Comparative pangenomic analyses and biotechnological potential of cocoa-related Acetobacter senegalensis strains.

Acetobacter senegalensis belongs to the group of acetic acid bacteria (AAB) that present potential biotechnological applications, for production of D-gluconate, cellulose and acetic acid. AAB can overcome heat and acid stresses by using strategies involving the overexpression of heat-shock proteins and enzymes from the complex pyrroquinoline-ADH, besides alcohol dehydrogenases (ADH). Nonetheless, the isolation of A. senegalensis and other AAB from food may be challenging due to presence of viable but non-culturable (VBNC) cells and due to uncertainties about nutritional requirements. To contribute for a better understanding of the ecology of AAB, this paper reports on the pangenome analysis of five strains of A. senegalensis recently isolated from a Brazilian spontaneous cocoa fermentation. The results showed biosynthetic clusters exclusively found in some cocoa-related AAB, such as those related to terpene pathways, which are important for flavour development. Genes related to oxidative stress were conserved in all the genomes, with multiple clusters. Moreover, there were genes coding for ADH and putative ABC transporters distributed in core, shell and cloud genomes, while chaperonin-encoding genes were present only in the core and soft-core genomes. Regarding quorum sensing, a response regulator gene was in the shell genome, and the gene encoding for acyl-homoserine lactone efflux protein was in the soft-core genome. There were quorum quenching-related genes, mainly encoding for lactonases, but also for acylases. Moreover, A. senegalensis did not have determinants of virulence or antibiotic resistance, which are good traits for strains intended to be applied in food fermentation.

Diagnosis of chronic alcohol consumption. Hair analysis for ethyl-glucuronide.

This paper describes a procedure for the detection and quantification of ethyl-glucuronide (EtG) in hair samples. During method development the efficacy of extraction of EtG from hair was compared in four extraction methods: (a) methanol; (b) methanol:water (1:1); (c) water; and (d) water:trifluoroacetic acid (9:1). In addition, three derivatizing agents were compared as well: N,O-bistrimethylsilyl-trifluoroacetamide (BSTFA): trimethylchlorosilane (TMCS) (99:1), pentafluoropropionic anhydride (PFPA) and heptafluorobutyric anhydride (HFBA). Water was found to be the best extracting solvent and PFPA the best derivatizing agent. Both provided the highest recoveries, with cleaner extracts and more stable derivatives. The final method is as follows: about 100mg of hair are sequentially washed with water and acetone. The decontaminated sample is finely cut with scissors, then the deuterated internal standard (EtG-d5) and 2 mL of water are added. After sonication for 2 h, the sample is maintained at room temperature overnight. Derivatization is performed with PFPA. Derivatives are injected into a GC-MS system in the electronic impact mode. The method shows linearity over the range of concentrations from 0.050 to 5 ng/mg. Detection and quantification limits are 0.025 and 0.050 ng/mg, respectively. Mean recoveries for the three studied concentrations (low, medium and high) are higher than 87%. The coefficients of variation in intra- and inter-assay precision are always lower than 7%. The method is being routinely applied in our lab for the diagnosis of chronic alcohol consumption.

Simultaneous quantification of opiates, cocaine and cannabinoids in hair.

The present paper describes a sensitive method developed in our laboratory for the simultaneous analysis of opiates (morphine, codeine and monoacetylmorphine), cocainis (cocaine and benzoylecgonine) and cannabinoids (delta 9-tetrahydrocannabinol and 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid) in hair samples. After decontaminating the sample with dichloromethane, two consecutive hydrolyses were performed in order to achieve the best conditions for extracting the three kinds of drugs from the protein matrix. First the opiate and cocainic compounds were extracted by means of a soft acidic hydrolysis with 0.1 N HCl at 50 degrees C overnight and organic solvent extraction at pH 9.2. The cannabinoids need a stronger basic hydrolysis with 11.8 N KOH for 10 min at laboratory temperature. After adding maleic acid, the cannabinoids were extracted with an organic solvent. The derivatization was carried out with heptafluorobutyric anhydride and hexafluoropropanol. Calibration curves were linear between 0.5-100 ng/mg of hair. Recovery and reproducibility were assured. The quantification limits ranged between 0.04-0.26 ng/mg of hair. Seventy hair samples from known drug abusers were cut into 1-cm segments and analyzed by this method. The ranges of measured concentrations (ng/mg) were 0.31-89 for cocaine, 0.1-5.76 for benzoylecgonine, 0.34-45.79 for morphine, 0.45-39.59 for codeine, 0.09-48.18 for monoacetylmorphine, 0.06-7.63 for THC and 0.06-3.87 for THC-COOH. The results of sectional analyses agreed with the self reported drug histories. The usefulness of this method is in assessing earlier drug consumption, and also at the same time obtaining a chronological profile of the consumption of these three types of drugs.

