A New Microarray Substrate for Ultra-Sensitive Genotyping of KRAS and BRAF Gene Variants in Colorectal Cancer

Molecular diagnostics of human cancers may increase accuracy in prognosis, facilitate the selection of the optimal therapeutic regimen, improve patient outcome, reduce costs of treatment and favour development of personalized approaches to patient care. Moreover sensitivity and specificity are fundamental characteristics of any diagnostic method. We developed a highly sensitive microarray for the detection of common KRAS and BRAF oncogenic mutations. In colorectal cancer, KRAS and BRAF mutations have been shown to identify a cluster of patients that does not respond to anti-EGFR therapies; the identification of these mutations is therefore clinically extremely important. To verify the technical characteristics of the microarray system for the correct identification of the KRAS mutational status at the two hotspot codons 12 and 13 and of the BRAF^V600E mutation in colorectal tumor, we selected 75 samples previously characterized by conventional and CO-amplification at Lower Denaturation temperature-PCR (COLD-PCR) followed by High Resolution Melting analysis and direct sequencing. Among these samples, 60 were collected during surgery and immediately steeped in RNAlater while the 15 remainders were formalin-fixed and paraffin-embedded (FFPE) tissues. The detection limit of the proposed method was different for the 7 KRAS mutations tested and for the V600E BRAF mutation. In particular, the microarray system has been able to detect a minimum of about 0.01% of mutated alleles in a background of wild-type DNA. A blind validation displayed complete concordance of results. The excellent agreement of the results showed that the new microarray substrate is highly specific in assigning the correct genotype without any enrichment strategy.     

Should we use proportional sampling for species–area studies?

Aim In this paper we aim to show that proportional sampling can detect species–area relationships (SARs) more effectively than uniform sampling. We tested the contribution of alpha and beta diversity in ant communities as explanations for the SAR. Location Tropical forest remnants in Viçosa, Minas Gerais, Brazil (20 °45′ S, 42 °50′ W). Methods We sampled 17 forest remnants with proportional sampling. To disentangle sampling effects from other mechanisms, species richness was fitted in a model with remnant size, number of samples (sampling effects) and an interaction term. Results A SAR was observed independent of the number of samples, discarding sampling effects. Alpha diversity was not influenced by remnant size, and beta diversity increased with remnant size; evidence to the fact that habitat diversity within remnants could be the dominant cause of the SAR. Such a relationship between beta diversity and remnant area may have also arisen due to the combined effects of territoriality and aggregation of ant species. Main conclusions The proposed model, together with proportional sampling, allowed the distinction between sampling effects and other mechanisms.     

Erythrocyte glutathione transferase: a potential new biomarker in chronic kidney diseases which correlates with plasma homocysteine

The erythrocyte glutathione S-transferase (e-GST) is a member of a superfamily of inducible enzymes involved in cell detoxification that shows an increased expression in chronic kidney disease (CKD) patients. We propose a new automated analysis procedure for e-GST activity that has been validated in 72 CKD patients and 62 maintenance hemodialysis patients (MHD). Regression analysis was carried out to assess association between e-GST activity data, main clinical variables, and plasma homocysteine (Hcy), a modified sulfur amino acid known as potential risk factor for cardiovascular disease that is increased above normal levels in more than 90% of the uremic patients. An increased e-GST activity was confirmed in MHD patients (N=62; 10.2±0.4 U/gHb) compared with healthy subjects (N=80; 5.8±0.4 U/gHb), and as an original finding, a significant increase of e-GST activity was observed in pre-dialysis CKD patients with a positive correlation with disease severity weighted according to the four stages of "Kidney Disease Outcomes Quality Initiative" classification (7.4±0.5, 8±1, 9.5±0.6, 12±1 U/gHb, respectively). No correlation was found between e-GST activity and hemoglobin, transferrin, blood iron and the markers of systemic inflammation and renal function such as alpha-1 acid glycoprotein and high-sensitive C-Reactive Protein, beta-2 microglobulin and the index of malnutrition-inflammation PINI, while a significant correlation was observed for the first time between plasma Hcy and e-GST activity (r2=0.64, P<0.0001) in MHD patients. Hcy, however, was not identified as an inhibitor of e-GST enzyme. The results in this study suggest the potential for automated e-GST analysis as a valuable tool to further explore phase II-related uremic toxicity in CKD and MHD patients.     

