Simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene using quenched-FRET real-time PCR

Hereditary hemochromatosis (HH) is a common autosomal disorder of iron metabolism mainly affecting Caucasian populations. Three recurrent disease-associated mutations have been detected in the hemochromatosis gene (HFE): C282Y, H63D, and S65C. Although HH phenotype has been associated with all three mutations, C282Y is considered the most relevant mutation responsible for hemochromatosis. Clinical complications of HH include cirrhosis of the liver, congestive cardiac failure and cardiac arrhythmias, endocrine pancreatic disease, which can be prevented by early diagnosis and treatment. Therefore, a reliable genotyping method is required for presymptomatic diagnosis. We describe the simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene by real-time PCR followed by melting curve analysis using fluorescence resonance energy transfer (FRET) probes. The acceptor fluorophore may be replaced by a quencher, increasing multiplex possibilities. Real-time PCR results were compared to the results of sequencing and conventional PCR followed by restriction digestion and detection by agarose gel electrophoresis (PCR-RFLP). Genotypes from 80 individuals obtained both by the conventional PCR-RFLP method and quenched-FRET real-time PCR were in full agreement. Sequencing also confirmed the results obtained by the new method, which proved to be an accurate, rapid and cost-effective diagnostic assay. Our findings demonstrate the usefulness of real-time PCR for the simultaneous detection of mutations in the HFE gene, which allows a reduction of a significant amount of time in sample processing compared to the PCR-RFLP method, eliminates the use of toxic reagents, reduces the risk of contamination in the laboratory, and enables full process automation.

Quantification of ruminal Clostridium proteoclasticum by real-time PCR using a molecular beacon approach

Aims: All members of the ruminal Butyrivibrio group convert linoleic acid (cis-9,cis-12-18 : 2) via conjugated 18 : 2 metabolites (mainly cis-9,trans-11-18 : 2, conjugated linoleic acid) to vaccenic acid (trans-11-18 : 1), but only members of a small branch, which includes Clostridium proteoclasticum, of this heterogeneous group further reduce vaccenic acid to stearic acid (18 : 0, SA). The aims of this study were to develop a real-time polymerase chain reaction (PCR) assay that would detect and quantify these key SA producers and to use this method to detect diet-associated changes in their populations in ruminal digesta of lactating cows. Materials and Results: The use of primers targeting the 16S rRNA gene of Cl. proteoclasticum was not sufficiently specific when only binding dyes were used for detection in real-time PCR. Their sequences were too similar to some nonproducing strains. A molecular beacon probe was designed specifically to detect and quantify the 16S rRNA genes of the Cl. proteoclasticum subgroup. The probe was characterized by its melting curve and validated using five SA-producing and ten nonproducing Butyrivibrio-like strains and 13 other common ruminal bacteria. Analysis of ruminal digesta collected from dairy cows fed different proportions of starch and fibre indicated a Cl. proteoclasticum population of 2-9% of the eubacterial community. The influence of diet on numbers of these bacteria was less than variations between individual cows. Conclusion: A molecular beacon approach in qPCR enables the detection of Cl. proteoclasticum in ruminal digesta. Their numbers are highly variable between individual animals. Signifance and Impact of the Study: SA producers are fundamental to the flow of polyunsaturated fatty acid and vaccenic acid from the rumen. The method described here enabled preliminary information to be obtained about the size of this population. Further application of the method to digesta samples from cows fed diets of more variable composition should enable us to understand how to control these bacteria in order to enhance the nutritional characteristics of ruminant-derived foods, including milk and beef.

“Financial alchemy” or a zero sum game? Real estate finance, securitisation and the UK property market

Following the US model, the UK has seen considerable innovation in the funding, finance and procurement of real estate in the last decade. In the growing CMBS market asset backed securitisations have included $2.25billion secured on the Broadgate office development and issues secured on Canary Wharf and the Trafford Centre regional mall. Major occupiers (retailer Sainsbury’s, retail bank Abbey National) have engaged in innovative sale & leaseback and outsourcing schemes. Strong claims are made concerning the benefits of such schemes – e.g. British Land were reported to have reduced their weighted cost of debt by 150bp as a result of the Broadgate issue. The paper reports preliminary findings from a project funded by the Corporation of London and the RICS Research Foundation examining a number of innovative schemes to identify, within a formal finance framework, sources of added value and hidden costs. The analysis indicates that many of the gains claimed conceal costs – in terms of market value of debt or flexibility of management – while others result from unusual firm or market conditions (for example utilising the UK long lease and the unusual shape of the yield curve). Nonetheless, there are real gains resulting from the innovations, reflecting arbitrage and institutional constraints in the direct (private) real estate market

Can carbon surface oxidation shift the pore size distribution curve calculated from Ar, N(2) and CO(2) adsorption isotherms? Simulation results for a realistic carbon model

Using the virtual porous carbon model proposed by Harris et al, we study the effect of carbon surface oxidation on the pore size distribution (PSD) curve determined from simulated Ar, N(2) and CO(2) isotherms. It is assumed that surface oxidation is not destructive for the carbon skeleton, and that all pores are accessible for studied molecules (i.e., only the effect of the change of surface chemical composition is studied). The results obtained show two important things, i.e., oxidation of the carbon surface very slightly changes the absolute porosity (calculated from the geometric method of Bhattacharya and Gubbins (BG)); however, PSD curves calculated from simulated isotherms are to a greater or lesser extent affected by the presence of surface oxides. The most reliable results are obtained from Ar adsorption data. Not only is adsorption of this adsorbate practically independent from the presence of surface oxides, but, more importantly, for this molecule one can apply the slit-like model of pores as the first approach to recover the average pore diameter of a real carbon structure. For nitrogen, the effect of carbon surface chemical composition is observed due to the quadrupole moment of this molecule, and this effect shifts the PSD curves compared to Ar. The largest differences are seen for CO2, and it is clearly demonstrated that the PSD curves obtained from adsorption isotherms of this molecule contain artificial peaks and the average pore diameter is strongly influenced by the presence of electrostatic adsorbate-adsorbate as well as adsorbate-adsorbent interactions.

