Urinary and plasma proteomics to discover biomarkers for diagnosing between diabetic nephropathy and minimal change nephrotic syndrome or membranous nephropathy

The choice of treatment for primary nephrotic syndrome depends on the pathologic type of the disorder. Renal biopsy is necessary for a definitive diagnosis, but it is burdensome for the patients, and can be avoided if tests could be performed using urine or plasma. In this study, we analyzed 100 urinary proteins, 141 plasma proteins, and 57 urine/plasma ratios in cases of diabetic nephropathy (DN; n = 11), minimal change nephrotic syndrome (MCNS; n = 14), and membranous nephropathy (MN; n = 23). We found that the combination of urinary retinol-binding protein 4 and SH3 domain-binding glutamic acid-rich-like protein 3 could distinguish between MCNS and DN, with an area under the curve (AUC) of 0.9740. On the other hand, a selectivity index (SI) based on serotransferrin and immunoglobulin G, which is often used in clinical practice, distinguished them with an AUC of 0.9091. Similarly, the combination of urinary afamin and complement C3 urine/plasma ratio could distinguish between MN and DN with an AUC of 0.9842, while SI distinguished them with an AUC of 0.8538. Evidently, the candidates identified in this study were superior to the SI method. Thus, the aim was to test these biomarkers for accurate diagnosis and to greatly reduce the burden on patients.     

Schistosome infection intensity is inversely related to auto-reactive antibody levels

In animal experimental models, parasitic helminth infections can protect the host from auto-immune diseases. We conducted a population-scale human study investigating the relationship between helminth parasitism and auto-reactive antibodies and the subsequent effect of anti-helminthic treatment on this relationship. Levels of antinuclear antibodies (ANA) and plasma IL-10 were measured by enzyme linked immunosorbent assay in 613 Zimbabweans (aged 2-86 years) naturally exposed to the blood fluke Schistosoma haematobium. ANA levels were related to schistosome infection intensity and systemic IL-10 levels. All participants were offered treatment with the anti-helminthic drug praziquantel and 102 treated schoolchildren (5-16 years) were followed up 6 months post-antihelminthic treatment. ANA levels were inversely associated with current infection intensity but were independent of host age, sex and HIV status. Furthermore, after allowing for the confounding effects of schistosome infection intensity, ANA levels were inversely associated with systemic levels of IL-10. ANA levels increased significantly 6 months after anti-helminthic treatment. Our study shows that ANA levels are attenuated in helminth-infected humans and that anti-helminthic treatment of helminth-infected people can significantly increase ANA levels. The implications of these findings are relevant for understanding both the aetiology of immune disorders mediated by auto-reactive antibodies and in predicting the long-term consequences of large-scale schistosomiasis control programs.     

Design, synthesis, and evaluation of novel 4-thiazolylimidazoles as inhibitors of transforming growth factor-β type I receptor kinase

A novel series of 4-thiazolylimidazoles was synthesized as transforming growth factor-β (TGF-β) type I receptor (also known as activin receptor-like kinase 5 or ALK5) inhibitors. These compounds were evaluated for their ALK5 inhibitory activity in an enzyme assay and their TGF-β-induced Smad2/3 phosphorylation inhibitory activity in a cell-based assay. N-{[5-(1,3-benzothiazol-6-yl)-4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazol-2-yl]methyl}butanamide 20, a potent and selective ALK5 inhibitor, exhibited good enzyme inhibitory activity (IC(50)=8.2nM) as well as inhibitory activity against TGF-β-induced Smad2/3 phosphorylation at a cellular level (IC(50)=32nM).     

The Influence of a High‐Fat Dietary Environment in the Fetal Period on Postnatal Metabolic and Immune Function

Few reports show whether a high‐fat (HF) dietary environment in the fetal period affects immune function or the development of lifestyle‐related disease at maturity. We examined the influence of an HF dietary environment in the fetal period on postnatal metabolic and immune function. A total of 16 pregnant mice were given control (CON) diet and 16 were given HF diet in the gestational period, from mating to delivery. After delivery lactating mice were given either CON or HF diet, resulting in four groups. After weaning, the offspring mice were given the same diet that their mothers received during lactation. HF dietary intake in the postnatal period increased fat pad weights, serum glucose, and leptin levels. An HF diet in the fetal period resulted in fewer splenic lymphocytes, a thinner thymic cortex, and impaired antigen‐specific immune reactions. Furthermore, tumor necrosis factor (TNF)‐α production and serum triglyceride levels were elevated in the fetal HF group. In addition, the HF‐HF group showed a consistent decrease in ovalbumin (OVA)‐specific IgG and elevation of IgE, associated with advanced fatty changes in the liver. Results from this study suggest that HF environment during the fetal period induces epigenetic propensity toward obesity and immunological burden in part due to increased adipose tissue mass, significant reduction in the number of immune cells and decreased activities of immune cells.     

