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Resumen En los ultimos anos la genetica ha adquirido una gran importancia en los procesos de identificacion masiva de victimas y constituye, en muchos casos, la unica herramienta util. Algunas instituciones externalizan estos analisis en laboratorios especializados. Es el caso de la Unidad de Derechos Humanos del Servicio Medico Legal (SML) de Chile, creada con el objetivo de identificar y restituir a las familias los restos de las victimas de la dictadura civico-militar instaurada en el pais entre 1973 y 1990, que provoco mas de 1.300 desaparecidos y muertos sin entrega. La externalizacion de los analisis impone la necesidad de establecer una rigurosa sistematica de revision y control de calidad de los analisis realizados por el laboratorio externo, lo que incluye asegurar la trazabilidad de las muestras y los analisis, ademas de reproducir tanto la comparacion de los perfiles geneticos como su valoracion estadistica. En este trabajo se presenta la experiencia del SML en esta materia y se establecen una serie de recomendaciones que pueden ser utilizadas como guia por otras instituciones que decidan externalizar los analisis geneticos en procesos de identificacion masiva de victimas.

The EuroForMix model has been extended to create a new open-source software called EFMrep which enables the combination of STR DNA mixture samples from different multiplexes. In addition to calculating combined likelihood ratios and carrying out deconvolution, the software also includes the capability to specify related unknown individuals. A graphical user interface has been implemented to ease the analysis for practitioners in real case work. The effect of combining multiple samples based on the PROVEDIt dataset was investigated, either from the same or different multiplexes. The information gain increases when more samples are combined. A head-to-head comparison against EuroForMix shows the benefit of a more general model. Guidelines are provided. A real case example was used to demonstrate how EFMrep could be used to combine multiple samples when a proposition includes kinship.

DNA analyses can be used for both investigative (crime scene-focused), or evaluative (suspect-focused) reporting. Investigative, DNA-led exploration of serious crimes always involves the comparison of hundreds of biological samples submitted by the authorities for analysis. Crime stain comparisons include both evidence to evidence profiles and reference to evidence profiles. When many complex DNA results (mixtures, low template LT-DNA samples) are involved in the investigation of a crime, the manual comparison of DNA profiles is very time-consuming and prone to manual errors. In addition, if the person of interest is a minor contributor, the classical approach of performing searches of national DNA databases is problematic because it is realistically restricted to clear major contributors and the occurrence of masking and drop-out means that there will not be a definitive DNA profile to perform the search with. CaseSolver is an open source expert system that automates analysis of complex cases. It does this by three sequential steps: a) simple allele comparison b) likelihood ratio (LR) based on a qualitative model (forensim) c) LR based on a quantitative model (EuroForMix). The software generates a list of potential match candidates, ranked according to the LRs, which can be exported as a report. The software can also identify contributors from small or large databases (e.g., staff database or 1 mill. individuals). In addition, an informative graphical network plot is generated that easily identifies contributors in common to multiple stains. Here we describe recent improvements made to the software in version v1.5.0, made in response to user requirements during intensive casework usage.

For very serious crimes, reporting scientists often have to contend with complex cases where literally hundreds of items are submitted by investigators for analysis. In order to efficiently expedite the challenge of comparing reference profiles to evidence profiles, many of which are mixtures, we have developed an investigative open source expert system CaseSolver. We have analysed a real case based on GlobalFiler involving 119 evidence profiles and 3 reference profiles. To provide a demonstration of the power of the system we also added the three references to a fictive large database of 1 million individuals in order to test subsequent recovery of the presumed true contributors. CaseSolver was used on a Fusion 6C validation study involving 25 two- to four-person mixture profiles based on 14 reference profiles. The sequential use of simple allele comparison, the qualitative model ( forensim) and the quantitative model ( EuroForMix) makes the analysis very fast and accurate – and finally, the software generates a list of potential match candidates which can be exported as a report. From these two studies we found that the resolution of match candidates from CaseSolver was the same as that reported by a scientist who worked manually through the samples, except that CaseSolver highlighted two manual errors. For the validation study we found low template DNA samples giving negative results, which demonstrate the limitations of the tool; but overall our assessment shows that CaseSolver will benefit all analyses involving mixture interpretation and screening. Importantly, CaseSolver removes the very time-consuming aspect of manual comparison and gives improved quality by preventing manual errors.

