Francisco J. Burguillo

EVALUATION OF MODEL DISCRIMINATION, PARAMETER ESTIMATION AND GOODNESS OF FIT IN NONLINEAR REGRESSION PROBLEMS BY TEST STATISTICS DISTRIBUTIONS

Abstract There are many programs for fitting nonlinear models to experimental data, and the use of this type of software is now widespread. After fitting a model or sequence of models, these programs usually calculate χ2, run, sign, F and t (statistics as an aid to model discrimination and parameter estimation. The distribution of such statistics from linear regression is well known, but these random variables do not have the stated named distribution after fitting nonlinear models. First we describe a set of programs that can be used to study the distribution of these well known test statistics from nonlinear regression. Then we present the results from a study of two models that are frequently employed in the life-sciences, and summarize our results from more extensive simulations. Finally, we explain how these programs can be used to create the appropriate cumulative distribution functions, so that exact probability levels can be calculated, given the models of interest, the design points and error structure of a data set.

Systematic Review and Meta-Analysis of Artemisinin Based Therapies for the Treatment and Prevention of Schistosomiasis

Background Chemotherapy based on repeated doses of praziquantel is still the most effective control strategy against Schistosomiasis, however artemisinin derivatives emerged as a family of compounds with schistomicide activity. The aim of the present work is to compare the efficacy of artemisinin-based therapies in the treatment and prophylaxis of human schistosomiasis. The design of this work involved a quantitative systematic review and meta-analysis. Methodology/Principal Findings Retrieval of published studies was carried out through an electronic search of the PubMed (MEDLINE), EMBASE, Cochrane Library and CINAHL databases. This included reports comparing the therapeutic efficacy of artesunate alone, artesunate plus sulfadoxine-pyrimethamine and a combination of artemisinin derivatives plus praziquantel against praziquantel alone on different types of schistosomiasis. Moreover, studies on artesunate and artemether used as preventive drugs were also analyzed against placebo. The primary outcome measure for schistosomiasis treatment was “parasitological cure”, whereas for the prophylaxis the outcome evaluated was “infection rate”. Our results show that patients treated with artesunate alone have significantly lower cure rates than those treated with praziquantel (OR = 0.27 (95% C.I. 0.13–0.53; p<0.001)) and that the combined therapy of artesunate plus sulfadoxine-pyrimethamine is also significantly less effective than praziquantel treatment (OR = 0.14 (95% C.I. 0.02–0.92; p = 0.04)). However, the combination of an artemisinin derivatives plus praziquantel showed a higher cure rate than praziquantel monotherapy with OR = 2.07 (95% C.I. 1.27–3.36; p = 0.003). Finally, chemoprophylaxis with either artesunate (RR = 0.11 (95% C.I. 0.06–0.22; p<0.001)) or artemether (RR = 0.25 (95% C.I. 0.16–0.40; p<0.001)) was significantly better than a placebo in both cases. Conclusions/Significance This meta-analysis confirms that artemisinin derivatives used in combination with praziquantel have the potential to increase the cure rates in schistosomiasis treatment, but not artesunate alone. It is also confirmed that repeated doses of artemisinin derivatives play a prophylactic role, significantly reducing the incidence of Schistosoma japonicum infections compared with placebo.

Comparative kinetic study of lipases A and B from Candida rugosa in the hydrolysis of lipid p-nitrophenyl esters in mixed micelles with Triton X-100.

(1) Lipases A and B from Candida rugosa catalyzing the hydrolysis of esters in micellar media have been characterized kinetically by studies on substrate specificity, rate equation forms and modeling of enzyme mechanisms. (2) The study on specificity revealed that both lipases are non-specific esterases with similar activity against lipid p-nitrophenyl esters micellized with Triton X-100. The slight difference was that lipase A has its maximum activity centered in the caprylate while that of lipase B is in the laurate. (3) Kinetic studies for both lipases were carried out with p-nitrophenyl laurate under three experimental conditions: (I) the molar fraction of substrate is fixed and the bulk concentration of substrate and Triton X-100 are varied; (II) the bulk concentration of substrate is held constant and the molar fraction of substrate and bulk concentration of Triton X-100 are varied; and (III) the bulk concentration of Triton X-100 is held constant but the bulk concentration of substrate and molar fraction of substrate are varied. (4) In case I, a similar Michaelis-Menten behaviour was observed with both lipases; the curve fitting gave kappcat/Kappm values of 3.0.10(5) and 5.6.10(5) s-1 M-1 for lipases A and B respectively. In case II, for both lipases the relationship between rate and the molar fraction of substrate required a fitting equation of 2:2 degree polynomial quotient. In case III, both lipases showed non-Michaelian behaviour with concave-up curves in the Eadie-Hofstee plot, a minimum degree of 2:2 in substrate concentration being detected for the rate equation. (5) The above results are interpreted in terms of the hypothesis that the mechanism of both lipases must include at least two different inputs for the molecule of substrate which would explain the quadratic terms observed in the rate equation.