Tissue concentrations of MDMA and its metabolite MDA in three fatal cases of overdose.

The recreational use of amphetamine derivatives has become increasingly popular in our country in past recent years. Their use is especially common among young people participating in dance parties known as "raves." As a direct consequence of their increased use, the number of fatal cases in which these compounds have been involved have increased dramatically since the second half of the last decade. In our laboratory, we have registered 25 cases related to amphetamine derivatives use since 1996. Three of them were deeply studied and the results obtained are presented in this paper. This information may be useful for the interpretation of the results obtained in toxicological analysis in the cases in which death may be attributed to MDMA use.

Determination of benzodiazepines by derivative spectroscopy.

In this work, a rapid method is described for the quantitative determination of benzodiazepines, by means of derivative spectroscopy, and the application of this technique towards the study of the aforementioned substances in biological fluids. Diazepam, chlordiazepoxide, nitrazepam and clorazepate may be identified and quantitated at concentrations of 0.2 mg/L or greater.

Rapid analysis of amphetamine, methamphetamine, MDA, and MDMA in urine using solid-phase microextraction, direct on-fiber derivatization, and analysis by GC-MS.

A rapid, sensitive, and solvent-free procedure for the simultaneous determination of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), and 3,4-methylenedioxymethamphetamine (MDMA) in urine was developed using solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring mode. A headspace vial containing the urine sample, NaOH, NaCl, and amphetamine-d3 as the internal standard was heated at 100 degrees C for 20 min. A polydimethylsiloxane fiber was maintained in the vial headspace for 10 min in order to adsorb the amphetaminic compounds, which were subsequently derivatized by exposing the fiber to trifluoroacetic anhydride for 20 min in the headspace of another vial maintained at 60 degrees C for 20 min. The trifluoroacetyl derivatives were desorbed in the GC injection port for 5 min. Several parameters were considered during the method optimization process. These included a comparison of SPME with or without headspace, the required derivatization procedure, and the influence of temperature on the headspace extraction and derivatization methods. The optimized method was validated for the four compounds tested. Calibration curves showed linearity in the range 50-1000 ng/mL (r = 0.9946-0.9999). Recovery data were 71.89-103.24%. The quantitation limits were 10 ng/mL for amphetamine and methamphetamine and 20 ng/mL for MDA and MDMA. All of these data recommend the applicability of the method for use in the analytical routine of a forensic laboratory.

New method for determination of ten pesticides in human blood.

An analytical method was developed for precise identification and quantitation of 10 pesticides in human blood. The pesticides studied, which have appeared frequently in actual cases, were endosulfan, lindane, parathion, ethyl-azinphos, diazinon, malathion, alachlor, tetradifon, fenthion and dicofol (o-p' and p-p' isomers). The current method replaces an earlier method which involved liquid-liquid extraction with a mixture of n-hexane-benzene (1 + 1). The extraction is performed by solid-phase extraction, with C18 cartridges and 2 internal standards, perthane and triphenylphosphate. Eluates were analyzed by gas chromatography (GC) with nitrogen-phosphorus and electrochemical detectors. Results were confirmed by GC-mass spectrometry in the electron impact mode. Blood blank samples spiked with 2 standard mixtures and an internal standard were used for quantitation. Mean recoveries ranged from 71.83 to 97.10%. Detection and quantitation limits are reported for each pesticide. Examples are provided to show the application of the present method to actual samples.

A fatally mistaken fruit juice drink: an unordinary way of cocaine intoxication.

Cocaine is one of the drugs of abuse more frequently consumed in Spain. Furthermore, Spain due to its geographical position is used by trafficker's organizations as the port of entrance of cocaine in the European Union. We present here a case of a fatal intoxication caused by a mistake in the cocaine distribution net in our country. Cocaine was concealed in a tropical juice only sold by the Internet.

Determination of amphetamine and methylamphetamine by gas-liquid chromatography (head space).

