Solidago canadensis is an invasive plant that is capable of adapting to variable light conditions. To elucidate the shade tolerance mechanism in S. canadensis at the molecular level, transcriptome analyses were performed for leaves growing under natural light and three shade level conditions. Many differentially expressed genes (DEGs) were found in the comparative analysis, including those involved in photosynthesis, antioxidant, and secondary metabolism of phenol‐ and flavonoid‐related pathways. Most genes encoding proteins involved in photosynthesis, such as photosystem I reaction center subunit (Psa), photosystem II core complex protein (Psb), and light‐harvesting chlorophyll protein (Lhca and Lhcb), and reactive oxygen species (ROS) scavenging‐related enzymes, such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), were upregulated with the shade levels. Furthermore, most of the DEGs related to secondary metabolite synthesis were also upregulated in the shade conditions. Our study indicates that S. canadensis can respond to shade stress by modulating the expression of several photosynthesis‐related, free radical scavenging‐related, and secondary metabolism‐related genes; thus, this species has the ability to adapt to different light conditions. Solidago canadensis under different shading conditions, the genes that played an important role in responding to shading stress were screened out. The differential expression of these genes may play an important role in the response of S. canadensis plant to light avoidance environment. It is possible to cope with different light intensities, which are likely to play an important role in the process of S. canadensis expanding invasion and forming invasive species. At the same time, it also provides a theoretical basis for the distribution of S. canadensis.The invasive plants are widely distributed in different external environments and have a good adaptive ability. Through the analysis of gene expression in the leaves of  Many studies have revealed that invasive plants have the ability to adapt to the scarcity in resources such as availability of nutrients, water, light, and various environmental stresses. , native to North America, has successfully invaded many areas worldwide. The success of invasion can be related to the plant's high phenotypic plasticity , Hubei Province, China. After three months (from February to April) of cultivation in pots under natural conditions , 25% (L2), and 10% (L3), respectively, via black shade nets that were clamped to a metal frame at a height of 150 cm . The contents of chlorophyll a, b, and total carotenoid for all samples were calculated according to previous studies as follows: Ca = 13.95 × A665–6.88 × A645; Cb = 24.96 × A649–7.32 × A665; xC+c = /245  with a minimum integrity value of 7.5. The cDNA library of each sample was constructed as follows. First, mRNA was isolated from total RNA with magnetic beads containing Oligo (dT) primer. Subsequently, the enriched mRNAs were fragmented in fragmentation buffer, and then, first‐strand and second‐strand cDNA were synthesized and amplified using an ABI Step One Plus Real‐Time PCR System. Finally, these cDNA library preparations were sequenced using an Illumina HiSeq X Ten from both the 5′ and 3′ ends, and 150‐bp paired‐end reads were generated.2.4E‐value ≤ 10−5. Based on the NR annotation results, Blast2GO software was used to classify unigenes against the Gene Ontology (GO) database according to cellular component, biological process, and molecular function | ≥ 1 and adjusted p‐value < .05 as the threshold for significantly different expression  for network visualization  is used to construct the expressed gene network and identify stress‐related candidate key genes. This method has been widely applied in model organisms and nonmodel organisms for gene expression studies  was the average calculated from three biological replicates. Letters on the bars represent a significant difference at p‐value ≤ .05 (LSD test) among different treatments compared with the control group.In order to investigate whether the chlorophyll and carotenoid content of plants were changed under different light conditions, the experiment of chlorophyll content measurement for each treatment was repeated three times in this study. The data of chlorophyll and carotenoid content were analyzed by one‐way analysis of variance, and the mean  technique was used to validate gene expression from RNA‐Seq. RNA was isolated with TRIzol reagent from each independent biological sample. In order to prevent the presence of DNA in extracted RNA samples, all the RNA samples were treated with DNase I to digest DNA. cDNAs were obtained by reverse transcription of each RNA sample. Specific primers for 12 randomly selected genes were designed using Primer 5 software Lalitha, . The SYB33.1S. canadensis growth, changes in plant morphology was observed under three treatment groups in comparison with control plants. Three levels of shading treatment significantly increased the plant height, leaf angle ‐treated plants had the highest growth height and largest leaves. The plant height and leaf area of the three‐layer shading net (L3) were smaller than those of the L2 but higher than those of the one‐layer shading net (L1) treatment groups. In the three different shading treatment groups, the leaf angle and total content of chlorophyll increased with the reduction of light intensity and were significantly higher than the control group. In addition, there was a significant difference in total chlorophyll content between the L with L1, L2, and L3 groups  and single read length of 150 bp Table . The clep Figure . The exp3.3S. canadensis. There were 376 DEGs in the comparison of L1 versus L, with 124 being upregulated and 152 downregulated. Similarly, there were 1,649 DEGs (788 upregulated and 861 downregulated) in the L2 condition comparison and 2,087 DEGs  in the L3 condition compared with their light‐grown counterparts , such as the PsaD, PsaE, and PsaH gene, and photosystem II core complex protein‐related genes (Psb), such as the PsbO, PsbQ, and PsbR gene, were differentially expressed. Other photosynthesis‐related DEGs were as follows: cytochrome b6 complex and photosynthetic electron transport genes (Pet), such as PetC, PetE, and PetF gene; F‐type ATPase synthesis‐related genes, such as the ATP‐gamma gene , such as the Lhca1, Lhca2, and Lhcb2 gene , phosphoribulokinase gene (PRK), and glyceraldehyde‐3‐phosphate dehydrogenase gene (NADP). Furthermore, some Chl‐related genes, such as the ChlP, ChlH, and HemH gene, were also differentially expressed in the shade condition groups when compared with the control  encoding enzymes catalyzing the conversion of glutathione (GSH) to glutathione disulfide (GSSG) were upregulated. Among four ascorbate peroxidase genes (APX) encoding enzymes catalyzing the conversion of ascorbate to dehydroascorbate, three were upregulated and one downregulated. Of eight glutathione S‐transferase (GST) genes encoding enzymes catalyzing the conversion of glutathione (GSH) to R‐S‐glutathione, six were upregulated and two downregulated. Two ascorbate oxidase (AO) genes encoding enzymes catalyzing the conversion of dehydroascorbate to ascorbate, one was upregulated and another downregulated. These DEGs could encode related antioxidant enzymes in response to shading stress. Furthermore, the number of antioxidant enzyme‐related DEGs increased as the light intensity was reduced, and most were upregulated. These results indicated that the activity of antioxidant enzymes might be enhanced in S. canadensis in response to shade stress.In this study, some antioxidant enzyme‐related genes, such as d Figure . In addi3.82 versus L and L3 versus L. In contrast, the proportion of downregulated genes among these three comparisons increased in the “phenylpropanoid biosynthesis” pathway, in which 34 DEGs were collected and most were upregulated in the three shade treatment groups compared with the L group, such as the PAL, C4H, 4Cl, and C3′H genes , with three in the L group shown in turquoise, dark red, and red and two in the L3 group shown in dark green and blue  gene and ARF (auxin response factor) gene, and antioxidant‐related genes such as AO which encode ascorbate oxidase. A total of 297 DEGs, 267 upregulated and 30 downregulated in L3 versus L, were clustered in the blue module. Most of these DEGs were photosynthesis‐related genes, such as Lhc, chlorophyll biosynthesis, chloroplast thylakoid membrane, and carbon fixation. Meanwhile, antioxidant enzymes‐related genes, such as POD, SOD, CAT, APX, and GST, and secondary metabolism‐related genes, such as PAL, C4H, CAD and CHS, were also clustered into this module. In addition, some transcription factor genes, such as EREBP, ERF, WRKY, TIFY, and bHLH, may encode corresponding TFs and play a regulatory role in this module. Based on the network analysis for the blue module, the top 300 paired DEGs with a weight value above 0.71 were chosen for exhibition  analysis. A total of 12 randomly selected key candidate genes from each of four different treatment groups were used for validation. As anticipated, the qRT‐PCR results for the different groups Figure  were con4S. canadensis, an invasive plant, could adapt to low‐light conditions in a shade stress environment by adjusting the morphology, growth, photosynthetic physiology, and other characteristics, and it exhibits a certain plasticity in response to low light intensity  in the different treatment groups. These results were consistent with the linear increase in shade stress, concurrent with a significant decrease in the chl a/b ratio. The carotenoid content increased in the three treatment groups with no significant differences among them. The ratio of carotenoid content with total chlorophyll was decreased with the reduction of light radiation. This phenomenon indicates that the photosynthesis system in S. canadensis has a wide range of strategies to adapt to shading stress and increase its light use efficiency by regulating the content of chlorophyll.Chlorophyll is the most important photosynthetic pigment in photosynthetic processes, and the chlorophyll content is always significantly affected by shade stress , ferredoxin (Fdx) complex, and ribulose‐bisphosphate carboxylase large chain (RbcL) involved in the Calvin cycle can play important roles in the photosynthesis process , and lutein (Lut), playing a vital role in photosystem II  and II (LCHII)‐related genes encoding subunits consisting of the photosystem I‐antenna and photosystem II‐antenna complex, respectively. Photosystem II‐antenna complexes include major antenna complexes (LHCII), such as Lhcb1, Lhcb2, and Lhcb3 proteins, which bind several pigment molecules, such as chlorophyll rbcL, PGK, GAPDH, and ALDO, were upregulated, which might enhance carbon synthesis in S. canadensis under shade conditions.Most of enzymes, such as phosphoribulokinase (PRK), phosphoglyceratekinase (PGK), RuBP carboxylase (rubisco), fructose‐bisphosphatase aldolase (ALDO), and glyceraldehyde phosphate dehydrogenase (GAPDH), plays an important role in the carbon fixation process  can act as regulatory proteins that play an important role in these mechanisms ; data curation (lead); formal analysis (lead); investigation (lead); methodology (lead); project administration (lead); resources (lead); software (lead); supervision (lead); validation (lead); visualization (lead); writing – original draft (lead); writing – review and editing (lead). Zeyu Li: Data curation (supporting); methodology (supporting); resources (supporting); software (supporting). Jianbo Wang: Conceptualization (lead); data curation (lead); formal analysis (lead); funding acquisition (lead); investigation (lead); methodology (lead); project administration (lead); resources (lead); software ; supervision (lead); validation ; visualization (lead), writing – original draft ; writing – review and editing .Fig S1Click here for additional data file.Fig S2Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.Table S4Click here for additional data file.Table S5Click here for additional data file.Supplementary MaterialClick here for additional data file.


