Detection of terbufos in cases of intoxication by means of entomotoxicological analysis using ATR-FTIR spectroscopy combined with chemometrics.

The detection of toxic substances in larvae from carcasses in an advanced stage of decomposition may help criminal expertise in elucidating the cause of death in suspected cases of poisoning. Terbufos (Counter®) or O,O-diethyl-S-[(tert-butylsulfanyl)methyl] phosphorodithioate is an insecticide and systemic nematicide, which has very high toxicity from an acute point of view (oral LD50 in rodents ranging from 1.4 to 9.2 mg/kg) that has been marketed irregularly and indiscriminately in Brazil as a rodenticide, often being used to practice homicides. The present study aims to evaluate the use of attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy to detect traces of terbufos pesticide in fly larvae (Sarcophagidae). ATR-FTIR spectra of scavenger fly larvae from control (n = 31) and intoxicated (n = 80) groups were collected and submitted to chemometric analysis by means of multivariate classification using principal component analysis with quadratic discriminant analysis (PCA-QDA), successive projections algorithm with quadratic discriminant analysis (SPA-QDA) and genetic algorithm with quadratic discriminant analysis (GA-QDA) in order to distinguish between control and intoxicated groups. All discriminant models showed sensitivity and specificity above 90%, with the GA-QDA model showing the best performance with 98.9% sensitivity and specificity. The proposed methodology proved to be sensitive and promising for the detection of terbufos in scavenger fly larvae from intoxicated rat carcasses. In addition, the non-destructive nature of the ATR-FTIR technique may be useful in preserving the forensic evidence, meeting the precepts of the chain of custody and allowing for counter-proof.

Near infrared spectroscopy (NIRS) coupled with chemometric methods to identify and estimate taxonomic relationships of flies with forensic potential (Diptera: Calliphoridae and Sarcophagidae).

Infrared spectroscopy has been gaining prominence in entomology, such as for solving taxonomic problems, sexing adult specimens, determining the age of immature specimens, detecting drugs of abuse in fly larvae, and can be an important technique in Forensic Entomology. In order to help identify the species of Calliphoridae and Sarcophagidae families, the present study aimed to evaluate the use of near infrared spectroscopy (NIRS) coupled with chemometric methods for separating fly specimens into taxonomic categories and understanding the taxonomic relationship between them. Spectra collected from nine species of flies were subjected to unsupervised principal component analysis (PCA) and hierarchical cluster analysis (HCA), in which we sought to visualize the relationship between the samples (segregation of genera and families) with subsequent identification. In PCA, the best model was achieved using five principal components (PCs), which explained 99.16% of total variance of the original data set. The first principal component (PC1) and the fourth principal component (PC4) provided the best segregation, the latter being more important in the segregation of the species Chrysomya albiceps, Lucilia eximia, and Ravinia belforti from the others. In the HCA dendrogram, there was a clear separation between the specimens by family (Calliphoridae and Sarcophagidae) and genera (Chrysomya, Lucilia, Oxysarcodexia, Peckia and Ravinia). This study shows that NIRS is efficient to identify flies' taxonomic properties, such as family and genera, providing quick evidence for the tested species identity.

gamma-Zein secondary structure in solution by circular dichroism.

The proline-rich N-terminal domain of gamma-zein has been reported in relevant processes, which include its ability to cross the cell membranes. Evidences indicate that synthetic hexapeptide (PPPVHL), naturally found in N-terminal portion of gamma-zein, can adopt the polyproline II (PPII) conformation in aqueous solution. The secondary structure of gamma-zein in maize protein bodies had been analyzed by solid state Fourier transform infrared and nuclear magnetic resonance spectroscopies. However, it was not possible to measure PPII content in physiological environment since the beta-sheet and PPII signals overlap in both solid state techniques. Here, the secondary structure of gamma-zein has been analyzed by circular dichroism in SDS aqueous solution with and without ditiothreitol (DTT), and in 60% of 2-propanol and water with DTT. The results show that gamma-zein has high helical content in all solutions. The PPII conformation was present at about 7% only in water/DTT solution.

Study of the conformation of gamma-zeins in purified maize protein bodies by FTIR and NMR spectroscopy.

The gamma-zeins are a mixture of 16, 27, and 50-kDa polypeptides which are important in the formation and stabilization of protein bodies (PB). These organelles are used for deposition of zeins, the water-insoluble storage proteins in maize. The nature of the physical interaction between proteins in the assembly and stabilization of PB are fairly well known. It is suggested the repeated hexapeptide sequence (PPPVHL)(8) in the N-terminus is responsible for aggregation of the gamma-zeins on the PB surface. Despite this importance, there is little information about the native conformation of gamma-zeins. In this work, we have analyzed the secondary structures of gamma-zeins in purified protein bodies from two maize cultivars, in the solid state, by FTIR and NMR spectroscopy. The results revealed that gamma-zeins in their physiological state are comprise similar proportions of alpha-helix and beta-sheet, 33 and 31% as determined by FTIR. It was not possible to state if the polyproline II (PPII) conformation is present in the solid-state structure of gamma-zeins, as has been demonstrated for the hexapeptide in solution. Because of the similarity of the solid-state NMR spectra of gamma and alpha-zeins in the alpha carbon region we attributed their contributions to the beta-sheet structures rather than to the PPII conformation or a mixture of these extended structures.