1 A method for determining amphetamine and methylamphetamine in urine by gas-liquid chromatography is described. 2 Chromatography is performed on a 10% Apiezon L, 10% KOH column with alkali-flame detection (NPD). This method avoids the losses of these volatile substances which are known to occur when solvent extraction is used. An alkalinized urine sample (1 ml) is introduced into a sealed vial which is heated in a water bath at 70 degrees C for 20 min. A 1 ml portion of the gas phase in equilibrium with the liquid phase is chromatographed. 3 Amphetamine and methylamphetamine can be detected and measured in urine samples after single therapeutic doses of the drugs.

Deaths from pesticide poisoning in Spain from 1991 to 1996.

Data on 184 deaths from pesticide poisonings that occurred in Spain from 1991 to 1996 have been collated via a survey from the National Institute of Toxicology, Sevilla. Organophosphates and carbamates accounted for the majority of the cases. Other substances involved were organochlorines such as endosulfan and the herbicide paraquat.

Estudio médico-legal del homicidio en la provincia de Sevilla (2004-2007). Especial referencia a los homicidios de mujeres en el contexto de violencia de género / Medico-legal study of the homicide in the province of Seville (2004-07). Special reference to the female victims inside the gender violence

Este estudio retrospectivo analiza los 57 homicidiosocurridos en la provincia de Sevilla durante unperiodo de cuatro años (2004-2007).Se han estudiado diversas variables relacionadascon los mismos obteniéndose los siguientes resultados: latasa media de homicidios ha sido de 0.77 por 100.000habitantes; el arma blanca es el método más utilizadoseguido por el arma de fuego, contusiones y asfixia mecánica;las víctimas son en su mayoría varones (relaciónhombre/mujer 2:1) con una edad media de 46 ± 21.2años; el mayor número de casos se registra en fines desemana (sábado y domingo) y durante el mes de octubre.El análisis toxicológico de las víctimas detectó etanol enel 47.4%, benzodiacepinas en el 14%, cocaína en el 12.3%,opiáceos en el 10.5% y tetrahidrocannabinol en el 8.8%.Se ha hecho una valoración más detallada de loscasos de homicidio dentro del contexto de violencia degénero dada su alta incidencia en nuestro país. En 18 de19 casos, las mujeres murieron a manos de su pareja,ex-pareja o familiar en primer grado.Los resultados han sido comparados con estudiossimilares realizados tanto en el ámbito nacional comointernacional

This retrospective study analyzes the 57 homicidesoccurred in the province of Seville in a period of fouryears (2004-2007).Some variables related with the homicides havebeen studied with the following results: the meanhomicide rate was 0.77 per 100.000 inhabitants; sharpinjury was the most frequent mechanism followed by shotgun, contusions and mechanical asphyxia; victims are inmajor part of cases males (male/female ratio 2:1) with amean age of 46 ± 21.2 years; homicides are morefrequents in weekend (Saturday and Sunday) and duringOctober. Toxicological analysis of the victims showedethanol in 47.4%, benzodiazepines in 14%, cocaine in12.3%, opium derivatives in 10.5% andtetrahydrocannabinol in 8.8%.A detailed analyses was also carried out in the casesof homicides occurred in the setting of gender violencedue to its high prevalence in our country. In 18 out of 19cases, women died in the hands of her couple, ex-couple,or first-degree relative.Results have been compared with similar nationaland international studies

Estudio epidemiológico del suicidio en Sevilla en 2004 / Epidemiological study of suicide in Seville in 2004

En este artículo se realiza un estudio epidemiológico descriptivo de las muertes de etiología suicida ocurridas en Sevilla en el año 2004 y los datos obtenidos se comparan con otros estudios previos realizados en la misma zona. La tasa de suicidio fue de 8,36 por 100.000 habitantes con una relación hombre/mujer de 2,6:1. La edad media global ha sido de 54,3 años (52,7 años en varones y 58,7 años en mujeres). El mayor número de suicidios (58,5%) se producen en el segundo y tercer trimestre del año (primavera y verano). En cuanto al estado civil, la mayor frecuencia se produce en personas casadas (31,2%). Solo en el 9,5% de los suicidas existían antecedentes de intentos autolíticos previos. La ahorcadura ha sido el mecanismo más empleado en los varones (50,9%) mientras que en las mujeres el mecanismo más frecuente ha sido la precipitación (41,5%). Un 57,1% de los sujetos tenía antecedentes psiquiátricos siendo los trastornos más frecuentes la depresión o los síntomas depresivos (65,5%) y la esquizofrenia (15,5%). Se efectuaron análisis químico-toxicológicos en el 78,2% de los casos de los cuales fueron positivos el 69,6%. El alcohol etílico fue la sustancia más detectada (24,3%), seguido de las benzodiazepinas (18,3%) y los antidepresivos (11,3%). Se observa una cierta estabilización en la tasa de suicidios en Sevilla desde mediados del siglo XX hasta la actualidad