Vigna unguiculata (L.) Walp] in sub-Sahara Africa is curtailed by a lack of farmer-preferred and improved cultivars and modern production technologies. The objectives of the study were to determine the extent of genetic diversity present among a collection of cowpea accessions from Zambia and Malawi using phenotypic traits and single nucleotide polymorphism (SNP) markers and, to select distinct and complementary parental lines for cultivar development. One hundred cowpea genotypes were evaluated for agronomic traits in two selected sites in Zambia, using a 10 × 10 alpha lattice design with two replications. Ninety-four of the test genotypes were profiled with 14,116 SNP markers.Productivity of cowpea [− 1 (NPP), pod length (PDL), and number of seeds pod− 1 (NSP), were significantly (p < 0.05) affected by genotype × environment interaction effects. Genotypes such as CP411, CP421, CP645, CP732, Chimponongo, and MS1–8–1-4 exhibited higher grain yield of > 1200 kg/ha with excellent performance in yield components such as NSP, PDL, HSW and GYD. Grain yield had significant (p < 0.05) associations with NPP (r = 0.50), NSP (r = 0.46) and PDL (r = 0.42) useful for simultaneous selection for yield improvement in cowpea. The SNP markers revealed gene diversity and polymorphic information content of 0.22 and 0.17, respectively, showing that the tested cowpea accessions were genetically diverse. Test genotypes were classified into four genetic groups irrespective of source of collection allowing selection and subsequent crosses to develop breeding populations for cultivar development.Number of pods plantGenotypes Bubebe, CP411, CP421, CP645, Chimponogo and MS1–8–1-4 were identified to be the most genetically divergent and high yielding making them ideal parental lines for breeding. This study provided a baseline information and identified promising cowpea genetic resources for effective breeding and systematic conservation. Vigna unguiculata (L.) Walp., 2n = 2x = 22] is a relatively low cost source of plant-derived protein, amino acids and essential nutrients globally. It is the main food staple supporting millions of people in sub-Sahara Africa (SSA) . repon gains , 31.In the present study qualitative traits such as seed coat, pod and leaf colour were more important traits for selection. These traits affect the market value of cowpea in Africa given that farmer and consumer preference are based on these attributes. Seed coat colour is often associated with processing quality (e.g. cooking time) and farmers deliberately select varieties that have shorter cooking time . The inhIn this study, the heritability estimate for grain yield was high (64%), suggesting that the grain yield achieved by the accessions was highly repeatable ensuring genetic improvement through selection. The high heritability value for grain yield corroborates with the findings of  but loweThe relationships among yield and yield components are critical in devising a selection strategy. Selection of one trait may amplify or negatively affect performance in the other traits. The high contribution and strong association of PDL, NSP, GYD, and NPP to PC1 as well as DTF and DTM with the PC2 indicated that these traits were highly discriminatory explaining the variation among the genotypes , 39. fouGenetic analysis using SNP markers delineated the test populations in to four genetic groups. This demarcation was irrespective of source of collection, suggesting that geographical sources of collection are not the sole factor for classification of cowpea genotypes. The germplasm exhibited high heterozygosity of 0.30, which showed that alternative alleles were represented in this population. This could be attributed to genetic exchange between and among cowpea breeding programs in sub-Sahara Africa, particularly in southern Africa. Heterozygosity estimates were highest for mutant lines, followed by elite lines and landraces. This suggests that mutant lines exhibited more variability possibly due to continuous gene segregation and other chromosomal aberrations. Landrace varieties are not subjected to artificial selection by breeders. Hence, landraces of self-pollinating crops including cowpea consists of heterogeneous populations due to genetic variation until complete homozygosity is reached through selection . Mutant The PIC and GD values were essential to assess genetic diversity within the whole population, and sub-populations for identification selection of divergent parental lines useful for breeding. The moderate PIC and GD values exhibited by the whole population could be attributed to the inherent nature of self-pollination in cowpea, which limits genetic diversity. Self-pollinating species often lack genetic diversity due to higher levels of homozygosity at multiple loci. The within-population diversity in self-pollinating species is relatively low but varies among populations , 42. In Phaseolus spp) [Dioscorea spp) [Brassica spp. [The phenotypic and genotypic distance matrices exhibited low and non-significant correlation, which confirm that phenotypic and genotypic matrices were independent but complementary. The discordance between phenotypic and genotypic matrices is partly attributed to the significant environmental effect on the variable expression of the assessed phenotypic traits. Phenotypic performance is the result of the genotype, environment and genotype-by-environment interaction effects . In addilus spp) ), yam (Drea spp)  and Brasica spp. . Due to ica spp. , 48. Theica spp. .Phenotypic analysis using qualitative and quantitative traits and genotyping using high density SNP markers revealed the presence of significant variation among 100 cowpea germplasm collections of southern Africa. Trait association analysis revealed significant correlation between NPP, NSP, PDL and GYD that could allow direct selection to improve grain yield. The SNP markers used in the study were able to deduce genetic variation among the tested cowpea populations. The largest proportion of variation was attributable to individual genotype differences that is essential for improving grain yield by crossing lines from different divergent populations. Test genotypes were classified in to four genetic groups irrespective of source of collection allowing selection for subsequent cross combinations to develop breeding populations for cultivar development. Genotypes Bubebe, CP411, CP421, CP645, Chimponogo and MS1–8–1-4 were identified being the most genetically divergent and high yielding making them ideal parental lines for breeding. This study provided a baseline genetic profile and identified promising cowpea genetic resources for effective breeding and systematic conservation.The study evaluated 90 cowpea genotypes acquired from different sources (Supplementary Table 1). The germplasm included 61 elites lines from Malawi, International Institute for Tropical Agriculture (IITA), Nigeria, The National Gene Bank of Zambia and the University of Zambia, 15 landraces collected from smallholder farmers in Zambia and 14 mutant lines, which were derived from three parental lines; Lutembwe, Bubebe and Msandile. The accessions from IITA and the released cultivars were used as standard checks.o 32′S and a longitude of 28o11’E with an altitude of 1206 m above sea level. The total mean annual rainfall at the SSCI site is 1092 mm, while the mean daily minimum and maximum temperatures were 12 °C and 26 °C, respectively. The GART site is situated at a latitude of 14o 96′S and a longitude of 28o10’E and an altitude of 1103 m above sea level. The GART site receives a total mean annual rainfall of 884 mm with mean daily minimum and maximum temperatures of 10 °C and 30 °C, respectively. The soils at both sites are classified as Haplustalf clays with pH of 5.8 and 5.2 at SCCI and GART, respectively [The 100 genotypes were field evaluated during the 2017/2018 main crop season at the following two sites: the Seed Control and Certification Institute (SCCI) in Chilanga and Golden Valley Agricultural Research Trust (GART) in Chisamba/Zambia. The SCCI site is situated at a latitude of 15ectively .2. The inter-row and intra-row spacings were 75 and 45 cm, respectively. Two seeds were sown per station at a depth of 2 cm and later thinned to one plant two weeks after emergence. Basal fertiliser (N: P: K), containing 20% nitrogen, 10% phosphorus and 20% potassium, was applied at a rate of 200 kg ha− 1 prior to planting. All other agronomic practices for cowpea production were followed as recommended for Zambia [The experiments were laid out in a 10 × 10 alpha lattice design with two replications. Each genotype was sown in a plot with two rows of 5 m long. The plot area was 3.75mr Zambia . The cro− 1 based on the following formula:Data was collected from six qualitative and eight quantitative traits following the descriptors of the , 51. The legumes  and 10,0βijk = μ + Gi + Ej + Gi ∗ Ej + Ei (rk)(b) + εijk, where; βijk = observed response; μ = grand mean Gi = the effect of ith genotype; Ej = the effect of jth location, Gi ∗ Ej = the genotype x location interaction effect; Ej(rk)(b) = error associated with kth replication in blocks in the jth location and εijk = experimental error. The blocks within replications were considered as random factor, while genotypes and locations were fixed factors. Trait means of test genotypes were separated using the Fischers Unprotected LSD at p ≤ 0.05 significance level. Genotypic, genotype by location interaction and phenotypic variances were computed from the excepted mean squares of the analysis of variance as follows; σ2g = genotypic variance, σ2gl = genotype by location interaction variance, σ2 : p = phenotypic variance, msg = mean square of genotype, mse = mean square of error, l = number of location and r = number of replication. Variances below zero were adjusted to zero according to [2) was computed according to [σ2g is genotypic variance and σ2p is phenotypic variance. Heritability was categorized as low (0–0.30), moderate (0.30–0.60) and high (> 0.60) following [σ2g = genotypic variance, σ2p = phenotypic variance, k) (σp) (h2), where, GA = Genetic advance; k = selection differential at 5% selection intensity; σp = phenotypic standard deviation; h2 = broad sense heritability; Genetic advance as a per cent of mean (GAM) was computed following [The frequency of test genotypes displaying the assessed qualitative traits were summarised and statistical significant tests conducted using the cross tabulation procedure with the Statistical Package for the Social Sciences (SPSS) version 24 . The quarding to . Heritabrding to ; \documeollowing . A covarollowing  as folloollowing  as folloollowing : \documee 0.30–0. and highr) using the SPSS version 24 [The magnitude of traits relationship was determined using Pearson’s correlation coefficients  using the Lysis Buffer 1 (based on the CTAB method) according to the