Novel role of Vav1-Rac1 pathway in actin cytoskeleton regulation in interleukin-13-induced minimal change-like nephropathy.

Our established interleukin-13 (IL-13) overexpression rat model of minimal change-like nephropathy provided a platform to study the molecular signalling pathways in T-helper 2 (Th2) cytokine associated minimal change nephrotic syndrome (MCNS). We hypothesized that IL-13 may act directly on podocytes, causing podocyte foot process effacement and hence proteinuria in our rat model of minimal change-like nephropathy. The present study aimed firstly to delineate the glomerular 'gene signature' associated with IL-13-mediated dysregulation of podocyte-related proteins, and subsequently to investigate the role of the differentially regulated genes (DEGs) in IL-13-mediated podocyte injury. Glomerular transcriptional profile of IL-13-overexpressed rats showed characteristic features of podocyte injury with 87% of podocyte-related genes being significantly down-regulated. Gene expression of Vav1 was shown to be highly up-regulated in the glomeruli of IL-13-overexpressed rats and pathway analysis of the DEGs suggested a possible novel role of Vav1 in podocyte cytoskeleton remodelling. Immunofluorescence examination demonstrated glomerular expression of Vav1 in rats which co-localized with synaptopodin, confirming podocyte expression. However, positive staining for the phosphorylated form of Vav1 (p-Vav1) was only seen in IL-13-overexpressed rats. Moreover, in vitro IL-13 stimulation of human podocytes resulted in phosphorylation of Vav1. This was associated with Rac1 activation and actin cytoskeleton rearrangement, which was abrogated in Vav1 knockdown podocytes. In conclusion, we have demonstrated the role of Vav1-Rac1 pathway characterized by phosphorylation of Vav1, activation of Rac1 and the subsequent actin cytoskeleton rearrangement in IL-13-induced podocyte injury, possibly explaining the podocyte foot process effacement seen in our IL-13 overexpression rat model.

Enhancement of early cervical cancer diagnosis with epithelial layer analysis of fluorescence lifetime images.

This work reports the use of layer analysis to aid the fluorescence lifetime diagnosis of cervical intraepithelial neoplasia (CIN) from H&E stained cervical tissue sections. The mean and standard deviation of lifetimes in single region of interest (ROI) of cervical epithelium were previously shown to correlate to the gold standard histopathological classification of early cervical cancer. These previously defined single ROIs were evenly divided into layers for analysis. A 10-layer model revealed a steady increase in fluorescence lifetime from the inner to the outer epithelial layers of healthy tissue sections, suggesting a close association with cellular maturity. The shorter lifetime and minimal lifetime increase towards the epithelial surface of CIN-affected regions are in good agreement with the absence of cellular maturation in CIN. Mean layer lifetimes in the top-half cervical epithelium were used as feature vectors for extreme learning machine (ELM) classifier discriminations. It was found that the proposed layer analysis technique greatly improves the sensitivity and specificity to 94.6% and 84.3%, respectively, which can better supplement the traditional gold standard cervical histopathological examinations.

A non-synonymous single nucleotide polymorphism in an OPRM1 splice variant is associated with fentanyl-induced emesis in women undergoing minor gynaecological surgery.

BACKGROUND: Fentanyl-induced emesis (FIE) is a distressing adverse effect in the postoperative setting. The genetic basis of FIE remains largely unknown, therefore, we examined whether it was associated with specific genetic variants of OPRM1, the gene encoding the main receptor target of fentanyl. METHODS: In this prospective case-control study, 193 women undergoing gynaecological surgery under a standardized anaesthetic with a low emetogenic risk were enrolled. Inclusion and exclusion criteria were designed to select extreme phenotypes as well as to ensure that most major confounders for FIE were either excluded or present in all patients. To control for unforeseen intra- and postoperative confounders for FIE, only 161 patients were further analysed, out of which 10 were categorized as having FIE, defined by the presence of at least one of three symptoms: nausea, vomiting or retching that was likely to be fentanyl-related. To identify SNPs relevant to FIE in our population, DNA from 40 controls and 10 cases was sequenced at the following OPRM1 regions: 3 kbp of the promoter, main and alternative exons as well as 2 kbp of the 3' downstream region. The genotype of the significant SNP was further determined in the remaining 111 controls. RESULTS: The incidence of FIE was 6.2%. Initial sequencing of 10 cases and 40 controls identified 25 SNPs. Only rs540825, a non-synonymous SNP in the splice variant, MOR1X, showed a significant association with FIE post-Bonferroni correction. This SNP was further examined in the remaining 111 controls which confirmed its significant association with FIE (p = 0.019 post-Bonferroni, OR: 5.6, 95% CI: 1.42-21.91). CONCLUSIONS: This is the first report of an association between the occurrence of FIE in Chinese women undergoing gynaecological surgery and an OPRM1 splice variant SNP, rs540825.

Multiplexed genotyping of ABC transporter polymorphisms with the Bioplex suspension array.

We have developed and validated a consolidated bead-based genotyping platform, the Bioplex suspension array for simultaneous detection of multiple single nucleotide polymorphisms (SNPs) of the ATP-binding cassette transporters. Genetic polymorphisms have been known to influence therapeutic response and risk of disease pathologies. Genetic screening for therapeutic and diagnostic applications thus holds great promise in clinical management. The allele-specific primer extension (ASPE) reaction was used to assay 22 multiplexed SNPs for eight subjects. Comparison of the microsphere-based ASPE assay results to sequencing results showed complete concordance in genotype assignments. The Bioplex suspension array thus proves to be a reliable, cost-effective and high-throughput technological platform for genotyping. It can be easily adapted to customized SNP panels for specific applications involving large-scale mutation screening of clinically relevant markers.

Genetic variability of arylalkylamine-N-acetyl-transferase (AA-NAT) gene and human sleep/wake pattern.

The cyclic production and secretion of melatonin has been associated with the sleep/wake cycle as well as other circadian rhythms. Since arylalkylamine-N-acetyl-transferase (AA-NAT) is the rate-limiting enzyme responsible for the production of melatonin, it has been postulated to determine the circadian oscillations of melatonin. Genetic variability of the AA-NAT gene may therefore potentially influence sleep patterns in the normal population. In this study, a sleep pattern survey was performed in 210 students. Five subjects with early sleep onset time and long sleep duration (early/long sleepers), and 5 subjects with late sleep onset time and short sleep duration (late/short sleepers) were identified for genetic studies. All 10 subjects had identical sequences throughout the coding regions of the AA-NAT gene. In the promoter region, a -263G/C (relative to the transcription start site) single nucleotide polymorphism (SNP) was observed in 4 of the 5 late/short sleepers, but in only 1 of 5 early/long sleepers. The -263G/C SNP may therefore be an important determinant of the late/short sleep pattern.

A naturally occurring -263G/C variant of the human AA-NAT gene and overnight melatonin production.

Several lines of evidence show that the daily amount of melatonin produced differs greatly between individuals. Any polymorphism in the gene of arylalkylamine N-acetyltransferase (AA-NAT), a critical enzyme involved in melatonin biosynthesis, may contribute to the variability of melatonin production. The present study investigated the possible association between overnight melatonin excretion and a commonly occurring -263G/C polymorphism in the promoter region of the human AA-NAT gene. However, we found that -263G/C variant had no effect on the overnight 6-OHMS excretion. In this study, individual genotyping for -263G/C was determined by denaturing high performance liquid chromatography (DHPLC) and confirmed by sequencing. The overnight urinary 6-hydroxymelatonin sulfate (6-OHMS) excretion was quantified by enzyme-linked immunosorbent assay (ELISA).