 11.48 10Serum glucose, creatinine, and electrolyte levels were normal. Gamma-GT: 24 U/L; alkaline phosphatase: 162 U/L; LDH: 1768 U/L; amylase: 75 U/L; CRP: 1.59 mg/L.Cranial CT showed a 37 × 9 mm hypodensity in the right parietal region, at the posterior horn of the lateral ventricle, with marked cerebral and cerebellar atrophy .Cranial MR showed a solid epidural of 2 × 2.8 × 5 cm mass, originating from the bone, in the right parietal region, showing homogeneously low signal intensity on T1-weighted images  and homoμV with irregular waves of 3–4 Hz arising on the right side. Impression: organic dysfunction of the right hemisphere.EEG showed partially rhythmic activity of 7–9 Hz and 20–40 Clear and transparent; cell count: 5 WBC/microliter; total protein: 368 mg/L; CSF glucose: 3.8 mmol/L; serum glucose (simultaneous): 7.5 mmol/L.CSF cytology: few monocytoid cells and leukocytes. No tumor cells are detected in the sample.CSF immunology: intact blood-liquor barrier. Intrathecal IgG synthesis is not detected, but oligoclonal gammopathy (OGP) is found.Flow cytometry of CSF did not reveal metastasis of MCC. CLL was only present in peripheral blood, where 50% of the cells showed a corresponding flow cytometry phenotype.Based on the clinical picture, the patient was started on carbamazepine. She did not have further epileptic seizures and did not develop any neurological deficit. Metastasis of MCC to the skull  was diagnosed based on clinical and radiological presentation and patient history. No histology was performed from the skull mass.The patient died 3 months later. The cause of death was multiple metastases of MCC.MCC is a rare and extremely aggressive neuroendocrine malignancy, presenting as a solitary, painless, thick, livid, and erythematous nodule, mostly on skin areas exposed to sunlight. First, it gives metastases to regional lymph nodes and then to distant organs. MCC is often associated with malignant hematologic diseases, such as CLL . RecentlShe had a history of proven CLLThe seizure was not elicited by a cerebral metastasis, but by bone metastases compressing the brainThe case history of our patient is unique even among rare cases of MCC giving metastases to the brain , 4 due tIn addition to patient history, clinical presentation and radiological findings enabled the suspected diagnosis of skull metastasis of MCC compressing the brain, causing symptomatic epileptic seizures. Bone metastasis from CLL was unlikely, as solitary bone lesions in the context of CLL are believed to result from either Richter's transformation or metastasis from another primary malignancy , in this