manufacturer’s procedures. The DNA concentration of each sample was measured using a NanoDrop 1000 . For verifying DNA integrity, 2 μL of DNA were subjected to gel electrophoresis on 1.0% (w/v) agarose gel, stained with ethidium bromide. Subsequently, 40 μL of a 50 ng/μL DNA of each sample were genotyped with Illumina Cowpea iSelect Consortium Array using Diversity Arrays Technology (DArT) markers. In total, 94 cowpea genotypes were genotyped by the genotyping by sequencing (GBS) technology as described by  with 20,For quality control, DArTseq SNP derived markers were filtered to remove bad SNPs and genotypes using the software’s PLINK 1.9 in MS window and R statistical package. Markers and genotypes with > 20% missing data were eliminated. Rare SNPs with < 5% minor allele frequencies were also pruned from the data. Only 14,116 informative SNP markers and 90 genotypes were used for analysis after removal of 4240 SNPs and four genotypes. The four genotypes, CP1, CP2, CP479, and CP2223 were removed due to extreme heterozygosity (< 90%), duplication or high levels of missing data (> 20%). The chromosomal coverage of the 14,116 SNPs was presented in Supplementary Fig. 3.The Bayesian clustering method was used for infering the population structure of the germplasm using the STRUCTURE version 2.3 software . The STRA joint analysis based on a combination of phenotypic and genotypic dissimilarity matrices was conducted. A Gower’s distance matrix was generated from the phenotypic data while the genotype dissimilarity matrix was computed based on Jaccard’s coefficient. The correlations among the phenotypic, genotypic and joint dissimilarity matrices were compared using the Mantel’s test with 999 permutations  using thAdditional file 1. Statistical tests and distribution of 100 cowpea germplasm collections based on qualitative traits.Additional file 2. Correlations among phenotypic, genotypic and joint matrices based on Mantel test with 999 permutations showing the correlations  and p-values .Additional file 3. List, source, and description of 90 cowpea genotypes used in the study.Additional file 4. The Mantel test between phenotypic and genotypic matrices.Additional file 5. The Mantel test between phenotypic and joint matrices.Additional file 6. The Mantel test between genotypic and joint matrices.Additional file 7. Chromosomal distribution of the 14,166 SNPs used to evaluate 90 cowpea genotypes.


Crocus sativus L.). None has been performed with a galenic preparation of a standardised saffron extract. The aim of the present research work was to study the effect of in vitro digestion process on the main bioactive components of saffron extract tablets and the corresponding pharmacokinetic parameters in humans. Pharmacokinetics were calculated collecting blood samples every 30 min during the first 3 h and at 24 h after administration of two different concentrations (56 and 84 mg of the saffron extract) to 13 healthy human volunteers. Additionally, an in vitro digestion process was performed in order to determine the bioaccessibility of saffron main bioactive compounds. Identification and quantification analysis were performed by HPLC-PAD/MS. Digestion resulted in 40% of bioaccesibility for crocin isomers, whereas, safranal content followed an opposite trend increasing about 2 folds its initial concentration after the digestion process. Crocetin in plasma was detected in a maximum concentration (Cmax) in blood between 60 and 90 min after oral consumption with dose-dependent response kinetics, showing that crocin isomers from galenic preparation of saffron extract are rapidly transformed into crocetin. The results showed that this tested galenic form is an efficient way to administer a saffron extract, since the observed crocetin Cmax was similar and more quickly bioavailable than those obtained by other studies with much higher concentrations of crocetin.There are few studies about the pharmacokinetics of the low-molecular mass carotenoids crocetin or crocin isomers from saffron ( Crocus sativus L. (Iridaceae), was traditionally used as food spice worldwide to provide colour, taste, and odour in several food recipes. Saffron is characterised by a pool of bright yellow-coloured crocins, water-soluble carotenoids of 20 atoms of carbon [Saffron, dried stigmas of f carbon , a bittef carbon , and itsf carbon , 5.N-methyl-D-aspartate receptor (NMDA) antagonism, and gamma-aminobutyric acid (GABA) agonism [Besides the culinary role of saffron, safranal and crocin isomers show bioactive properties with several therapeutics and pharmacological applications –8, altog agonism , 18. In  agonism . The ant agonism .invitro experiments, cell models, and animal tests. During in vitro digestion of an aqueous saffron extract, a significant reduction of crocin concentration was observed, mainly trans-crocins, in a dependent process on the physiological pH, temperature, and time of digestion [in vitro studies results were confirmed in animals. After oral dosing of crocins or crocetin, no crocin but crocetin was measured in the rat and mouse plasma as free, mono-, and diglucuronide conjugates [in vitro digestion studies report that the bioaccessibility of crocins depends on different factors such as the dietary ingredient which are consumed with or its combination with other bioactive compounds [The published beneficial effects of saffron extracts led to better understand the role of its bioactive compounds, but their bioaccessibility and bioavailability were investigated mostly only in igestion . In expeigestion , 22. Thenjugates , 24. Mosompounds , 26.in vitro bioaccessibility of the main bioactive components of a commercial saffron extract (affron®) during the digestion process and the corresponding pharmacokinetics in humans after oral administration.Despite the growing interest in the use of saffron extracts as a functional product in mental health, there are few studies about the pharmacokinetics of its bioactive substances in humans. One of them determined the crocetin appearance in the plasma samples of 4 human volunteers after the intake of a cup of 200 mg saffron infusion . The twoCrocus sativus L.) stigmas were extracted in the factory of Pharmactive Biotech Products S. L. in Madrid (Spain) to produce the commercial saffron extract, branded as affron®, and dosed in tablets as preferred galenic form for the in vitro digestion and the clinical trial. Each tablet contained 14 mg of dried affron® standardised to ≥3.5% lepticrosalides®, defined as the content of bioactive compounds including safranal and crocin by HPLC [The saffron . Kaempferol was purchased from Extrasynthese , and trans-crocin-4 was purchased from Phytolab . All the solvents used for the chromatographic analyses were of HPLC grade and purchased from Merck and VWR .Safranal, crocetin, in vitro digestion was carried out according to Hollebeeck et al. in three stages  with 28 mg of affron® . Briefly, the salivary step runs for 5 min at pH 6.9 and 37°C, under aerobic conditions with 3.9 units α-amlylase/ml. Then, the samples were transferred for 90 min to the gastric step at pH 2 and 37°C with 71.2 units pepsin/ml under anaerobic conditions. Finally, the duodenal step was set for 150 min at pH 7 and 37°C, with 9.2 mg pancreatin/ml and 55.2 mg bile extract/ml under anaerobic conditions. Samples were taken from every steps and were centrifuged at 7000 g for 40 min at 4°C. The soluble fractions were frozen at −20°C, freeze-dried and the resulting dry powders were stored in a dark, dry place until analysis [Abiotic analysis . All samPeak identification of affron® compounds was performed by mass spectrometry (MS) using an Agilent series 1100 HPLC, coupled to a mass-quadrupole detector , with an electrospray ionization source (ESI) set at the following conditions: mass range, from 50 to 1500 amu; drying gas flow, 10 L/min; drying gas temperature, 340°C; nebulizer pressure, 40 psig; vaporizer temperature, 150°C; capillary tension, 2000 V; charge tension, 2000 V. Ion current acquisition was operated at both, positive and negative modes, with precision, accuracy, and detection limits as in .trans-crocin-4, kaempferol, and p-nitroaniline were used to plot external calibration curves for the quantification of safranal, crocetin-crocin isomers, kaempferol derivatives, and picrocrocin, respectively.High-performance liquid chromatography (HPLC) analysis of bioactive components from initial and digested saffron extract was performed using an Agilent Technologies 1220 Infinity series system equipped with a photodiode array detector (PAD) according to Kell et al. . The refTo estimate the percentage of bioaccessibility of the main bioactive compounds of affron®, an equation was used according to Ordoudi et al. .A single dose, randomized, double blinded study was used to evaluate the pharmacokinetics of two different saffron doses (56 mg and 84 mg). The randomization code was generated by Random Allocation Software, sealed envelope . Enrolled participants were then allocated to one of two groups: group 1: consumed 4 × 14 mg of affron® tablets (56 mg single dose); group: 2 : 6 × 14 mg of affron® tablets (84 mg single dose).This study was conducted in accordance with the ethical approval from Bellberry Limited ethics committee . All participants provided written informed consent and were screened for inclusion and exclusion criteria prior to the conduct of the study.n = 5) and female  healthy volunteers of 18–30 years age. All participants self-reported to be in normal physical health (BMI < 30) and passed the exclusion screening. Exclusion criteria included suffering clinically significant medical condition, such as, but not limited to, cardiovascular, neurological, psychiatric, renal, immunological, endocrine (including uncontrolled diabetes or thyroid disease), or uncontrolled haematological abnormalities. Other exclusion criteria were the use within the past 3 months of the tested compounds and/or antioxidants; no current use of prescription medications except the oral contraceptive pill in women; and known allergy to any tested compound and/or antioxidant.The subjects of the pharmacokinetics were adult male  and lunches  were served at the center. The meals were poor in carotenoids. Subjects remained onsite for the full first 6 hours of sample collection and returned to the center again for the 24 h samples. Subjects were monitored and asked to report any possible side-effects experienced due to intake of the tablets during the whole 24 h time after the intake of the tablets.