Transcriptome analysis reveals molecular profiles associated with evolving steps of monoclonal gammopathies

A multistep model has been proposed of disease progression starting in monoclonal gammopathy of undetermined significance continuing through multiple myeloma, sometimes with an intermediate entity called smoldering myeloma, and ending in extramedullary disease. To gain further insights into the role of the transcriptome deregulation in the transition from a normal plasma cell to a clonal plasma cell, and from an indolent clonal plasma cell to a malignant plasma cell, we performed gene expression profiling in 20 patients with monoclonal gammopathy of undetermined significance, 33 with high-risk smoldering myeloma and 41 with multiple myeloma. The analysis showed that 126 genes were differentially expressed in monoclonal gammopathy of undetermined significance, smoldering myeloma and multiple myeloma as compared to normal plasma cell. Interestingly, 17 and 9 out of the 126 significant differentially expressed genes were small nucleolar RNA molecules and zinc finger proteins. Several proapoptotic genes (AKT1 and AKT2) were down-regulated and antiapoptotic genes (APAF1 and BCL2L1) were up-regulated in multiple myeloma, both symptomatic and asymptomatic, compared to monoclonal gammopathy of undetermined significance. When we looked for those genes progressively modulated through the evolving stages of monoclonal gammopathies, eight snoRNA showed a progressive increase while APAF1, VCAN and MEGF9 exhibited a progressive downregulation. In conclusion, our data show that although monoclonal gammopathy of undetermined significance, smoldering myeloma and multiple myeloma are not clearly distinguishable groups according to their gene expression profiling, several signaling pathways and genes were significantly deregulated at different steps of the transformation process.

Kinetic and enantioselective behavior of the lipase from Candida cylindracea: A comparative study between the soluble enzyme and the enzyme immobilized on agarose and silica gels

Candida cylindracea lipase has previously been covalently immobilized on agarose by the tosylation method and on silica by the trichlorotiazine method. We describe a simultaneous study of the soluble enzyme and its counterpart immobilized derivatives and quantify the differences between the kinetic behavior of the three forms. The dependence of lipase activity on Na(I) and Ca(II) concentrations has been examined for the three forms of the enzyme, and results interpreted. Kinetic studies with p-nitrophenyl acetate (esterase activity) and tributyrine (lipase activity) as substrates revealed that the immobilized derivatives have lower kcatapp and Kmapp values than the soluble enzyme. With both substrates, the parallel decrease in kcatapp and Kmapp produces the favorable effect of maintaining the kcatappKmapp ratios of the supported enzymes at values comparable to those of the soluble enzyme. Kinetic studies with methyl (R)-(+) and methyl (S)-(−)-2-chloro-propionates (enantioselective activity) point to certain deviations in the Michaelis-Menten behavior. Accordingly, the v versus [S] curves were fitted by the cubic splines method and interpreted using the areas under the curves. A new type of average enantiomeric excess calculated from these areas varied from 6.4% for the soluble enzyme to 24 and 42% when immobilized on agarose and silica, respectively. This is interpreted in terms of a rigidification of the enzyme produced by the covalent immobilization.

Kinetic and enantioselective behaviour of isoenzymes A and B from Candida rugosa lipase in the hydrolysis of lipids and esters

The isoenzymes A and B from C. rugosa lipase have been kinetically characterized by non-linear regression methods measuring the hydrolysis of tributyrin (lipase activity), p-nitrophenyl acetate (esterase activity) and methyl 2-chloropropionate (enantioselectivity). With tributyrin as substrate, the specificity constant (k::f/K%P) for lipase A is about 2-fold that of the lipase B. This confirms the higher specificity of lipase A for substrates containing butyric acid moieties. However, the kinetic behaviour of both isoenzymes for the hydrolysis of pnitrophenyl acetate is quite similar, with lipase A showing a slight preference for this substrate. Kinetic analysis with the substrates methyl (R)-(+) and methyl (S)-(-) 2-chloropropionates showed that both isoenzymes exhibit stereospecificity for the enantiomer R and display nonMichaelian kinetics of a sigmoidal type. Since this behaviour precludes the usual treatment on the basis of the enantiomeric ratio [(Vmax/Km)&( Vmax/Km)s~ow], enantioselectivity was quantified using the areas under the v-[S] curves. Thus, the ratio between the areas under the curves for the two enantiomers assayed increases from 1.27 for isoenzyme A to 1.68 for isoenzyme B. This result clearly shows that isolation and purification of isoenzymes may enhance the enantioselective properties of an enzymatic system.