An engineered folded PLP-bound monomer of Treponema denticola cystalysin reveals the effect of the dimeric structure on the catalytic properties of the enzyme

Cystalysin, a dimeric pyridoxal 5'-phosphate (PLP)-dependent lyase, is a virulence factor of the human oral pathogen Treponema denticola. Guided by bioinformatic analysis, two interfacial residues (Leu57 and Leu62) and an active site residue (Tyr64*), hydrogen-bonded with the PLP phosphate group of the neighboring subunit, have been mutated. The wild-type and the L57A, L62A, Y64*A, L57A/L62A, L57A/Y64*A, L57A/L62A/Y64*A mutants, all having a C-terminal histidine tag, have been constructed, expressed, and purified. The impact of these mutations on the dimeric state of cystalysin in the apo- and holo-form has been analyzed by size-exclusion chromatography. The results demonstrate that (i) Leu57 is more critical than Leu62 for apodimer formation, (ii) Tyr64*, more than Leu62, interferes with dimerization of holocystalysin without affecting that of apoenzyme, (iii) while each single mutation is inadequate in significantly altering the extent of monomerization of both apo- and holo-cystalysin, their combination leads to species which remain in a folded monomeric state at a reasonably high concentration in both the apo- and holo-forms. Although L57A/L62A or L57A/Y64*A, even to a different extent, are stimulated to dimer formation in the presence of either unproductive or productive ligands, L57A/L62A/Y64*A remains prevalently monomer at a concentration up to 50 microM. Kinetic analyses show that in this monomeric species the alpha,beta-eliminase, alanine racemase, and D-alanine half-transaminase activities are almost abolished, while the L-alanine half-transaminase activity is slightly enhanced when compared with that of wild-type. The structural basis of the stereospecific transaminase activity displayed by the engineered folded PLP-bound monomer has been analyzed and discussed.     

Caveolin-1 expression sensitizes fibroblastic and epithelial cells to apoptotic stimulation

The potential role of caveolin-1 in apoptosis remains controversial. Here, we investigate whether caveolin-1 expression is proapoptotic or antiapoptotic using a well-defined antisense approach. We show that NIH/3T3 cells harboring antisense caveolin-1 are resistant to staurosporine-induced apoptosis, as assessed using cell morphology, DNA content, caspase 3 activation, and focal adhesion kinase cleavage. Importantly, sensitivity to apoptosis is recovered when caveolin-1 levels are restored. Conversely, recombinant stable expression of caveolin-1 in T24 bladder carcinoma cells sensitizes these cells to caspase 3 activation. Consistent with the observations using NIH/3T3 cells, downregulation of caveolin-1 in T24 cells substantially diminishes caspase 3-like activity. Loss of sensitivity to apoptotic stimulation is recovered by inhibition of the phosphatidylinositol 3-kinase pathway using LY-294002, suggesting a possible mechanism for the sensitizing effect of caveolin-1. Thus our results suggest that caveolin-1 may act as a coupling or sensitizing factor in signaling apoptotic cell death in both fibroblastic (NIH/3T3) and epithelial (T24) cells.     

No evidence of fetal DNA persistence in maternal plasma after pregnancy

Short- and long-term persistence of fetal DNA in maternal plasma has been investigated. Short-term persistence at very low concentration was detected in 47 out of 105 women within two days after delivery. Twelve out of 13 samples re-tested within three days scored negative. No long-term persistence was detected in 172 women who had previous sons or abortions. Molecular microchimerism due to circulating fetal DNA persisting from previous pregnancies should not hamper non-invasive plasma-based prenatal testing.     

Constitutive and growth factor-regulated phosphorylation of caveolin-1 occurs at the same site (Tyr-14) in vivo: identification of a c-Src/Cav-1/Grb7 signaling cassette

Caveolin-1 was first identified as a phosphoprotein in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblasts. Tyrosine 14 is now thought to be the principal site for recognition by c-Src kinase; however, little is known about this phosphorylation event. Here, we generated a monoclonal antibody (mAb) probe that recognizes only tyrosine 14-phosphorylated caveolin-1. Using this approach, we show that caveolin-1 (Y14) is a specific tyrosine kinase substrate that is constitutively phosphorylated in Src- and Abl-transformed cells and transiently phosphorylated in a regulated fashion during growth factor signaling. We also provide evidence that tyrosine-phosphorylated caveolin-1 is localized at the major sites of tyrosine-kinase signaling, i.e. focal adhesions. By analogy with other signaling events, we hypothesized that caveolin-1 could serve as a docking site for pTyr-binding molecules. In support of this hypothesis, we show that phosphorylation of caveolin-1 on tyrosine 14 confers binding to Grb7 (an SH2-domain containing protein) both in vitro and in vivo. Furthermore, we demonstrate that binding of Grb7 to tyrosine 14-phosphorylated caveolin-1 functionally augments anchorage-independent growth and epidermal growth factor (EGF)-stimulated cell migration. We discuss the possible implications of our findings in the context of signal transduction.