μl each) and stored at −20°C for less than 48 h before being finally transported and stored at −80°C.For crocetin pharmacokinetic analysis, blood samples (3 ml collected into tubes with EDTA) were drawn prior to affron® supplementation (time 0 h), between time 30 and 180 min at every 30 min and 24 h after supplementation. The blood tubes were briefly mixed by inversion and put on ice and centrifuged  to separate the plasma. The EDTA-plasma of every sample was carefully removed in 3 aliquots  = (AUC + 131.56)/9475). The intra-assay precision CV was 4.8%, and the interassay variability and precision CV were 7.3%, with a blood detection limit of 0.05 μg/ml.The crocetin concentration in biological samples was calculated using a standard linear curve at several crocetin concentration and the Shimadzu software for chromatogram analysis. All samples fell within the range of the linear standard curve (concentration (p ≤ 0.05 was determined by one-way ANOVA (Friedman test), and the Bonferroni test was used as post hoc analysis.Pharmacokinetic parameters were derived using GraphPad Prism 7.0 . Data are represented as the mean ± SD and analysed using the SPSS 17.0 statistical software . Significance of differences at Crocus sativus L.).Individual identification of each component of the commercial saffron extract affron® was done by their characteristic UV absorbance peaks at 250, 310, and 440 nm, and their mass/charge relation, as in m/z) and ESI− (609 m/z) modes. These results were in accordance with those obtained with saffron stigmas by Lech et al. [Among the main bioactive compounds found in affron®, at 250 nm, picrocrocin was detected, with characteristic quasimolecular (353.2 m/z) and fragment ions  in ESI + mode. Kaempferol diglucoside was also identified at 250 nm, with its characteristic UV-Vis spectrum (maximum absorbance at 266 and 347 nm), together with typical quasimolecular ions in ESI+As can be observed in in vitro digestion process  did not increase the concentration of crocetin compared to the initial composition of affron®, and its concentration remained 2 orders of magnitude lower compared with crocins. In addition, kaempferol derivatives content was also not affected during digestion, with high bioaccessibility data  and Cmax between the two doses were statistically significant according to the Friedman test (p < 0.05). The average maximum concentration (Cmax) of crocetin was between 0.26 and 0.39 μg/ml for the minimum (56 mg) and maximum (84 mg) dosages, obtained at 60 and 90 min, respectively, after oral intake , the obtained values of


IL13 rs20541, IL4 rs2243250, ADRB2 rs1042713, and FCER1B rs569108 associated with asthma and atopy in Chinese Han children. However, whether the gene model is associated with allergies in early life has yet to be determined. This study aimed to apply the gene model in a birth cohort to investigate its associations with the development of allergic diseases in Chinese Han toddlers.Our previous study has developed a four‐gene model involving Five hundred and ninety‐seven children from a birth cohort completing 2‐year follow‐up were included. Epidemiologic information and cord blood were collected. Children were genotyped for the above polymorphisms and divided into high or low genetic risk groups based on the genotypes. Subjects were followed at 6, 12, and 24 months, with information on allergic diseases collected via standard questionnaires and assessed by specialists.p = .025). After stratification for delivery mode, children at high risk were more likely to develop eczema  over 2 years of follow‐up compared with those at low risk. No significant associations were found between genetic risk and food allergy, wheezing and allergic rhinitis (p > .05).Two hundred and eighty‐four children were divided into a high‐risk group and 313 into a low‐risk group. Between the two groups, a significant difference was only found in delivery mode among all the subject characteristics (The gene model was significantly associated with the development of eczema in Chinese Han toddlers. Long‐term follow‐up along with functional and replication studies on the gene model are still needed in future. The four‐gene model involving IL13 rs20541, IL4 rs2243250, ADRB2 rs1042713, and FCER1B rs569108 was significantly associated with the development of eczema in Chinese Han toddlers. Long‐term follow‐up along with functional and replication studies on the gene model are still needed in future. At 6‐month follow‐up, an internet‐based questionnaire survey was conducted to collect children's medical history of allergic diseases along with information on breastfeeding, antibiotic use, and environmental exposures, including home pet sitting, secondhand smoke exposure, and home decoration. At 1‐ and 2‐year follow‐up visits, face‐to‐face questionnaire interviews were conducted to collect similar information as that at 6‐month follow‐up.At the three follow‐up surveys, children's caregivers were asked whether their children had been diagnosed with eczema, food allergy, wheezing, or allergic rhinitis by doctors in the past 6 months (at the 6‐month and 1‐year follow‐ups) or 1 year (at the 2‐year follow‐up). If the answer was yes, further information on eczema, wheezing or allergic rhinitis was collected by the standardized International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire, which had been validated and adapted in China.2.3Allergic diseases assessment was made by specialists based on the questionnaires. Two dermatologists diagnosed eczema independently for all the participants, with disagreements resolved by consensus. The diagnostic criteria for eczema were: itchy, scaly rash on the flexural sites , face, or around the neck or ears, and itchy rash coming and going for at least 6 months, based on the U.K. working party's diagnostic criteria.Two pediatricians specialized in respiratory medicine and allergy made diagnoses of wheezing, allergic rhinitis, and food allergy independently for all the children, and disagreements were also resolved by consensus. Wheezing was defined if children breathed with whistling sounds in the chest once or more. According to the guideline of the American Academy of Otolaryngology—Head and Neck Surgery, clinical diagnosis of allergic rhinitis was made if children had nasal congestion, runny nose, itchy nose, or sneezing when he/she did not have a cold or the flu, and it was accompanied by itchy watery eyes.2.4Genomic DNA was isolated from cord blood samples using the QIAamp DNA Blood Mini Kit (Qiagen). SNP genotyping was carried out with matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry (MALDI‐TOF MS)2.5IL13 rs20541, IL4 rs2243250, ADRB2 rs1042713, and FCER1B rs569108 are jointly associated with asthma and atopy in Chinese Han children, and each of the SNPs has a single effect on the diseases, with rs20541 GG, rs2243250 TT, rs1042713 AA, and rs569108 GG as the risk genotypes, respectively. More risk homozygotes of the four SNPs, higher risk for asthma and atopy.p < .05 and odds ratio [OR] > 1) compared with the control were categorized as high risk, otherwise as low risk.We have found that 2.6χ2 test. All the participants were divided into high‐ or low‐risk groups based on the genotypes of the four SNPs. The subject characteristics between the two different genetic risk groups were compared using Pearson's χ2 or Fisher's exact test, as appropriate. Associations between genetic risk and allergic diseases were assessed by χ2 tests, and p value, relative risk (RR), and 95% confidence interval (95% CI) were calculated. A two‐tailed p < .05 was considered statistically significant. The statistical analyses were performed using SPSS version 17.0 (IBM Corp.).Each SNP was examined for Hardy–Weinberg equilibrium in the subjects by the 33.1p > .10). Among the 597 study subjects, 284 were at high risk for asthma and atopy and the others were at low risk according to the genetic risk grouping method. The subject characteristics were presented in Table p = .025). More children in the high‐risk group were born by cesarean section than those in the low‐risk group . However, no significant differences were found between the two groups in gender, gestational age, birth weight, parity, breastfeeding, antibiotic use, home pet sitting, in‐home secondhand smoke exposure, home decoration, parental atopy, parental education, and family income (p > .05).All the SNPs met Hardy–Weinberg equilibrium criteria (3.2p = .044). No significant association was found between genetic risk and food allergy, wheezing, and allergic rhinitis (p > .05).As shown in Table 3.3p = .040). And the association was even stronger than that without stratification. Still no significant associations were found between genetic risk and food allergy, wheezing and allergic rhinitis (p > .05).Table 4IL13 rs20541, IL4 rs2243250, ADRB2 rs1042713, and FCER1B rs569108 for determining asthma susceptibility in Chinese Han children.In our previous study, we developed a four‐way gene model including In this study, a significant difference in delivery mode was found between the high and low genetic risk groups, while no differences were found in other subject characteristics between the two groups. After stratification for delivery mode, the positive association between the four‐locus gene–gene interaction model with the development of eczema was a little stronger than that without stratification. In this birth cohort study, 28.14% (168/597) of the subjects developed eczema while 39.03% (233/597) developed allergies  over 2 years of follow‐up. These prevalence data were similar with those reported in other studies.IL13, IL4, ADRB2, and FCER1B, which constitute the gene model, are all involved in IgE‐mediated allergic responses. Cytokines IL‐13 (IL‐13) and IL4 bind to the shared heterodimeric receptor on B cells and switch Ig production from IgM to IgE via a common signaling pathway.IL13 rs20541, IL4 rs2243250, ADRB2 rs1042713, and FCER1B rs569108) has been reported associated with increased IgE levels.Allergic diseases have been attributed to IgE‐mediated inflammatory reactions.In the present study, no associations were found between the gene model and food allergy/wheezing/allergic rhinitis, which may be partly attributed to the short‐time follow‐up of the cohort and allergic diseases assessments based on parental‐reported symptoms. Hence, long‐term follow‐up and disease assessment by symptoms plus objective measurements  are necessary in the future.IL13, IL4, ADRB2, and FCER1B) were chosen as candidate genes. However, the four genes are susceptible genes of asthma and atopy replicated in >10 different studies,Additionally, there are some other limitations in this study. First, only four genes (i.e., IL13 rs20541, IL4 rs2243250, ADRB2 rs1042713, and FCER1B rs569108 was significantly associated with the development of eczema in Chinese Han toddlers. The four SNPs may interact with each other via IgE‐mediated inflammatory pathways in the pathogenesis of allergic eczema. Long‐term follow‐up along with functional and replication studies on the gene model are still needed in future.In summary, the data presented in this study suggest that the four‐way gene model involving The authors declare that there are no conflict of interests.