Detection, diversity and expression of aerobic bacterial arsenite oxidase genes

The arsenic (As) drinking water crisis in south and south-east Asia has stimulated intense study of the microbial processes controlling the redox cycling of As in soil-water systems. Microbial oxidation of arsenite is a critical link in the global As cycle, and phylogenetically diverse arsenite-oxidizing microorganisms have been isolated from various aquatic and soil environments. However, despite progress characterizing the metabolism of As in various pure cultures, no functional gene approaches have been developed to determine the importance and distribution of arsenite-oxidizing genes in soil-water-sediment systems. Here we report for the first time the successful amplification of arsenite oxidase-like genes (aroA/asoA/aoxB) from a variety of soil-sediment and geothermal environments where arsenite is known to be oxidized. Prior to the current work, only 16 aroA/asoA/aoxB-like gene sequences were available in GenBank, most of these being putative assignments from homology searches of whole genomes. Although aroA/asoA/aoxB gene sequences are not highly conserved across disparate phyla, degenerate primers were used successfully to characterize over 160 diverse aroA-like sequences from 10 geographically isolated, arsenic-contaminated sites and from 13 arsenite-oxidizing organisms. The primer sets were also useful for confirming the expression of aroA-like genes in an arsenite-oxidizing organism and in geothermal environments where arsenite is oxidized to arsenate. The phylogenetic and ecological diversity of aroA-like sequences obtained from this study suggests that genes for aerobic arsenite oxidation are widely distributed in the bacterial domain, are widespread in soil-water systems containing As, and play a critical role in the biogeochemical cycling of As.     

Basal cells express functional TRPV4 channels in the mouse nasal epithelium

Basal cells in the nasal epithelium (olfactory and airway epithelia) are stem/progenitor cells that are capable of dividing, renewing and differentiating into specialized cells. These stem cells can sense their biophysical microenvironment, but the underlying mechanism of this process remains unknown. Here, we demonstrate the prominent expression of the transient receptor potential vanilloid type 4 (TRPV4) channel, a Ca²⁺-permeable channel that is known to act as a sensor for hypo-osmotic and mechanical stresses, in the basal cells of the mouse nasal epithelium. TRPV4 mRNA was expressed in the basal portions of the prenatal mouse nasal epithelium, and this expression continued into adult mice. The TRPV4 protein was also detected in the basal layers of the nasal epithelium in wild-type but not in TRPV4-knockout (TRPV4-KO) mice. The TRPV4-positive immunoreactions largely overlapped with those of keratin 14 (K14), a marker of basal cells, in the airway epithelium, and they partially overlapped with those of K14 in the olfactory epithelium. Ca²⁺ imaging analysis revealed that hypo-osmotic stimulation and 4α-phorbol 12,13 didecanoate (4α-PDD), both of which are TRPV4 agonists, caused an increase in the cytosolic Ca²⁺ concentration in a subset of primary epithelial cells cultured from the upper parts of the nasal epithelium of the wild-type mice. This response was barely noticeable in cells from similar parts of the epithelium in TRPV4-KO mice. Finally, there was no significant difference in BrdU-labeled proliferation between the olfactory epithelia of wild-type and TRPV4-KO mice under normal conditions. Thus, TRPV4 channels are functionally expressed in basal cells throughout the nasal epithelium and may act as sensors for the development and injury-induced regeneration of basal stem cells.     

Zizimin2: a novel, DOCK180-related Cdc42 guanine nucleotide exchange factor expressed predominantly in lymphocytes

A novel superfamily of guanine nucleotide exchange factors for Rho GTPases includes DOCK180 and zizimin1. The zizimin subfamily includes three genes of which only zizimin1 has been cloned. We report here the cloning of zizimin2, identified in a screen for genes enriched in germinal center B cells. Zizimin2 and zizimin1 have similar primary structures and both proteins bound and activated Cdc42 but not the Cdc42-related proteins TC10 or TCL. Their tissue distributions are distinct, however, with zizimin2 expressed predominantly in lymphocytes and an opposite pattern for zizimin1. Zizimin3 was also analyzed and showed distinct GTPase specificity and tissue distribution.     

DNA display selection of peptide ligands for a full-length human G protein-coupled receptor on CHO-K1 cells

The G protein-coupled receptors (GPCRs), which form the largest group of transmembrane proteins involved in signal transduction, are major targets of currently available drugs. Thus, the search for cognate and surrogate peptide ligands for GPCRs is of both basic and therapeutic interest. Here we describe the application of an in vitro DNA display technology to screening libraries of peptide ligands for full-length GPCRs expressed on whole cells. We used human angiotensin II (Ang II) type-1 receptor (hAT1R) as a model GPCR. Under improved selection conditions using hAT1R-expressing Chinese hamster ovary (CHO)-K1 cells as bait, we confirmed that Ang II gene could be enriched more than 10,000-fold after four rounds of selection. Further, we successfully selected diverse Ang II-like peptides from randomized peptide libraries. The results provide more precise information on the sequence-function relationships of hAT1R ligands than can be obtained by conventional alanine-scanning mutagenesis. Completely in vitro DNA display can overcome the limitations of current display technologies and is expected to prove widely useful for screening diverse libraries of mutant peptide and protein ligands for receptors that can be expressed functionally on the surface of CHO-K1 cells.