Trajectory of vitamin D status during pregnancy in relation to neonatal birth size and fetal survival: a prospective cohort study

Background: We investigated the associations between vitamin D status in early and late pregnancy with neonatal small for gestational age (SGA), low birth weight (LBW) and preterm delivery. Furthermore, associations between vitamin D status and pregnancy loss were studied. Methods: Serum 25-hydroxyvitamin D (25OHD) was sampled in gestational week ≤ 16 (trimester 1 (T1), N = 2046) and > 31 (trimester 3 (T3), N = 1816) and analysed using liquid chromatography tandem mass spectrometry. Pregnant women were recruited at antenatal clinics in south-west Sweden at latitude 57–58°N. Gestational and neonatal data were retrieved from medical records. Multiple gestations and terminated pregnancies were excluded from the analyses. SGA was defined as weight and/or length at birth < 2 SD of the population mean and LBW as < 2500 g. Preterm delivery was defined as delivery < 37 + 0 gestational weeks and pregnancy loss as spontaneous abortion or intrauterine fetal death. Associations between neonatal outcomes and 25OHD at T1, T3 and change in 25OHD (T3-T1) were studied using logistic regression. Results: T1 25OHD was negatively associated with pregnancy loss and 1 nmol/L increase in 25OHD was associated with 1% lower odds of pregnancy loss (OR 0.99, p = 0.046). T3 25OHD ≥ 100 nmol/L (equal to 40 ng/ml) was associated with lower odds of SGA (OR 0.3, p = 0.031) and LBW (OR 0.2, p = 0.046), compared to vitamin D deficiency (25OHD < 30 nmol/L, or 12 ng/ml). Women with a ≥ 30 nmol/L increment in 25OHD from T1 to T3 had the lowest odds of SGA, LBW and preterm delivery. Conclusions: Vitamin D deficiency in late pregnancy was associated with higher odds of SGA and LBW. Lower 25OHD in early pregnancy was only associated with pregnancy loss. Vitamin D status trajectory from early to late pregnancy was inversely associated with SGA, LBW and preterm delivery with the lowest odds among women with the highest increment in 25OHD. Thus, both higher vitamin D status in late pregnancy and gestational vitamin D status trajectory can be suspected to play a role in healthy pregnancy

Extra-Renal Elimination of Uric Acid via Intestinal Efflux Transporter BCRP/ABCG2

Urinary excretion accounts for two-thirds of total elimination of uric acid and the remainder is excreted in feces. However, the mechanism of extra-renal elimination is poorly understood. In the present study, we aimed to clarify the mechanism and the extent of elimination of uric acid through liver and intestine using oxonate-treated rats and Caco-2 cells as a model of human intestinal epithelium. In oxonate-treated rats, significant amounts of externally administered and endogenous uric acid were recovered in the intestinal lumen, while biliary excretion was minimal. Accordingly, direct intestinal secretion was thought to be a substantial contributor to extra-renal elimination of uric acid. Since human efflux transporter BCRP/ABCG2 accepts uric acid as a substrate and genetic polymorphism causing a decrease of BCRP activity is known to be associated with hyperuricemia and gout, the contribution of rBcrp to intestinal secretion was examined. rBcrp was confirmed to transport uric acid in a membrane vesicle study, and intestinal regional differences of expression of rBcrp mRNA were well correlated with uric acid secretory activity into the intestinal lumen. Bcrp1 knockout mice exhibited significantly decreased intestinal secretion and an increased plasma concentration of uric acid. Furthermore, a Bcrp inhibitor, elacridar, caused a decrease of intestinal secretion of uric acid. In Caco-2 cells, uric acid showed a polarized flux from the basolateral to apical side, and this flux was almost abolished in the presence of elacridar. These results demonstrate that BCRP contributes at least in part to the intestinal excretion of uric acid as extra-renal elimination pathway in humans and rats

Ribonucleotide reductases of Salmonella Typhimurium : transcriptional regulation and differential role in pathogenesis

Ribonucleotide reductases (RNRs) are essential enzymes that carry out the de novo synthesis of deoxyribonucleotides by reducing ribonucleotides. There are three different classes of RNRs (I, II and III), all having different oxygen dependency and biochemical characteristics. Salmonella enterica serovar Typhimurium (S. Typhimurium) harbors class Ia, class Ib and class III RNRs in its genome. We have studied the transcriptional regulation of these three RNR classes in S. Typhimurium as well as their differential function during infection of macrophage and epithelial cells. Deletion of both NrdR and Fur, two main transcriptional regulators, indicates that Fur specifically represses the class Ib enzyme and that NrdR acts as a global repressor of all three classes. A Fur recognition sequence within the nrdHIEF promoter has also been described and confirmed by electrophoretic mobility shift assays (EMSA). In order to elucidate the role of each RNR class during infection, S. Typhimurium single and double RNR mutants (as well as Fur and NrdR mutants) were used in infection assays with macrophage and epithelial cell lines. Our results indicate class Ia to be mainly responsible for deoxyribonucleotide production during invasion and proliferation inside macrophages and epithelial cells. Neither class Ib nor class III seem to be essential for growth under these conditions. However, class Ib is able to maintain certain growth in an nrdAB mutant during the first hours of macrophage infection. Our results suggest that, during the early stages of macrophage infection, class Ib may contribute to deoxyribonucleotide synthesis by means of both an NrdR and a Fur-dependent derepression of nrdHIEF due to hydrogen peroxide production and DNA damage associated with the oxidative burst, thus helping to overcome the host defenses