Micronutrients have been implicated as an important factor in regulating various metabolic processes and thus playing a role in the aetiology of obesity. Many studies have been conducted worldwide that clearly show a direct link between obesity and micronutrient deficiencies. The aim of this study was to assess the nutritional status of overweight and obese Australian adults to see if there were any associations between BMI and serum micronutrient levels.2 and aged between 18 and 65 years was compared to the clinical micronutrient reference ranges for associations between BMI and micronutrient status.Baseline serum micronutrient data of overweight and obese individuals with a body mass index (BMI) between 25 and 40 kg/mp = 0.044), folate (p = 0.025), magnesium (p = 0.010) and potassium (p = 0.023).There were significant negative associations between BMI and serum vitamin D , metabolism , increased physiologic requirements, and lower absolute total dietary intake being the current theory for observed differences. While vitamins D, folate, magnesium and potassium showed a negative relationship to BMI, other micronutrients did not. This may be explained by the fortification of certain processed foods, or the possibility of overweight and obese people eating more to satisfy their nutritional requirements. In most cases, overweight or obesity is due to a positive energy balance stemming from excessive energy intake - compared to energy expenditure - from diets that are typically nutritionally poor . Excess Micronutrient interactions within the food matrix can impact absorption and bioavailability by a number of mechanisms. Minerals with chemical similarities can compete for transport proteins or other uptake mechanisms, as well as for chelating organic substances, facilitating or hindering absorption . The oveNutrient reference values as provided by the National Health and Medical Research Centre (NHMRC) are ‘Recommended Dietary Intake’ (RDI) or Adequate Intake (AI), which includes the amounts of specific nutrients required on average each day for sustenance or avoidance of deficiency states . FurtherFew studies have examined the nutritional status of overweight and obese adults. One such study suggested vitamin D, chromium, biotin, thiamine and vitamin C levels are significantly lower in persons who are obese and that insufficiency in these micronutrients has the ability to impact glucose metabolism and cause insulin resistance . Biochem12, C, D, E and B9 as well as minerals, iron, iodine, calcium, potassium, sodium, magnesium and zinc were evaluated.The current study examined baseline blood micronutrient levels of a group of overweight and obese Australian adults with the most up-to-date serum nutrient reference ranges for Australian adults. The aim of this study was to assess the nutritional status of overweight and obese Australian adults prior to the commencement of a high fibre weight loss clinical trial, to determine if there were any associations between BMI and poor serum micronutrient levels. Vitamins A, BWhat is the baseline micronutrient status of a sample of overweight and obese Australian adults?n = 127) with a BMI between 25 and 40 kg/m2 and aged between 18 and 65 years, were recruited from the community in Perth, Australia via advertisements in newspapers, flyers posted around the University and community noticeboards, as well as radio advertising on Curtin FM. Potential participants were screened by telephone or online using a Qualtrics Survey and attended an orientation session at Curtin University to assess suitability for the study, at which time the details of the study were also explained. Exclusion criteria included smoking, lipid lowering medication, use of steroids and other agents that may influence lipid metabolism, diuretics, use of warfarin, diabetes mellitus, hypo and hyperthyroidism, cardiovascular events within the last 6 months, psychological unsuitability, major systemic diseases, gastrointestinal problems, proteinuria, liver, renal failure, weight fluctuations over the past 6 months, vegetarianism or veganism and participation in any other clinical trials within the last 6 months.Overweight and obese individuals . Participants in all groups were asked to maintain their usual diet for the duration of the study. For the purposes of this study only serum and food diary data obtained from week 0 was used for comparison.12, B9, C, D and E and minerals  for analysis. All blood samples were collected by a trained phlebotomist. Blood samples were then centrifuged at 2500 rpm at 4 °C for 10 min using an Eppendorf centrifuge and prepared for storage at -80 °C. Micronutrients were analysed systematically, after all the participants had completed the 52-week study. Vitamins A, E, D, as well as vitamin B9, and B12 and thyroglobulin as a measure of Iodine status were measured using Enzyme-Linked Immunosorbent Assay kits (ELISA). ELISA combines antibody binding with enzymatic detection to identify molecules of interest; the result is a colour change that is measured by spectrophotometry at a particular wavelength [For the purposes of this present study, baseline blood samples were analysed for 127 participants. Volunteers attended Curtin University, after an overnight fast of 10–12 h, where blood samples were drawn by venepuncture. Samples were collected into lithium heparin or serum separator tubes (5 ml) for antioxidant/vitamins A, Bvelength .12, B9, C, D E, folate and minerals calcium, iron, iodine, magnesium, potassium, sodium and zinc. Other outcome measures that formed part of the larger study, such as weight/BMI and metabolic syndrome risk factors, have been analysed and discussed elsewhere [The present study was designed to assess participants’ nutritional status in relation to: vitamins A, Blsewhere .n = 127) of overweight/obese individuals, baseline micronutrients were observed with the clinical reference intervals (serum) sourced from the Stedman’s Medical Dictionary ) or, where there was an absence of data, The Merck Manual [To determine the nutritional status of this sample .One sample t-tests were conducted, with the lower limit from the clinical reference range for each micronutrient used as the test value, with the exception of thyroglobulin (an indirect measurement of iodine status), where the upper limit was used . The bas2. The gender of the study group was mostly female with 73 women versus 54 men. Of the 127 participants 33.1% were classified as overweight and 66.9% fell within the obese category according to BMI.Blood samples, for the 127 overweight and obese participants, were analysed for baseline micronutrient levels. The group results showed that the average age of the study participant was 49.3 ± 1.0 years, the average weight was 94.0 ± 1.5 kg and average BMI was 32.3 ± 0.4 kg/m12 and direct comparison between the NRVs and vitamin A, measured as retinol equivalents (RE) was not possible either as FoodWorks breaks vitamin A into retinol and beta carotene  did not reach required levels for vitamin D, deficiency is described at <20–24 ng/mL and the sample mean was 10.9 ± 0.6 ng/mL. Only 28% were within the reference range for folate, with deficiency <3 μg/L, the sample mean was 2.5 ± 0.2 μg/L. However, thyroglobulin levels were mostly (84.9%) within range 2–50 ng/ml with a mean of 8.8 ± 1.3 ng/mL. 100% of the sample did not meet the NRV for potassium with deficiency at <3.5 mEq/L; and the sample mean: 2.5 ± 0.02 mmol/L. Sample population serum values for sodium (Mean 118.8 ± 0.9 mmol/L), zinc (Mean 27.9 ± 1.2 μg/dL) and calcium (3.4 ± 0.1 mg/dL) levels were all lower than the clinical reference interval. The majority of participants were within the clinical reference interval for iron with a reference of 30 to 300 ng/mL for serum ferritin and mean values for men 114.4 ± 4.8 μg/dL and women 95.2 ± 4.4 μg/dL respectively. Serum magnesium levels were significantly lower than the clinical reference interval with a mean value of 0.7 ± 0.01 mg/dL compared to 1.8–2.6 mg/dL.The baseline serum values were compared with the clinical reference intervals for nutrients in Australia (AACB) – shown iTable s = − 0.152, p = 0.044), folate , potassium , and magnesium . Vitamin D, folate, magnesium and potassium all showed that as BMI increased the serum concentration for these micronutrients decreased slightly  between serum vitamin D status and body mass index  have been implicated as an important factor in regulating various metabolic processes and thus playing a role in the aetiology of obesity. Many studies are being conducted worldwide that clearly show a direct link between obesity and micronutrient deficiencies , 42. DefIn an ideal world, micronutrient requirements would be met by a varied diet high in fruit and vegetables. However several of studies have shown that people simply are not consuming the amounts required to achieve and maintain nutritional sufficiency . In many12 intake accounted for between 5 and 8% of females dependent on age and less than 1 % of males; 40% of 14–18 year old females and 38% of 19–50 year-old females had inadequate iron intakes compared to only 3% of males; less than 5% of the population did not meet their dietary needs for vitamin C and vitamin E; and, from age 14 males have a much higher requirement of zinc than females due to its key role in the male reproductive system [Key findings from the 2011–2012 Australian health Survey support that notion. Usual Nutrient Intake data showed that 73% of females and 50% of males aged 2 years and over did not meet their calcium requirements; 17% of males and 14% of females had inadequate usual intakes of vitamin A. One in eleven adult females (aged 19 and over) did not meet the requirement for folate, however almost all males let the dietary requirement through intake; insufficient Be system . Accordie system . One in e system .12. All of the study participants either met or exceeded vitamin C levels and for vitamin A every person had less than adequate serum levels. Eighty nine percent of participants had vitamin D levels that would put them in the ‘severe’ deficiency category. Seventy two percent were below the lower limit for folate, however 84.9% were within range for thyroglobulin which is a more accurate biomarker of folate status. For potassium, all participants measured had inadequate serum levels and 94.5% had lower than normal sodium levels. Approximately 90%  were within reference range values for iron, however 99.2% had less than adequate serum values for zinc. Calcium values of the participants were also lower than the clinical reference values with 91.3% having a serum calcium level lower than < 4.64 mg/dL.When comparing the current study with the Australian Health survey findings Table , 31% of The exact mechanisms that explain the relationship between folate metabolism and obesity are still being established. Previous studies have reported people with higher BMI have high erythrocyte folate concentrations, as well as high levels of circulating serum folate oxidation products, but can also have low fasting serum levels . OverweiA negative association with BMI for vitamin D, potassium and magnesium, implies people with higher BMI are at risk of deficiency and deficiency related conditions. Vitamin D, potassium and magnesium all play essential roles in bone health and