Public real estate and the term structure of interest rates: a cross-country study

Using a variation of the Nelson-Siegel term structure model we examine the sensitivity of real estate securities in six key global markets to unexpected changes in the level, slop and curvature of the yield curve. Our results confirm the time-sensitive nature of the exposure and sensitivity to interest rates and highlight the importance of considering the entire term structure of interest rates. One issue that is of particular of interest is that despite the 2007-9 financial crisis the importance of unanticipated interest rate risk weakens post 2003. Although the analysis does examine a range of markets the empirical analysis is unable to provide definitive evidence as to whether REIT and property-company markets display heightened or reduced exposure.

The curve selection lemma and the Morse-Sard theorem

We use an inequality due to Bochnak and Lojasiewicz, which follows from the Curve Selection Lemma of real algebraic geometry in order to prove that, given a C(r) function f : U subset of R(m) -> R, we have lim(y -> xy is an element of crit(f)) vertical bar f(y) - f(x)vertical bar/vertical bar y - x vertical bar(r) = 0, for all x is an element of crit(f)` boolean AND U, where crit( f) = {x is an element of U vertical bar df ( x) = 0}. This shows that the so-called Morse decomposition of the critical set, used in the classical proof of the Morse-Sard theorem, is not necessary: the conclusion of the Morse decomposition lemma holds for the whole critical set. We use this result to give a simple proof of the classical Morse-Sard theorem ( with sharp differentiability assumptions).

Comparing equilibrium real interest rates : different approaches to measure Brazilian rates

Despite the difficulties involved in the precise determination of equilibrium real interest rates, it seems clear that nominal interest rates has been higher in Brazil than in similar emerging economies. This paper aims to shed light on the possible reasons for this feature of the Brazilian economy. We extend Miranda and Muinhos (2003) one-country study to a sample of 20 countries, using many methods to compare measures of the real interest: (i) extracting equilibrium interest rates from IS curves; (ii) extracting steady state interest rates from marginal product of capital; (iii) capturing relevant variables and the fixed effects having real interest rates as dependent variable in a panel for emerging countries; and (iv) extracting inflation expectation from the spread between fixed rate and inflation-indexed treasure notes.

A structuralist Philips curve

This paper presents a structuralist model of the Philips curve and applies it to the US and Brazilian economies. The theoretical model starts from a simple markup rule to build a Philips curve based on the assumptions that firms have a desired rate of profit and wokers have a target real wage. Inflation expectations are modeled in terms of current inflation and the governments’ target, and the model shows that relative prices can have both a short-run and long-run influence on inflation. When applied to the US, the structuralist Philips curve results in a nonlinear model in which there are two steady states for inflation, and where the wageshare of income becomes the main instrument to drive inflation to the governments’ target. When applied to Brazil, the structuralist Philips curve reveals a nonlinear relationship between long-run inflation and the real exchange rate, so that the same inflation target can be consistent with more than one value of the exchange rate. The main conclusion of the paper is that a structuralist specification of the Philips curve is a useful instrument to model many macroeconomic topics as well as alternative theoretical closures.

Determination of malic acid in real samples by using enzyme immobilized reactors and amperometric detection

The malate dehydrogenase (MDH) and ascorbate oxidase were immobilized independently, onto silanized controlled porous silica and packed in a tygon tube. The reactors were inserted in the flow system, and the malic acid was determined by measurement of NADH produced by enzymatic reaction. The NADH was reoxidized in a wall jet cell that consisted of spectrographic graphite, Ag/AgCl, KCl(sat), and steel needle as work, reference, and counter electrodes, respectively. The current intensities were measured at 390 mV. The malate calibration curve shows a linear range from 5.0 x 10(-6) to 1.0 x 10(-4) molL(-1), the lifetime was 40 analyses, after that a decrease of 20% on the response is observed. Three different citric juices were analyzed and a good correlation between the proposed method and spectrophotometric commercial kit were obtained.

Real-time monitoring and kinetic parameter estimation of the affinity interaction of jArtinM and rArtinM with peroxidase glycoprotein by the electrogravimetric technique

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Application of dissociation curve analysis to radiation hybrid panel marker scoring: generation of a map of river buffalo (B-bubalis) chromosome 20

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Design, construction, and performance of a real-time holographic refractometry prototype for liquid analysis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Characteristics of the double-torsion test to determine the R-curve of ceramic materials

Double-torsion tests were carried out on a commercial ceramic floor tile to verify whether this test is suitable for determining the R-curve of ceramics. The instantaneous crack length was obtained by means of compliance calibration, and it was found that the experimental compliance underestimates the real crack length. The load vs. displacement curves were also found to drop after maximum loading, causing the stress intensity factor to decline. The R-curves were calculated by two methods: linear elastic fracture mechanics and the energetic method. It was obtained that the average values of crack resistance, R, and the double of the work of fracture, 2 · γwof, did not depend on notch length, a0, which is a highly relevant finding, indicating that these parameters were less dependent on the test specimen's geometry. The proposal was to use small notches, which produce long stable crack propagation paths that in turn are particularly important in the case of coarse microstructures.

Desenvolvimento da técnica de RT-PCR em tempo real para detecção e diferenciação de estirpes do vírus da bronquite infecciosa das galinhas

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Detecção de Cryptosporidium serpentis em amostras fecais de serpentes utilizando PCR em tempo real

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)