Research article: Meta-analysis of microarray data: The case of imatinib resistance in chronic myelogenous leukemia

With the proliferation of related microarray studies by independent groups, a natural approach to analysis would be to combine the results across studies. In this article, we address a meta-analysis of the gene expression data on imatinib resistance in chronic myelogenous leukemia. First, an analysis of the overlapping among 6 published studies revealed that only 3 genes were coincident between 2 studies. A later reprocessing using different methods on 4 publicly available datasets revealed that 2 extra genes were overlapped between two sets. Both poor overlappings may be due to large differences in the sample source, the microarray platforms used, and a small difference in gene expression between the imatinib non-responder and responder patients. A search of common genes inside 4 public datasets afforded 404 well defined genes. Nevertheless, this necessary condition for meta-analysis caused the loss of many genes of possible interest. The expression signals of the common genes in the four datasets were reanalyzed using three summary statistical methods for combining quantitative information: Fisher, Stouffer and effect-size. Taking the three methods together and using an FDR<0.10 threshold, a gene-list with 33 differentially expressed genes was found. Considering all the reanalysis approaches used in this work, a final gene-list with 38 differentially expressed genes is reported. Despite the important limitations to this microarray meta-analysis, the presented procedures and integrated gene-list may have some potential value as regards imatinib resistance in CML patients since it is the first attempt to integrate evidence about gene-lists in this area.

Kinetic properties of derepressible acid phosphatase from the yeast form of Yarrowia lipolytica

(1) An acid phosphatase from depressed cells of the yeast form of Yarrowia lipolytica has been characterized kinetically by studies on specificity, inhibition, rate equation forms and modelling of the enzyme mechanisms. (2) The study on specificity revealed that the acid phosphatase is a rather unspecific phosphohydrolase that has similar activity on several different phosphate esters. A very weak transphosphorylating activity was also detected. (3) Among the reversible inhibitors, phosphate and vanadate were outstanding, whereas EDTA behaved as an activator. (4) v vs. [S] studies with o-carboxyphenyl phosphate as substrate show that the acid phosphatase of Y. lipolytica exhibits non-Michaelian behaviour, a minimum degree of 2:2 being detected for the rate equation in [S]. (5) The inhibition by phosphate and vanadate seems to have the same pattern of partial inhibition with a certain non-competitive nature, 1:1 being the minimum degree of the rate equation detected in (I).

A Partial Least Squares Algorithm for Microarray Data Analysis Using the VIP Statistic for Gene Selection and Binary Classification

A package for simulation, curve fitting, statistical analysis, and graph plotting. 2. Meta-analysis of microarray data: The case of imatinib resistance in chronic myelogenous leukemia. 3. Modelling the simultaneous processes occurring during the BOD5 test using synthetic waters as a pattern. 4. Hyper-adhesion in desmosomes: its regulation in wound healing and possible relationship to cadherin crystal structure. 5. Directed enzyme evolution guided by multidimensional analysis of substrate-activity space. 7. Uso del paquete Estadístico Simfit en la enseñanza del análisis de datos en ciencias experimentales.-Using the SIMFIT Statistical Package to teach Data Analysis in Experimental Sciences. 9. The photoreactivity of the retinal age pigment lipofuscin. 10. Intravitreal growth factors in proliferative diabetic retinopathy: correlation with neovascular activity and glycaemic management. 12. Using ASCII text files in post-fix notation (reverse Polish) to define mathematical models and systems of differential equations for simulation and nonlinear regression.

Kinetic behaviour and reaction mechanism of the hydrolysis of p-nitrophenyl palmitate in mixed micelles with Triton X-100 catalyzed by lipase from Candida rugosa

Abstract The physicochemical properties of lipase from Candida rugosa in the hydrolysis of micellized p -nitrophenyl palmitate, such as thermal stability, enzyme concentration and the effect of ionic strength on the rate of catalysis, have been characterized. As regards the specificity for a series of p -nitrophenyl esters ( p -NPC n ), n = 2, 4, 8, 12 and 16 being the number of carbon atoms of the hydrophobic tail, the lipase from Candida rugosa proved to be non-specific, although it did hydrolyze them at different rates, depending on n and the physicochemical nature of the substrate (mixed micelles with surfactant or simple solution). At Triton X-100 levels above the critical micelle concentration (c.m.c.), the kinetic behaviour of the hydrolysis of p -nitrophenyl palmitate in Triton X-100 mixed micelles catalyzed by Candida rugosa lipase was consistent with the Michaelis—Menten rate equation under three different experimental conditions: (i) the molar fraction of substrate held constant and the Triton X-100 concentration varied; (ii) the bulk substrate concentration held constant and the Triton X-100 concentration varied, and (iii) the Triton X-100 concentration held constant and the bulk substrate concentration varied. Kinetic analysis performed in the above conditions revealed that the simple model described by Verger et al. [ J. Biol. Chem. , 248 (1973), 4023] correctly interprets the kinetic behaviour of the commercial lipase from Candida rugosa used in the study and highlights the advantage that this classic mechanism may have in current lipase modelling in biocatalysis.