calcium storage, resorption and regulation , 50. A sThis study is unique in the fact that not many studies have examined the nutritional status of overweight and obese individuals, as most focus on over-nutrition in terms of macronutrients, and interventions to treat this disease. The baseline figures suggest show that serum micronutrient levels for vitamin D, calcium, folate, potassium, sodium, magnesium and vitamin A were all below the NRV recommendation. The study participants who fall below the clinical reference intervals for these micronutrients could be said to have high calorie malnutrition. There is an emerging theory that proposes overweight and obese individuals who consume a large amount of highly processed food within the standard ‘Western Diet’ have a physiological drive to eat excessive amounts due to the poor nutritional content of these foods, and the body’s innate need to achieve (micro) nutritional sufficiency . This stPossible limitations of the current study may include that the blood samples and dietary intake data from 3-day food diaries were only collected at baseline whereas multiple time periods throughout the year may have been of more significance. Many deficiency states happen over a period of time depending on the micronutrient, so analysis of a ‘snap shot’ on the time scale may not be the most representative of micronutrient levels. Some also argue serum measurements aren’t the most accurate assessment of nutritional status for every micronutrient. Calcium status for example, bone mineral density is potentially a more accurate measure than serum calcium levels as serum calcium is so tightly regulated within the blood. Consequently if serum calcium levels fluctuate this is not necessarily an indication of status, but rather something is affecting bone calcium release or renal processing and reabsorption . Also inWith studies of overweight and obese individuals, it is not often that their baseline nutritional status is examined. By comparing serum micronutrient levels against the clinical reference intervals for Australia, it shows that dietary intake affects nutritional status and not just body weight, further highlighting the importance of following dietary recommendations for fruit and vegetables.There is limited research examining micronutrient status in individuals with overweight and obesity, and it is probable that different mechanisms explain nutrient-specific increases or decreases among the overweight and obese. Several hypotheses have been proposed to explain the variability of serum micronutrient levels in this subset of the population, including: how a vitamin or mineral is absorbed, excreted, stored/distributed; whether its sequestered by fat or dispersed in tissue, metabolic processes , increased physiologic requirements, and lower absolute total dietary intake , 9. OverThis study aimed to identify potential relationships between BMI and baseline micronutrient status, and found that participants within this sample with a higher BMI tended to have lower serum vitamin D, folate, magnesium and potassium levels. Serum micronutrient levels compared to the clinical reference intervals and dietary intake data compared to the NRVs also supported the theory that diet quality in this subset of the population is poor. More research is needed to further clarify the associated health implications of micronutrient deficiency or surplus in overweight and obese individuals.


Tenofovir disoproxil fumarate (TDF) has been efficacious in treating chronic hepatitis B (CHB), but long‐term use is accompanied by a decline in renal function and bone mineral density (BMD). Tenofovir alefanamide (TAF) is a prodrug of tenofovir, with similar efficacy in CHB but with fewer side effects than TDF. Recent studies on patients who underwent the switch from TDF to TAF have shown improved bone and renal profiles from 24 to 48 weeks of follow‐up.This study provides follow‐up at 72 weeks in a real‐world cohort of 61 Asian CHB patients who were switched from TDF to TAF. All patients had been treated with TDF for at least 12 months with hepatitis B virus DNA <21 IU/mL prior to switch.P < 0.01). Renal function showed decline at 72 weeks compared to baseline . Improvement in hip BMD was sustained at 72 weeks . However, spine BMD showed discordance, with initial improvement at 24 weeks  but regression at 72 weeks . Interestingly, there was a slight increase in weight and BMI after 72 weeks (P < 0.01).Improvements in proximal tubular function, measured by urine beta‐2‐microglobulin to creatinine and retinol‐binding protein to creatinine ratios, were sustained at 72 weeks (CHB patients who switch from long‐term TDF to TAF therapy show sustained improvement in proximal tubular function and hip BMD. Weight gain was noted, and long‐term studies are needed to evaluate its effect on patient outcomes. Long‐term follow‐up of CHB patients who switched from tenofovir disoproxil fumarate to tenofovir alefanamide continued to show efficacy in hepatitis B virus suppression along with an improved renal and bone safety profile. Improvement in proximal renal tubular function and bone density was seen as early as week 12 after the switch, and it appeared to have a “ceiling effect” at week 24. However, this improvement was sustained at week 72. Each patient provided informed consent prior to enrollment. Adults (≥18 years) with CHB who were treated with TDF for at least 1 year with HBV DNA < 20 IU/mL for >6 months were invited to participate. Patients previously treated with adefovir or had a coinfection with human immunodeficiency virus (HIV) or hepatitis C virus were excluded. Patients with a history of organ transplantation were also excluded. Patients had an initial follow‐up of 24 weeks after the switch, with results reported in a previous study.4/GFR). These tests were obtained at day 0, week 12, week 24, and week 72. Patients underwent BMD testing at screening, week 12, week 24, and week 72 of treatment. BMD in the anteroposterior view of the lumbar spine (from L1 to L4) and hip was measured using a Hologic QDR Discovery C enhanced‐array whole‐body DEXA scanner and version 12.1 software . All scans were conducted on the same machine with a single operator. No major changes were made to other medications during the time of switch.Patients who met the inclusion criteria were switched from TDF to 25 mg of TAF orally once daily, administered with food. Postbaseline study visits occurred at weeks 12, 24, and 72. At each visit, a history and physical examination were obtained, while compliance was determined by pill count. Laboratory tests included a comprehensive metabolic panel, serum phosphorus level, uric acid level, and HBV DNA. Markers of tubular function obtained included urine albumin to creatinine ratio, fractional excretion of uric acid, fractional excretion of phosphate, and tubular maximum for phosphate corrected for glomerular filtration rate (GFR) , medians (range), or frequencies (percentages). Laboratory values were compared by treatment status . BMD values were also reported by mean percentage changes from baseline. Paired sample t‐test or Wilcoxon signed‐rank test was used to compare differences as appropriate. McNemar's test was used to compare proportions as appropriate. All statistical analyses were performed using R 3.6.1 .From the previous cohort of 75 patients,CG > 90 mL/min, while 19 patients (31%) had GFRCG between 60 and 90 mL/min, and 6 patients (10%) had GFRCG between 30 and 59 mL/min. When stratified by age, those 50 years or older had lower GFRCG compared to younger patients (89.4 vs 111.5 mL/min). There were no differences in the prevalence of diabetes, hypertension, or time on TDF prior to switch between GFRCG groups. Creatinine clearance by Cockcroft‐Gault was unchanged at week 24 but declined at week 72   had normal renal function as estimated by GFRvs 1.2 μg/g, P < 0.01) and urine retinol‐binding protein (RBP)  at 24 weeks compared to baseline.P < 0.01)   with an associated increase in fractional excretion of phosphate compared to baseline . The phosphate threshold for renal tubular reabsorption (TmPO4) also declined at week 24 compared to baseline . At week 72, all parameters of renal phosphate handling were reverted and were no longer significantly different from baseline  were overweight, and 3 patients (5%) were obese. At week 24, there was an increase in BMI , which remained elevated at week 72 compared to BMI at screening  (Table The mean body mass index (BMI) at screening was 24.1 ± 3.4 kg/mAt baseline, 16 patients (26%) had abnormal serum alanine aminotransferase (ALT) levels. Of 16 of those with abnormal ALT, 6 were overweight/obese, while 1 patient was diabetic. There were no significant changes in ALT throughout the study. Of these 16 patients, 4 had normalization of serum ALT at 72 weeks. None of the patients with normal ALT level at baseline developed abnormal ALT level at 72 weeks. After 24 weeks of switch from TDF to TAF therapy, there were two patients with detectable HBV DNA levels. In both patients, virologic relapse was due to noncompliance that was corroborated by pill count.Due to its antiviral potency and high genetic barrier, TDF is one of the recommended first‐line agents for treatment of CHBversus TAF with a follow‐up of 92 weeks, CHB patients receiving TDF compared to TAF had greater mean percentage changes at week 96 for urine RBP to creatinine ratio  and urine β2M to creatinine ratio .Compared to baseline, proximal renal tubular dysfunction, including urine β2M to creatinine ratios and RBP to creatinine ratios, improved significantly at week 24 from the initial switch. However, there was no further improvement between weeks 24 and 72, although the improvement from baseline was sustained. Our study suggests that the improvement in renal tubular parameters following the switch occurred during the first few months, and there may be a “ceiling effect” in recovery after the initial toxicity of TDF. In a study by Agarwal and colleagues comparing TDF 4/GFR) is another parameter used to assess proximal tubular function.4 at week 24 after switching despite improvement in other markers of proximal renal tubular function. These changes were concurrent with a decrease in serum phosphorus levels and increase in fractional excretion of phosphate. However, after 72 weeks, this effect was no longer present, and renal parameters of phosphate handling and serum phosphate levels were no longer significantly different from baseline. At the end of follow‐up, there were no episodes of Fanconi syndrome in the study cohort.Renal tubular absorption of phosphate  was not assessed in this study. Thus, our results cannot be extrapolated to this patient population.Long‐term follow‐up of CHB patients who switched from TDF to TAF continued to show efficacy in HBV suppression along with an improved renal and bone safety profile. Improvement in proximal renal tubular function and bone density was seen as early as week 12 after switch, and it appeared to have a “ceiling effect” at week 24. However, this improvement was sustained at week 72. Our study of a cohort of real‐world patients with CHB shows the long‐term benefit in switching from TDF to TAF. Future studies should evaluate the aspect of weight gain and its clinical implications after switching from TDF to TAF.