Many watershed models simulate overland and instream microbial fate and transport, but few provide loading rates on land surfaces and point sources to the waterbody network. This paper describes the underlying equations for microbial loading rates associated with 1) land-applied manure on undeveloped areas from domestic animals; 2) direct shedding (excretion) on undeveloped lands by domestic animals and wildlife; 3) urban or engineered areas; and 4) point sources that directly discharge to streams from septic systems and shedding by domestic animals. A microbial source module, which houses these formulations, is part of a workflow containing multiple models and databases that form a loosely configured modeling infrastructure which supports watershed-scale microbial source-to-receptor modeling by focusing on animal- and human-impacted catchments. A hypothetical application – accessing, retrieving, and using real-world data – demonstrates how the infrastructure can automate many of the manual steps associated with a standard watershed assessment, culminating in calibrated flow and microbial densities at the watershed’s pour point.

The Spanish and Portuguese-Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG) has organized a second collaborative exercise on a simulated case of Disaster Victim Identification (DVI), with the participation of eighteen laboratories. The exercise focused on the analysis of a simulated plane crash case of medium-size resulting in 66 victims with varying degrees of fragmentation of the bodies (with commingled remains). As an additional difficulty, this second exercise included 21 related victims belonging to 6 families among the 66 missings to be identified. A total number of 228 post-mortem samples were represented with aSTR and mtDNA profiles, with a proportion of partial aSTR profiles simulating charred remains. To perform the exercise, participants were provided with aSTR and mtDNA data of 51 reference pedigrees —some of which deficient—including 128 donors for identification purposes. The exercise consisted firstly in the comparison of the post-mortem genetic profiles in order to re-associate fragmented remains to the same individual and secondly in the identification of the re-associated remains by comparing aSTR and mtDNA profiles with reference pedigrees using pre-established thresholds to report a positive identification. Regarding the results of the post-mortem samples re-associations, only a small number of discrepancies among participants were detected, all of which were from just a few labs. However, in the identification process by kinship analysis with family references, there were more discrepancies in comparison to the correct results. The identification results of single victims yielded fewer problems than the identification of multiple related victims within the same family groups. Several reasons for the discrepant results were detected: a) the identity/non-identity hypotheses were sometimes wrongly expressed in the likelihood ratio calculations, b) some laboratories failed to use all family references to report the DNA match, c) In families with several related victims, some laboratories firstly identified some victims and then unnecessarily used their genetic information to identify the remaining victims within the family, d) some laboratories did not correctly use “prior odds” values for the Bayesian treatment of the episode for both post-mortem/post-mortem re-associations as well as the ante-mortem/post-mortem comparisons to evaluate the probability of identity. For some of the above reasons, certain laboratories failed to identify some victims. This simulated “DNA-led” identification exercise may help forensic genetic laboratories to gain experience and expertize for DVI or MPI in using genetic data and comparing their own results with the ones in this collaborative exercise., This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