Ensemble Analysis of Angiogenic Growth in Three-Dimensional Microfluidic Cell Cultures

We demonstrate ensemble three-dimensional cell cultures and quantitative analysis of angiogenic growth from uniform endothelial monolayers. Our approach combines two key elements: a micro-fluidic assay that enables parallelized angiogenic growth instances subject to common extracellular conditions, and an automated image acquisition and processing scheme enabling high-throughput, unbiased quantification of angiogenic growth. Because of the increased throughput of the assay in comparison to existing three-dimensional morphogenic assays, statistical properties of angiogenic growth can be reliably estimated. We used the assay to evaluate the combined effects of vascular endothelial growth factor (VEGF) and the signaling lipid sphingoshine-1-phosphate (S1P). Our results show the importance of S1P in amplifying the angiogenic response in the presence of VEGF gradients. Furthermore, the application of S1P with VEGF gradients resulted in angiogenic sprouts with higher aspect ratio than S1P with background levels of VEGF, despite reduced total migratory activity. This implies a synergistic effect between the growth factors in promoting angiogenic activity. Finally, the variance in the computed angiogenic metrics (as measured by ensemble standard deviation) was found to increase linearly with the ensemble mean. This finding is consistent with stochastic agent-based mathematical models of angiogenesis that represent angiogenic growth as a series of independent stochastic cell-level decisions

EphA2-receptor deficiency exacerbates myocardial infarction and reduces survival in hyperglycemic mice

Background We have previously shown that EphrinA1/EphA expression profile changes in response to myocardial infarction (MI), exogenous EphrinA1-Fc administration following MI positively influences wound healing, and that deletion of the EphA2 Receptor (EphA2-R) exacerbates injury and remodeling. To determine whether or not ephrinA1-Fc would be of therapeutic value in the hyperglycemic infarcted heart, it is critical to evaluate how ephrinA1/EphA signaling changes in the hyperglycemic myocardium in response to MI. Methods Streptozotocin (STZ)-induced hyperglycemia in wild type (WT) and EphA2-receptor mutant (EphA2-R-M) mice was initiated by an intraperitoneal injection of STZ (150 mg/kg) 10 days before surgery. MI was induced by permanent ligation of the left anterior descending coronary artery and analyses were performed at 4 days post-MI. ANOVAs with Student-Newman Keuls multiple comparison post-hoc analysis illustrated which groups were significantly different, with significance of at least p < 0.05. Results Both WT and EphA2-R-M mice responded adversely to STZ, but only hyperglycemic EphA2-R-M mice had lower ejection fraction (EF) and fractional shortening (FS). At 4 days post-MI, we observed greater post-MI mortality in EphA2-R-M mice compared with WT and this was greater still in the EphA2-R-M hyperglycemic mice. Although infarct size was greater in hyperglycemic WT mice vs normoglycemic mice, there was no difference between hyperglycemic EphA2-R-M mice and normoglycemic EphA2-R-M mice. The hypertrophic response that normally occurs in viable myocardium remote to the infarct was noticeably absent in epicardial cardiomyocytes and cardiac dysfunction worsened in hyperglycemic EphA2-R-M hearts post-MI. The characteristic interstitial fibrotic response in the compensating myocardium remote to the infarct also did not occur in hyperglycemic EphA2-R-M mouse hearts to the same extent as that observed in the hyperglycemic WT mouse hearts. Differences in neutrophil and pan-leukocyte infiltration and serum cytokines implicate EphA2-R in modulation of injury and the differences in ephrinA1 and EphA6-R expression in governing this are discussed. Conclusions We conclude that EphA2-mutant mice are more prone to hyperglycemia-induced increased injury, decreased survival, and worsened LV remodeling due to impaired wound healing

Transcriptome analysis of Bupleurum chinense focusing on genes involved in the biosynthesis of saikosaponins