To examine mediation and moderation of racial/ethnic all‐cause mortality disparities among Veteran Health Administration (VHA)‐users by neighborhood deprivation and residential segregation.Electronic medical records for 10/2008‐9/2009 VHA‐users linked to National Death Index, 2000 Area Deprivation Index, and 2006‐2009 US Census.Racial/ethnic groups included American Indian/Alaskan Native (AI/AN), Asian, non‐Hispanic black, Hispanic, Native Hawaiian/Other Pacific Islander, and non‐Hispanic white (reference). We measured neighborhood deprivation by Area Deprivation Index, calculated segregation for non‐Hispanic black, Hispanic, and AI/AN using the Isolation Index, evaluated mediation using inverse odds‐weighted Cox regression models and moderation using Cox regression models testing for neighborhood*race/ethnicity interactions.P = .047). Black segregation's mediating effect was limited to VHA‐users living in counties with low black segregation. AI/AN segregation also mediated AI/AN mortality disparities in counties that included or were near AI/AN reservations.Mortality disparities existed for AI/ANs  but no other groups after covariate adjustment. Neighborhood deprivation and Hispanic segregation neither mediated nor moderated AI/AN disparities. Non‐Hispanic black segregation both mediated and moderated AI/AN disparities. The AI/AN vs. non‐Hispanic white disparity was attenuated for AI/ANs living in neighborhoods with greater non‐Hispanic black segregation (Neighborhood characteristics, particularly black and AI/AN residential segregation, may contribute to AI/AN mortality disparities among VHA‐users, particularly in communities that were rural, had greater black segregation, or were located on or near AI/AN reservations. This suggests the importance of neighborhood social determinants of health on racial/ethnic mortality disparities. Living near reservations may allow AI/AN VHA‐users to maintain cultural and tribal ties, while also providing them with access to economic and other resources. Future research should explore the experiences of AI/ANs living in black communities and underlying mechanisms to identify targets for intervention. Even within health care systems that strive to provide equal access to care to all patients, such as the Veterans Health Administration, racial/ethnic disparities in mortality existResearch in the general population suggests that neighborhood deprivation and segregation contribute to racial/ethnic disparities, but it is unknown whether these neighborhood characteristics also account for racial/ethnic disparities among Veteran Health Administration (VHA) users.This study identified all‐cause mortality disparities among American Indian/Alaskan Native (AI/AN) VHA‐users versus non‐Hispanic whites and that non‐Hispanic black and AI/AN neighborhood segregation both mediated these disparities.The disparity in all‐cause mortality among AI/AN VHA‐users was attenuated in neighborhoods with greater non‐Hispanic black segregation, suggesting the importance of neighborhood‐level social determinants of health on racial/ethnic mortality disparities.1Health care is an important—but not sole—determinant of health outcomes.The Veterans Health Administration (VHA) is an example of a health system that has made strides in addressing racial/ethnic disparities. Within VHA, non‐Hispanic blacks  had similar or lower mortality from prostate cancer, lung cancer, and chronic kidney disease relative to non‐Hispanic whites .The neighborhoods in which people live are an important social determinant of health. Methodological advances have allowed researchers to disentangle the neighborhood's compositional effects  from its contextual effects on health .While neighborhoods encompass complex social and physical environments, identifying specific neighborhood characteristics associated with health disparities may guide policy and practice, such as community‐based partnerships, initiatives to improve healthy food access, and comprehensive neighborhood revitalization efforts.Racial/ethnic disparities may arise from racial/ethnic groups living in different neighborhoods that expose them to different contexts and risk factors. In the US general population, racial/ethnic minority groups at higher risk for mortality, such as blacks, are more likely to live in segregated and more deprived neighborhoods.Segregation effects on health may vary by race/ethnicity. Segregation of different racial/ethnic groups arose through different processes and historical forces. Black segregation formed through discriminatory practices and policies .It is unknown whether neighborhood deprivation and segregation account for observed disparities among racial/ethnic minority VHA‐users. Understanding whether such relationships exist in this population can yield important insights for the VHA and the broader health care system. A key difference between VHA‐users and the US general population is that VHA‐users have access to a health care system that provides comprehensive physical and mental health services and has initiatives to improve access .This study has specific implications to VHA, given its broad mission to improve the health of all Veterans. Compared to other health care systems, VHA may be better positioned to address social determinants of health, as they are already targeting some determinants including housing and employment. For example, VHA’s permanent supportive program is one of the largest programs in the nation and has been credited with substantially reducing Veteran homelessness nationally.In this study, we examined the role of two neighborhood‐level social determinants of health on racial/ethnic all‐cause mortality disparities among a cohort of Veteran VHA‐users—neighborhood deprivation and residential segregation. Our first aim explored racial/ethnic differences in all‐cause mortality from 2008 to 2016 in a national cohort of VHA‐users. Our second aim examined whether neighborhood deprivation a) mediated or b) moderated racial/ethnic all‐cause mortality disparities among VHA‐users. Our third aim similarly examined whether residential segregation a) mediated or b) moderated these relationships. We hypothesized that blacks and AI/ANs would experience disparities in all‐cause mortality, that both neighborhood deprivation and segregation would mediate the racial/ethnic all‐cause mortality disparities, and that racial/ethnic disparities would be greater in neighborhoods with greater deprivation and segregation and attenuated in less deprived and segregated neighborhoods.22.1n = 13), invalid dates of index ambulatory care visits , and missing data on covariates . Mortality was ascertained through 12/2016.Our analytic sample consisted of a national cohort of Veterans (age ≥ 18 years) with VHA ambulatory care use from 10/2008 to 9/2009 . Data came from VHA’s electronic medical records, linked to the Centers for Disease Control and Prevention (CDC)’s National Death Index (NDI) to provide mortality data, the 2000 Area Deprivation Index (ADI)2.22.2.1We examined time to all‐cause mortality, calculated as the difference in years between date‐of‐death and qualifying date‐of‐initial FY2009 health care use. Deaths were classified as either observed on the date of death or administratively censored at the end of the mortality ascertainment period.2.2.2Our main independent variable was a categorical indicator of patient race/ethnicity. We created the race/ethnicity variable using a validated algorithm that combines multiple data sources and years of data in a hierarchy ordered by data quality of the source data files, with self‐identified race/ethnicity considered the highest.2.2.3We examined two neighborhood characteristics: neighborhood deprivation and neighborhood segregation. Neighborhood deprivation was captured through the ADI.∑i=1n[(xi/X)(xi/ti)], where n is the number of census tracts in the county, xi is the comparison group population in census tract i, X is the sum of all xi , and ti is the total population of census tract i. The isolation index ranges from 0 to 1, where higher isolation indices indicate greater racial/ethnic group isolation. We calculated separate isolation indices for blacks, Hispanics, and AI/ANs.While numerous measures of neighborhood segregation have been developed to measure residential segregation,2.2.4We included the following covariates in our analysis: age , sex, medical comorbidity, mental health comorbidity , individual‐level SES , and rurality indicator . The medical comorbidity index was adapted from the Seattle Index of Comorbidity, based on a weighted count of smoking status and seven chronic medical conditions associated with increased mortality .2.3We calculated descriptive statistics of race/ethnicity stratified means and proportions of the VHA population's demographics, health characteristics, and neighborhood characteristics, and crude race/ethnicity‐stratified mortality rates (number of deaths/100 000 population).2.3.1We used three Cox regression models to calculate all‐cause mortality hazard ratios comparing each racial/ethnic minority group to whites (reference group). Model 1 adjusted for age and sex. Model 2 further adjusted for individual SES and urban/rural status, which are important factors when considering neighborhood‐level social determinants of health. Model 3 further adjusted for comorbidity and mental health comorbidity, which are on the causal pathway of mortality disparities. All models included clustered standard errors at the VHA facility level to account for within‐VHA facility correlation.2.3.2P < .05, we subsequently estimated the direct