DNA is a powerful tool available for forensic investigations requiring identification of species. However, it is necessary to develop and validate methods able to produce results in degraded and or low quality DNA samples with the high standards obligatory in forensic research. Here, we describe a voluntary collaborative exercise to test the recently developed Species Identification by Insertions/Deletions (SPInDel) method. The SPInDel kit allows the identification of species by the generation of numeric profiles combining the lengths of six mitochondrial ribosomal RNA (rRNA) gene regions amplified in a single reaction followed by capillary electrophoresis. The exercise was organized during 2014 by a Working Commission of the Spanish and Portuguese-Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG), created in 2013. The 24 participating laboratories from 10 countries were asked to identify the species in 11 DNA samples from previous GHEP-ISFG proficiency tests using a SPInDel primer mix and control samples of the 10 target species. A computer software was also provided to the participants to assist the analyses of the results. All samples were correctly identified by 22 of the 24 laboratories, including samples with low amounts of DNA (hair shafts) and mixtures of saliva and blood. Correct species identifications were obtained in 238 of the 241 (98.8%) reported SPInDel profiles. Two laboratories were responsible for the three cases of misclassifications. The SPInDel was efficient in the identification of species in mixtures considering that only a single laboratory failed to detect a mixture in one sample. This result suggests that SPInDel is a valid method for mixture analyses without the need for DNA sequencing, with the advantage of identifying more than one species in a single reaction. The low frequency of wrong (5.0%) and missing (2.1%) alleles did not interfere with the correct species identification, which demonstrated the advantage of using a method based on the analysis of multiple loci. Overall, the SPInDel method was easily implemented by laboratories using different genotyping platforms, the interpretation of results was straightforward and the SPInDel software was used without any problems. The results of this collaborative exercise indicate that the SPInDel method can be applied successfully in forensic casework investigations. Fil: Alves, Cíntia. Universidad de Porto; Portugal Fil: Pereira, Rui. Universidad de Porto; Portugal Fil: Prieto, Lourdes. Universidad de Santiago de Compostela; España Fil: Aler, Mercedes. Instituto de Medicina Legal y Ciencias Forenses de Valencia; España Fil: Amaral, Cesar R. L.. Universidade do Estado do Rio de Janeiro; Brasil Fil: Arévalo, Cristina. Universidad de Alcalá; España Fil: Berardi, Gabriela. Fundación Favaloro; Argentina Fil: Di Rocco, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina Fil: Caputo, Mariela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Carmona, Cristian Hernandez. Poder Judicial. Departamento de Ciencias Forenses. Sección de Bioquímica; Costa Rica Fil: Catelli, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Equipo Argentino de Antropología Forense; Argentina Fil: Costa, Heloísa Afonso. Instituto Nacional de Medicina Legal e Ciências Forenses; Portugal Fil: Coufalova, Pavla. Institute of Criminalistics Prague; República Checa Fil: Furfuro, Sandra Beatriz. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Laboratorio de Análisis de ADN; Argentina Fil: García, Óscar. Polícia del País Vasco. Sección de Genética Forense; España Fil: Gaviria, Anibal. Cruz Roja Ecuatoriana; Ecuador Fil: Goios, Ana. Universidad de Porto; Portugal Fil: Gómez, Juan José Builes. Universidad de Antioquia; Colombia Fil: Hernández, Alexis. Instituto Nacional de Toxicología y Ciencias Forenses; España Fil: Betancor Hernández, Eva del Carmen. Instituto de Medicina Legal de Las Palmas. Laboratorio Genética Forense; España Fil: Miranda, Luís. Universidade de Aveiro; Portugal Fil: Parra, David. Servicio de Criminalística de la Guardia Civil. Departamento de Química y Medio Ambiente; España Fil: Pedrosa, Susana. Unidad de Laboratorio de Navarra de Servicios y Tecnologías; España Fil: Porto, Maria João Anjos. Instituto Nacional de Medicina Legal e Ciências Forenses; Portugal Fil: Rebelo, Maria de Lurdes. Instituto Nacional de Medicina Legal e Ciências Forenses; Portugal Fil: Spirito, Matteo. Università Cattolica del Sacro Cuore; Italia Fil: Torres, María del Carmen Villalobos. Universidad Autónoma de Nuevo León; México Fil: Amorim, António. Universidad de Porto; Portugal Fil: Pereira, Filipe. Universidad de Porto; Portugal

The demonstrated functional interaction of metabotropic glutamate 5 (mGlu₅) receptors with N-methyl-d-aspartate (NMDA) receptors has prompted speculation that their activation may offer a potential treatment for aspects of schizophrenia. Development of selective mGlu₅ agonists has been difficult, but several different positive allosteric modulator (PAM) molecules have now been identified. This study describes two novel mGlu₅ PAMs, LSN2463359 (N-(1-methylethyl)-5-(pyridin-4-ylethynyl)pyridine-2-carboxamide) and LSN2814617 [(7S)-3-tert-butyl-7-[3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl]-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-A]pyridine], which are useful tools for this field of research. Both compounds are potent and selective potentiators of human and rat mGlu₅ receptors in vitro, displaying curve shift ratios of two to three fold in the concentration-response relationship to glutamate or the glutamate receptor agonist, DHPG, with no detectable intrinsic agonist properties. Both compounds displaced the mGlu₅ receptor antagonist radioligand, [³H]MPEP in vitro and, following oral administration reached brain concentrations sufficient to occupy hippocampal mGlu₅ receptors as measured in vivo by dose-dependent displacement from the hippocampus of intravenously administered MPEPy. In vivo EEG studies demonstrated that these mGlu₅ PAMs have marked wake-promoting properties but little in the way of rebound hypersomnolence. In contrast, the previously described mGlu₅ PAMs CDPPB and ADX47273 showed relatively poor evidence of in vivo target engagement in either receptor occupancy assays or EEG disturbance. Wake-promoting doses of LSN2463359 and LSN2814617 attenuated deficits in performance induced by the competitive NMDA receptor antagonist SDZ 220,581 in two tests of operant behaviour: the variable interval 30 s task and the DMTP task. These effects were lost if the dose of either compound extended into the range which disrupted performance in the baseline DMTP task. However, the improvements in response accuracy induced by the mGlu₅ potentiators in SDZ 220,581-treated rats were not delay-dependent and, therefore, perhaps more likely reflected optimization of general arousal than specific beneficial effects on discrete cognitive processes. The systematic profiling of LSN2463359 and LSN2814617 alongside other previously described molecules will help determine more precisely how mGlu₅ potentiator pharmacology might provide therapeutic benefit. This article is part of a Special Issue entitled 'Cognitive Enhancers'.

