Co-culture of human fibroblasts,smooth muscle and endothelial cells promotes osteopontin induction in hypoxia

Arteriovenous fistulas (AVFs) are the preferred vascular access for haemodialysis of patients suffering from end-stage renal disease, a worldwide public health problem. However, they are prone to a high rate of failure due to neointimal hyperplasia and stenosis. This study aimed to determine if osteopontin (OPN) was induced in hypoxia and if OPN could be responsible for driving AVF failure. Identification of new factors that participate in remodelling of AVFs is a challenge. Three cell lines representing the cells of the three layers of the walls of arteries and veins, fibroblasts, smooth muscle cells and endothelial cells, were tested in mono- and co-culture in vitro for OPN expression and secretion in normoxia compared to hypoxia after silencing the hypoxia-inducible factors (HIF-1α, HIF-2α and HIF-1/2α) with siRNA or after treatment with an inhibitor of NF-kB. None of the cells in mono-culture showed OPN induction in hypoxia, whereas cells in co-culture secreted OPN in hypoxia. The changes in oxygenation that occur during AVF maturation up-regulate secretion of OPN through cell-cell interactions between the different cell layers that form AVF, and in turn, these promote endothelial cell proliferation and could participate in neointimal hyperplasia.     

CRB2 Mutations Produce a Phenotype Resembling Congenital Nephrosis,Finnish Type,with Cerebral Ventriculomegaly and Raised Alpha-Fetoprotein

We report five fetuses and a child from three families who shared a phenotype comprising cerebral ventriculomegaly and echogenic kidneys with histopathological findings of congenital nephrosis. The presenting features were greatly elevated maternal serum alpha-fetoprotein (MSAFP) or amniotic fluid alpha-fetoprotein (AFAFP) levels or abnormalities visualized on ultrasound scan during the second trimester of pregnancy. Exome sequencing revealed deleterious sequence variants in Crumbs, Drosophila, Homolog of, 2 (CRB2) consistent with autosomal-recessive inheritance. Two fetuses with cerebral ventriculomegaly and renal microcysts were compound heterozygotes for p.Asn800Lys and p.Trp759Ter, one fetus with renal microcysts was a compound heterozygote for p.Glu643Ala and p.Asn800Lys, and one child with cerebral ventriculomegaly, periventricular heterotopias, echogenic kidneys, and renal failure was homozygous for p.Arg633Trp in CRB2. Examination of the kidneys in one fetus showed tubular cysts at the corticomedullary junction and diffuse effacement of the epithelial foot processes and microvillous transformation of the renal podocytes, findings that were similar to those reported in congenital nephrotic syndrome, Finnish type, that is caused by mutations in nephrin (NPHS1). Loss of function for crb2b and nphs1 in Danio rerio were previously shown to result in loss of the slit diaphragms of the podocytes, leading to the hypothesis that nephrosis develops from an inability to develop a functional glomerular barrier. We conclude that the phenotype associated with CRB2 mutations is pleiotropic and that the condition is an important consideration in the evaluation of high MSAFP/AFAFP where a renal cause is suspected.     

Design, synthesis and biological activity of novel dimethyl-[2-[6-substituted-indol-1-yl]-ethyl]-amine as potent, selective, and orally-bioavailable 5-HT(1D) agonists

A novel series of highly potent human 5-HT_1D agonists, dimethyl-{2-[6-substituted-indol-1-yl]-ethyl}-amine, was synthesized. Structure–activity relationship (SAR) investigation revealed 4-[1-(2-dimethylamino-ethyl)-1H-indol-6-yl]-tetrahydro-thiopyran-4-ol, 11b (ALX-2732), as a potent (K_i=2.4 nM) agonist at the human 5-HT_1D receptor with good selectivity over the other serotonin receptor subtypes. This compound demonstrated favorable in vitro metabolic stability in human and rat liver microsomes and was found to be orally bioavailable in rats (F_po=51%).     

Differential expression of a gene for a methionine-rich storage protein in maize

Summary A methionine-rich 10 kDa zein storage protein from maize was isolated and the sequence of the N-terminal 30 amino acids was determined. Based on the amino acid sequence, two mixed oligonucleotides were synthesized and used to probe a maize endosperm cDNA library. A fulllength cDNA clone encoding the 10 kDa zein was isolated by this procedure. The nucleotide sequence of the cDNA clone predicts a polypeptide of 129 amino acids, preceded by a signal peptide of 21 amino acids. The predicted polypeptide is unique in its extremely high content of methionine (22.5%). The maize inbred line BSSS-53, which has increased seed methionine due to overproduction of this protein, was compared to W23, a standard inbred line. Northern blot analysis showed that the relative RNA levels for the 10 kDa zein were enhanced in developing seeds of BSSS-53, providing a molecular basis for the overproduction of the protein. Southern blot analysis indicated that there are one or two 10 kDa zein genes in the maize genome.     