and indirect effects of race/ethnicity with Tchetgen Tchetgen's inverse odds weighting approach (IOW)P < .05 as evidence of mediation. Finally, we transformed the total, direct and indirect effect coefficients into hazard ratios and transformed the indirect effect coefficients into proportions of the total effect explained by the neighborhood characteristic mediator of interest .Mediation analysis involves estimating the total effect of race/ethnicity on mortality, and decomposing this effect into its direct (unmediated) and indirect (mediated) effect components. We tested mediation by neighborhood deprivation and segregation separately in the following way. First, we estimated the total effect of race/ethnicity on mortality for each mediator through Cox regression while adjusting for potential confounders and the neighborhood mediator of interest. For each total effects model showing evidence of a racial/ethnic disparity relative to whites at 2.3.3P < .05, we considered the neighborhood characteristic to be a mediator.We examined whether neighborhood deprivation or neighborhood segregation modified racial/ethnic mortality differences. We modeled the neighborhood‐level modifiers in separate Cox regression models that included a product term between the moderator of interest and race/ethnicity, while controlling for all covariates described above in model 3’s specifications. If the interaction term was statistically significant at 2.3.4We conducted two sensitivity analyses. First, urban‐rural differences may be important to consider because AI/ANs are more likely to reside in rural and highly rural communities, and AI/ANs residing in urban versus rural communities may differ in ways that influence mortality. Thus, for neighborhood characteristics that statistically significantly mediated or moderated AI/AN disparities, we also stratified analyses by urban vs. rural . Second, since small population sizes can affect the stability of the segregation indices,3P < .05 for all variables). Whites were the oldest in the sample . Blacks had the largest proportion of female Veterans (9.98 percent). Asians had the lowest medical comorbidity . AI/ANs had the highest levels of depression without SMI (23.41 percent) and other mental disorders without SMI or depression (13.52 percent). Whites were most likely to have high individual SES (28.79 percent). AI/ANs were most likely to reside in highly rural areas (10.43 percent). Neighborhood deprivation was lowest among Asians (87.48). Blacks and Hispanics were more likely to live in higher black‐segregated (mean: 0.45 SD: 0.22) and Hispanic‐segregated neighborhoods , respectively. Crude mortality proportions were highest among whites (4509 per 100 000).Within the cohort of 5,032,009 Veterans who were observed for 34,859,260 person‐years, there were 1,374,356 deaths. Cohort characteristics stratified by Veteran race/ethnicity are presented in Table 3.1After adjusting for age and sex, AI/ANs  and blacks  had higher mortality versus whites Table . With fuAge‐ and sex‐adjusted mortality was lower in Asians  and Hispanics  and similar in NH/OPIs  versus whites. Differences for Asians and Hispanic remained after individual SES, rurality, and medical and mental health comorbidity adjustment. With additional medical and mental health comorbidity adjustment, NH/OPIs had lower mortality compared to whites .3.2P‐value = .03), reducing the total effect of the AI/AN mortality disparity by 35.4% . There was no evidence of medication by neighborhood deprivation  or Hispanic segregation .Analysis of whether neighborhood characteristics mediated the relationship between AI/AN race/ethnicity and mortality revealed that black segregation mediated AI/AN mortality disparities  Table . When stFor the racial/ethnic groups with mortality advantages relative to whites, we found that associations between race/ethnicity and mortality varied by neighborhood characteristics. As neighborhood deprivation increased, mortality advantages increased for Asian and Hispanics, but were attenuated for blacks. The positive association between mortality and neighborhood deprivation was stronger among blacks than whites . In more Hispanic‐segregated neighborhoods, mortality advantages were accentuated among Hispanics  and NH/OPIs .3.4P‐value = .09), but not among urban Veterans    , but did mediate AI/AN disparities  Tables ‐S3.3.5P‐value < .01), but not in high black‐segregated counties  and low (≤33 percent black isolation) black segregation counties. We determined high and low segregation based on the level of black isolation where AI/AN and white mortality hazard ratios were equivalent. We found that black segregation mediated AI/AN disparities among Veterans living in counties with low black segregation , it can also consider creative ways to look beyond health care to impact health outcomes.Future research can explore the experience of AI/ANs residing in black‐segregated communities, including their experiences of acculturation and discrimination, underlying mechanisms through which black segregation is related to AI/AN health disparities, especially in high black‐segregated communities, and other neighborhood‐level social determinants of health that may contribute to urban AI/AN disparities. Research on social determinants influencing AI/AN disparities should consider urban‐rural differences. These efforts can inform future social determinant‐oriented interventions, such as improving AI/AN social support from other Veterans , and ways that health systems might partner with community‐based organizations.Health care systems, including VHA, are the primary entity charged with improving health of populations and increasingly held accountable to address health disparities. Closing this gap may require health care systems to explore novel collaborations with social services to address neighborhood‐level social determinants of health, such as community partnerships, financing strategies that reimburse social services, and co‐locating resources  in or near health care settings.The authors declare no conflicts of interest.Supplementary MaterialClick here for additional data file.Table S1‐S7Click here for additional data file.


We found that the separation between vesicle and target membranes strongly correlates with the organisation of protein complexes at junctions. At larger membrane separations, protein complexes assume a ‘clustered’ distribution at the docking site, inducing a protrusion in the target membrane. As the membrane separation decreases, protein complexes become displaced radially outwards and assume a ‘ring‐like’ arrangement. Our findings indicate that docked vesicles can possess a wide range of protein complex numbers and be heterogeneous in their protein arrangements.Synaptic vesicle proteins, including N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs), Synaptotagmin‐1 and Complexin, are responsible for controlling the synchronised fusion of synaptic vesicles with the presynaptic plasma membrane in response to elevated cytosolic calcium levels. A range of structures of SNAREs and their regulatory proteins have been elucidated, but the exact organisation of these proteins at synaptic junction membranes remains elusive. Here, we have used cryoelectron tomography to investigate the arrangement of synaptic proteins in an   Cpx, ComplexinCCs, correlation coefficientscryo‐EM, cryo‐electron microscopycryo‐ET, cryo‐electron tomographyGUVs, giant unilamellar vesicles2PIP, phosphatidylinositol‐4,5‐bisphosphateSNAREs, N‐ethylmaleimide‐sensitive factor attachment protein receptorsSUVs, small unilamellar vesiclesSyt1, Synaptotagmin 1WT, wild‐typeVesicle fusion occurs when the membrane of a vesicle merges with the membrane of an organelle or plasma membrane. In doing so, fusion allows the contents of vesicles and lipids to be transferred between intracellular and extracellular compartments . In neur2+ regulation requires Synaptotagmin 1 (Syt1) and Complexin (Cpx) , o, oin vitin vitro reconstitution, we also observed highly variable numbers of protein densities at primed prefusion junctions, and highly heterogeneous morphologies at fusion sites. In this reconstituted system, nearly all primed prefusion SUVs fuse upon calcium addition [in vivo, in particular at the neuronal synapse, with synaptic vesicles of defined diameters and very fast exocytosis, other regulators including Munc13 likely coordinate with Munc‐18 and Syt1 to more precisely define the copy number and arrangement of SNARE protein assemblies.The number of protein complexes at fusion junctions has also been a subject of intense debate. Recent cryo‐ET studies of vitrified synapses suggested a possible sixfold symmetrical arrangement of fusion protein complexes at primed junctions . Anotheraddition . This leJAGB is an academic editor of FEBS Letters.LG, JM, and AFPS performed experiments. LG and DM analysed images. LG, JM and AS prepared reagents. JM, THS and JAGB designed experiments. LG, JM, AFPS, THS and JAGB interpreted data. LG, JM, THS and JAGB wrote the paper with input from all authors.Fig. S1. Slices from subtomograms depicting junctions from the ring‐like protein distribution class.Fig. S2. Total number of protein electron densities at ring‐like, intermediate and clustered fusion sites.Click here for additional data file.Video S1. Animation of slices through subtomogram volumes of ring‐like, intermediate and scattered fusion sites.Click here for additional data file.Video S2. Animation of averaged junction volumes from the ‘Clustered’, ‘Intermediate’ and ‘Ring‐like’ junctions.Click here for additional data file.