There is growing evidence that the histone-DNA complexes found in nucleosomes offer protection from DNA degradation processes, including apoptotic events in addition to bacterial and environmental degradation. We sought to locate human nucleosome regions and build a catalogue of SNPs sited near the middle of these genomic segments that could be combined into a single PCR multiplex specifically for use with extremely degraded human genomic DNA samples. Using recently optimized bio-informatics tools for the reliable identification of nucleosome sites based on sequence motifs and their positions relative to known promoters, 1395 candidate loci were collected to construct an 18-plex single base extension assay. Genotyping performance of the nucleosome SNPs was tested using artificially degraded DNA and 24 casework samples where the likely state of degradation of DNA was established by comparison to profile completeness in four other forensic assays: a standard 15-plex STR identification test, a miniaturized STR multiplex and two autosomal SNP multiplexes. The nucleosome SNP assay gave genotyping success rates 6% higher than the best existing forensic SNP assay: the SNPforID Auto-2 29-plex and significantly higher than the mini-STR assay. The nucleosome SNPs we located and combined therefore provide a new type of marker set that can be used to supplement existing approaches when the analysed DNA is likely to be extremely degraded and may fail to give sufficient STR genotypes for a reliable identification. © 2011 Elsevier Ireland Ltd. All rights reserved.

Made available in DSpace on 2013-09-27T14:53:31Z (GMT). No. of bitstreams: 1 WOS000288248400016.pdf: 119361 bytes, checksum: 9af8c0ce8fdb208234453f9523e8aa1b (MD5) Previous issue date: 2011-03-01 Made available in DSpace on 2013-09-30T19:24:06Z (GMT). No. of bitstreams: 1 WOS000288248400016.pdf: 119361 bytes, checksum: 9af8c0ce8fdb208234453f9523e8aa1b (MD5) Previous issue date: 2011-03-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T15:34:26Z No. of bitstreams: 1 WOS000288248400016.pdf: 119361 bytes, checksum: 9af8c0ce8fdb208234453f9523e8aa1b (MD5) Made available in DSpace on 2014-05-20T15:34:26Z (GMT). No. of bitstreams: 1 WOS000288248400016.pdf: 119361 bytes, checksum: 9af8c0ce8fdb208234453f9523e8aa1b (MD5) Previous issue date: 2011-03-01 Mitochondrial DNA (mtDNA) population data for forensic purposes are still scarce for some populations, which may limit the evaluation of forensic evidence especially when the rarity of a haplotype needs to be determined in a database search. In order to improve the collection of mtDNA lineages from the Iberian and South American subcontinents, we here report the results of a collaborative study involving nine laboratories from the Spanish and Portuguese Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG) and EMPOP. The individual laboratories contributed population data that were generated throughout the past 10 years, but in the majority of cases have not been made available to the scientific community. A total of 1019 haplotypes from Iberia (Basque Country, 2 general Spanish populations, 2 North and 1 Central Portugal populations), and Latin America (3 populations from São Paulo) were collected, reviewed and harmonized according to defined EMPOP criteria. The majority of data ambiguities that were found during the reviewing process (41 in total) were transcription errors confirming that the documentation process is still the most error-prone stage in reporting mtDNA population data, especially when performed manually. This GHEP-EMPOP collaboration has significantly improved the quality of the individual mtDNA datasets and adds mtDNA population data as valuable resource to the EMPOP database (www.empop.org). (C) 2010 Elsevier B.V. All rights reserved. Innsbruck Med Univ, Inst Legal Med, Innsbruck, Austria Univ Inst Res Forens Sci IUICP, Madrid, Spain Univ Porto IPATIMUP, Inst Mol Pathol & Immunol, Oporto, Portugal Univ Estadual Paulista, UNESP, Lab Patern, São Paulo, Brazil Natl Inst Toxicol & Forens Sci INTCF, Madrid, Spain Univ São Paulo, Sch Med, Dept Legal Med Bioeth & Occupat Hlth, BR-05508 São Paulo, Brazil Univ Basque Country, BIOMICs Res Grp, Ctr Invest & Estudios Avanzados Lucio Lascaray, Vitoria, Spain Natl Inst Legal Med, N Branch, Oporto, Portugal Natl Inst Legal Med, Ctr Branch, Coimbra, Portugal Genom Engn Mol, São Paulo, Brazil Univ Estadual Paulista, UNESP, Lab Patern, São Paulo, Brazil