In this paper, we present an epidemiological descriptive study of the suicides occurred in Seville in 2004 and the results are compared with data obtained in previous studies in the same area. Suicide rate was 8,36 per 100.000 inhabitants with a male/female ratio of 2,6:1. The medium age has been 54,3 years (52,7 years in males and 58,7 years in females). Suicides predominate in the second and third trimester of the year (spring and summer). Taking into account the civil status, the mayor frequency appears in married (31,2%). Only 9,5% of the cases had antecedents of previous autolysis intents. Hanging was the most frequent mechanism to commit suicide in men (50.9%) while in women was jumping (41,5%). In 57,1% of cases there were antecedents of mental disorder mainly depression of depressive symptoms (65,5%) followed by schizophrenia (15,5%). Toxicological analyses were performed in 78,2% of cases with positive results in 69,6%. Ethanol was the toxic most detected (24,3%) followed by benzodiazepines (18,3%) and antidepressants (11,3%). According to the results of the study, we observe a certain stabilization of the suicide rates in Seville from the middle of the XXth century up to now

Incidencia del alcohol etílico y de sustancias psicoactivas en las muertes acaecidas por accidente de tráfico en el sur de España durante 2004 / The incidence of alcohol and psicoactive substances in fatal accidents in the south of Spain during 2004

El Instituto Nacional de Toxicología y Ciencias Forenses, la Delegación del Gobierno para el Plan Nacional sobre Drogas y la Dirección General de Tráfico tienen firmado un Convenio de colaboración para establecer la incidencia del consumo de alcohol y sustancias psicoactivas en las muertes en accidentes de tráfico, tanto conductores como peatones. En el estudio, objeto de este trabajo, se analizan los resultados de las muestras de sangre obtenidas en las autopsias practicadas a los conductores en el Sur de España durante el año 2004. Los parámetros a considerar son: sexo, edad, fecha, concentración de etanol y consumo simultáneo de sustancias psicoativas que afecten la capacidad de conducción. Los análisis se han rea liza do s iguiendo los procedi mie ntos em pl eados habitualmente en el Instituto Nacional de Toxicología y Ciencias Forenses para este tipo de determinaciones, consistente en cromatografía de gases con detector FID para la determinación de alcoholemia; mientras que para las sustancias psicoactivas se realiza screening m ediante CEDIA y posterior c onfirmac ión y cuantificación mediante cromatografía de gases con detector NPD y cromatografía de gases-espectrometría de masas. De los 495 casos recibidos, 232 correspondían a conductores, la mayoría de los cuales eran varones accidentados en la comunidad andaluza. Cabe destacar la incidencia de muertes en menores de 30 años, así como el consumo de alcohol entre los fallecidos, observándose en más de la mitad de estos casos una concentración de alcohol en sangre superior al límite permitido por la Dirección General de Tráfico, de 0,5 gramos de etanol por litro de sangre, superándose incluso, en muchos de ellos, la concentración de 1,5 g/l. Los resultados obtenidos confirman la importante relación entre los accidentes de tráfico mortales y el consumo de alcohol y sustancias psicoactivas(AU)

The Instituto Nacional de Toxicología y Ciencias Forenses, the Delegación del Gobierno para el Plan Nacional sobre Drogas and the Dirección General de Tráfico have an agreement of collaboration to establish the incidence of psycoactive substances and alcohol consumption in traffic accident related deaths, both in drivers and pedestrians. The objective of this paper is to study the results obtained in blood analyses from autopsies performed on drivers in the South of Spain during 2004. The following parameters have been considered: sex, age, date of the accident, blood alcohol concentration (BAC) and simultaneous consumption of affecting driving ability. Analyses were performed following the procedures routinely applied in our lab. Briefly: blood alcohol concentration was determined by headspace CG-FID. For the psycoactive substances were firstly screened by CEDIA and then confirmed and quantified by CG-NPD and CG-MS. 495 cases were received. 232 were from drivers, the majority of whom were Andalussian men. The high incidence of fatal accidents in drivers younger than 30, as well as previous alcohol consumption should be noticed. In more than 50% of the cases BAC was higher than 0.5 g/L, which is the limit allowed by the Dirección General de Tráfico, in many of them BAC was even higher than 1.5 g/L. In conclusion, from this study we can deduce a significant relationship among fatal traffic accidents and alcohol and psycoactive substances consumption(AU)