<p>Abstract</p> <p>Background</p> <p><it>Bupleurum chinense </it>DC. is a widely used traditional Chinese medicinal plant. Saikosaponins are the major bioactive constituents of <it>B. chinense</it>, but relatively little is known about saikosaponin biosynthesis. The 454 pyrosequencing technology provides a promising opportunity for finding novel genes that participate in plant metabolism. Consequently, this technology may help to identify the candidate genes involved in the saikosaponin biosynthetic pathway.</p> <p>Results</p> <p>One-quarter of the 454 pyrosequencing runs produced a total of 195, 088 high-quality reads, with an average read length of 356 bases (NCBI SRA accession SRA039388). A <it>de novo </it>assembly generated 24, 037 unique sequences (22, 748 contigs and 1, 289 singletons), 12, 649 (52.6%) of which were annotated against three public protein databases using a basic local alignment search tool (E-value ≤1e-10). All unique sequences were compared with NCBI expressed sequence tags (ESTs) (237) and encoding sequences (44) from the <it>Bupleurum </it>genus, and with a Sanger-sequenced EST dataset (3, 111). The 23, 173 (96.4%) unique sequences obtained in the present study represent novel <it>Bupleurum </it>genes. The ESTs of genes related to saikosaponin biosynthesis were found to encode known enzymes that catalyze the formation of the saikosaponin backbone; 246 cytochrome P450 (<it>P450</it>s) and 102 glycosyltransferases (<it>GT</it>s) unique sequences were also found in the 454 dataset. Full length cDNAs of 7 <it>P450</it>s and 7 uridine diphosphate <it>GT</it>s (<it>UGT</it>s) were verified by reverse transcriptase polymerase chain reaction or by cloning using 5' and/or 3' rapid amplification of cDNA ends. Two <it>P450</it>s and three <it>UGT</it>s were identified as the most likely candidates involved in saikosaponin biosynthesis. This finding was based on the coordinate up-regulation of their expression with <it>β-AS </it>in methyl jasmonate-treated adventitious roots and on their similar expression patterns with <it>β-AS </it>in various <it>B. chinense </it>tissues.</p> <p>Conclusions</p> <p>A collection of high-quality ESTs for <it>B. chinense </it>obtained by 454 pyrosequencing is provided here for the first time. These data should aid further research on the functional genomics of <it>B. chinense </it>and other <it>Bupleurum </it>species. The candidate genes for enzymes involved in saikosaponin biosynthesis, especially the <it>P450</it>s and <it>UGT</it>s, that were revealed provide a substantial foundation for follow-up research on the metabolism and regulation of the saikosaponins.</p

Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference

Effects of fluoxetine on functional outcomes after acute stroke (FOCUS): a pragmatic, double-blind, randomised, controlled trial

Background Results of small trials indicate that fluoxetine might improve functional outcomes after stroke. The FOCUS trial aimed to provide a precise estimate of these effects. Methods FOCUS was a pragmatic, multicentre, parallel group, double-blind, randomised, placebo-controlled trial done at 103 hospitals in the UK. Patients were eligible if they were aged 18 years or older, had a clinical stroke diagnosis, were enrolled and randomly assigned between 2 days and 15 days after onset, and had focal neurological deficits. Patients were randomly allocated fluoxetine 20 mg or matching placebo orally once daily for 6 months via a web-based system by use of a minimisation algorithm. The primary outcome was functional status, measured with the modified Rankin Scale (mRS), at 6 months. Patients, carers, health-care staff, and the trial team were masked to treatment allocation. Functional status was assessed at 6 months and 12 months after randomisation. Patients were analysed according to their treatment allocation. This trial is registered with the ISRCTN registry, number ISRCTN83290762. Findings Between Sept 10, 2012, and March 31, 2017, 3127 patients were recruited. 1564 patients were allocated fluoxetine and 1563 allocated placebo. mRS data at 6 months were available for 1553 (99·3%) patients in each treatment group. The distribution across mRS categories at 6 months was similar in the fluoxetine and placebo groups (common odds ratio adjusted for minimisation variables 0·951 [95% CI 0·839–1·079]; p=0·439). Patients allocated fluoxetine were less likely than those allocated placebo to develop new depression by 6 months (210 [13·43%] patients vs 269 [17·21%]; difference 3·78% [95% CI 1·26–6·30]; p=0·0033), but they had more bone fractures (45 [2·88%] vs 23 [1·47%]; difference 1·41% [95% CI 0·38–2·43]; p=0·0070). There were no significant differences in any other event at 6 or 12 months. Interpretation Fluoxetine 20 mg given daily for 6 months after acute stroke does not seem to improve functional outcomes. Although the treatment reduced the occurrence of depression, it increased the frequency of bone fractures. These results do not support the routine use of fluoxetine either for the prevention of post-stroke depression or to promote recovery of function. Funding UK Stroke Association and NIHR Health Technology Assessment Programme