We report the results of the seventh edition of the GEP-ISFG mitochondrial DNA (mtDNA) collaborative exercise. The samples submitted to the participant laboratories were blood stains from a maternity case and simulated forensic samples, including a case of mixture. The success rate for the blood stains was moderate (∼77%); even though four inexperienced laboratories concentrated about one-third of the total errors. A similar success was obtained for the analysis of mixed samples (78.8% for a hair-saliva mixture and 69.2% for a saliva-saliva mixture). Two laboratories also dissected the haplotypes contributing to the saliva-saliva mixture. Most of the errors were due to reading problems and misinterpretation of electropherograms, demonstrating once more that the lack of a solid devised experimental approach is the main cause of error in mtDNA testing. © 2007 Elsevier Ireland Ltd. All rights reserved.

Herein we describe a series of potent and selective PPARgamma agonists with moderate PPARalpha affinity and little to no affinity for other nuclear receptors. In vivo studies in a NIDDM animal model (ZDF rat) showed that these compounds are efficacious at low doses in glucose normalization and plasma triglyceride reduction. Compound 1b (LY519818) was selected from our SAR studies to be advanced to clinical evaluation for the treatment of type II diabetes.

The Spanish and Portuguese ISFG Working Group (GEP-ISFG) carried out a collaborative exercise in order to asses the performance of two Y chromosome STR tetraplexes, which include the loci DYS461, GATA C4, DYS437 and DYS438 (GEPY I), and DYS460, GATA A10, GATA H4 and DYS439 (GEPY II). The groups that reported correct results in all the systems were also asked to analyse a population sample in order to evaluate the informative content of these STRs in different populations. A total of 1020 males out of 13 population samples from Argentina, Brazil, Costa Rica, Macao, Mozambique, Portugal and Spain were analysed for all the loci included in the present study. Haplotype and allele frequencies of these eight Y-STRs were estimated in all samples. The lowest haplotype diversity was found in the Lara (Argentina) population (95.44%) and the highest (99.90%) in Macao (China). Pairwise haplotype analysis showed the relative homogeneity of the Iberian origin samples, in accordance with what was previously found in the European populations for other Y-STR haplotypes (http://www.ystr.org). As expected, the four non-Caucasian samples, Macao (Chinese), Mozambique (Africans), Costa Rica (Africans) and Argentina (Lara, Amerindians), show highly significant Φst values in the pairwise comparisons with all the Caucasian samples. © 2003 Elsevier Ireland Ltd. All rights reserved.

Since 1998, the Spanish Scientific Police has been analyzing both hypervariable segments (HVR1 and HVR2) of the mitochondrial control region by sequencing analysis. During this period, different sample types (e.g., blood, saliva, telogen hairs, hair shafts, nails, tissues, bone and dental remains) related to murders, sexual assaults, robberies and identification of human remains have been studied. Some problematic findings detected in routine forensic casework involving sequence heteroplasmy and one single nucleotide mismatch between forensic specimens and reference samples are described in this paper. In specimens which, in principle, due to apparent features should not give any problems when being analysed, a lack of amplification was detected within some of the subregions studied. Subsequently, it was proven that this was caused by point mutations in the primer binding sites. A substitution of the primers by non-conventional ones overcame the absence of reactions. Given that a proper statistical evaluation of the results using a large database is needed, samples from 120 individuals living in Spain have been analysed. The most frequent sequence found was 263-G; 315.1-C (differences compared to the reference sequence), being consistent with other Caucasian population studies.