Playing it safe? Solitary vervet monkeys (Chlorocebus pygerythrus) choose high‐quality foods more than those in competition

An important goal in foraging ecology is to determine how biotic and abiotic variables impact the foraging decisions of wild animals and how they move throughout their multidimensional landscape. However, the interaction of food quality and feeding competition on foraging decisions is largely unknown. Here we examine the importance of food quality in a patch on the foraging decisions of wild vervet monkeys (Chlorocebus pygerythrus) at Lake Nabugabo, Uganda using a multidestination platform array. The overall nutritional composition of the vervet diet was assessed and found to be low in sodium and lipids, thus we conducted a series of experimental manipulations in which the array was varied in salt and oil content. Although vervets prioritized platforms containing key nutrients (i.e., sodium and lipids) overall, we found that solitary vervets prioritized nutrient‐dense platforms more strongly than competing vervets. This finding was opposite to those in a similar experiment that manipulated food site quantity, suggesting that large, salient rewards may be worth competing over but slight differences in nutritional density may be only chosen when there are no potentially negative social consequences (i.e., aggression received). We also found that vervets chose platforms baited with oil‐only, and oil combined with salt, but not salt‐only, suggesting that energy was an important factor in food choice. Our findings demonstrate that when wild vervets detect differences in feeding patches that reflect nutritional composition, they factor these differences into their navigational and foraging decisions. In addition, our findings suggest that these nutritional differences may be considered alongside social variables, ultimately leading to the complex strategies we observed in this study.     

Fluorescence lifetime imaging microscopy reveals sodium pump dimers in live cells

The sodium-potassium ATPase (Na/K-ATPase, NKA) establishes ion gradients that facilitate many physiological functions including action potentials and secondary transport processes. NKA comprises a catalytic subunit (alpha) that interacts closely with an essential subunit (beta) and regulatory transmembrane micropeptides called FXYD proteins. In the heart, a key modulatory partner is the FXYD protein phospholemman (PLM, FXYD1), but the stoichiometry of the alpha–beta–PLM regulatory complex is unknown. Here, we used fluorescence lifetime imaging and spectroscopy to investigate the structure, stoichiometry, and affinity of the NKA-regulatory complex. We observed a concentration-dependent binding of the subunits of NKA–PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit as well as lower affinity alpha–alpha and alpha–PLM interactions. These data provide the first evidence that, in intact live cells, the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits. Docking and molecular dynamics (MD) simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha–alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with the stoichiometry (alpha-beta-PLM)₂.     

Sheep-urine-induced changes in soil microbial community structure

Soil microbial communities play an important role in nutrient cycling and nutrient availability, especially in unimproved soils. In grazed pastures, sheep urine causes local changes in nutrient concentration which may be a source of heterogeneity in microbial community structure. In the present study, we investigated the effects of synthetic urine on soil microbial community structure, using physiological (community level physiological profiling, CLPP), biochemical (phospholipid fatty acid analysis, PLFA) and molecular (denaturing gradient gel electrophoresis, DGGE) fingerprinting methods. PLFA data suggested that synthetic urine treatment had no significant effect on total microbial (total PLFA), total bacterial or fungal biomass; however, significant changes in microbial community structure were observed with both PLFA and DGGE data. PLFA data suggested that synthetic urine induced a shift towards communities with higher concentrations of branched fatty acids. DGGE banding patterns derived from control and treated soils differed, due to a higher proportion of DNA sequences migrating only to the upper regions of the gel in synthetic urine-treated samples. The shifts in community structure measured by PLFA and DGGE were significantly correlated with one another, suggesting that both datasets reflected the same changes in microbial communities. Synthetic urine treatment preferentially stimulated the use of rhizosphere-C in sole-carbon-source utilisation profiles. The changes caused by synthetic urine addition accounted for only 10-15% of the total variability in community structure, suggesting that overall microbial community structure was reasonably stable and that changes were confined to a small proportion of the communities.