The Species Identification by Insertions/Deletions (SPInDel) multiplex PCR allows the identification of species by the generation of numeric profiles combining the lengths of six mitochondrial ribosomal RNA (rRNA) gene regions. Here, we describe a collaborative exercise for the taxonomic identification of forensic samples using the SPInDel kit carried out in 2014 by the GHEP-ISFG. A total of 24 laboratories from 10 countries were supplied with a SPInDel primer mix and control samples of the 10 target species needed to perform genotyping of 11 samples from previous GHEP-ISFG Intercomparison Exercises. Overall, correct identifications were reported by 22 of the 24 laboratories. The errors were concentrated in a few laboratories, with one laboratory reporting errors in all profiles. The success rate in the identification of species with the SPInDel kit was 100% in 8 of the 11 samples. The level of concordance in identifications was always higher than 93%, including in samples with low amounts of DNA (hair shafts) and mixtures of saliva and blood. When considering all cases together, 98.6% of the reported profiles yielded correct species identifications. The frequency of wrong (5.8%) and missing (2.4%) alleles was low and did not interfere with the correct species identification, mainly because the SPInDel method relies on the analysis of multiple loci. In summary, the SPInDel method was easily implemented by different laboratories and genotyping platforms and the interpretation of results was straightforward. The method proved to be efficient in the identification of species in diverse forensic samples.

Ethyl 4,4-dimethylpyroglutamate has been used as a chiral auxiliary for α,β-unsaturated acids in Michael addition reactions. The conjugate addition of Grignard reagents to the amides in the presence of copper iodide, tetramethylene diamine (TMEDA) and trimethylsilyl chloride, proceeded with high yields and excellent stereoselectivities, yielding after hydrolysis enantiomerically pure β-substituted carboxylic acid derivatives.

( 1S,2S,5R,6S )-2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740) was synthesised enantiospecifically from a sugar derived enantiomerically pure cyclopentenone. The α-amino acid stereogenic centre was formed by reacting the ketone with chloroform anion and then the alcohol so formed was reacted with sodium azide/DBU in methanol to give an azido ester. Critically, this modified Corey-Link reaction gives the opposite stereochemical outcome to the traditional Bucherer-Bergs and Strecker reactions. The azide was reduced and acylated, the 1,2 diol deoxygenated and the protecting groups removed to give LY354740 with an e.e.>98%.

The mitochondrial DNA (mtDNA) working group of the GEP-ISFG carried out an inter-laboratory exercise consisting of the study of mixture stains (saliva/semen and blood/semen) in order to investigate the behaviour of these common forensic samples when analysing their mtDNA using standard sequencing methodology. All labs extracted the DNA by preferential lysis and amplified and sequenced the first hypervariable region I (HVS-I). The results showed high consensus between labs for the first fraction of the lysis but not for the second one. We also observed differences between mixtures prepared from different donors and different body fluids. The present study has important consequences for the analysis and interpretation of mtDNA mixtures. © 2005 Elsevier B.V. All rights reserved.

The asymmetric synthesis of (+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740) 1, a potent and selective group 2 mGluR agonist, has been accomplished starting from the readily available enantiomerically pure cyclopentenone 4. Thus, cyclopropanation with ethyl(dimethylsulfuranylidene)acetate generated in situ with DBU, followed by deketalization gave rise to the dihydroxy bicyclic ketone 9. After protecting the ketone as 1,3-dioxolane and its transformation to the orthoformate 11, this was pyrolytically deoxygenated in a sealed tube to the bicyclic enone 13. The synthesis was completed after hydrogenation, stereoselective Bucherer-Bergs reaction and hydantoin hydrolysis, yielding LY354740 (+)-1 with an e.e. ≥98%.

We describe a method to estimate the proportion of length variants in samples carrying mtDNA homopolymeric C-stretch (303 to 315) originated by a T310C transition. Sequencing analysis of these samples produces blurred electropherograms and the exact number of C-residues remains ambiguous. This has important consequences in forensic casework, since one of the aims in the interpretation of mtDNA profiles is the determination of the existence of a match or a mismatch between two or more sequences under study. HV-2B region (positions 172 to 408) was amplified with two sets of labelled primers. Electrophoresis of fluorescently labelled amplicons was performed on an ABI310 or ABI377. Results were reproducible in repeated PCR experiments, using the two sets of primers and using either capillary or slab-gel electrophoresis.

We studied the role of alpha1-adrenoceptors in the modulation of ventricular tachycardia and fibrillation in chloralose-anesthetized dogs subjected to 30 min left anterior descending coronary artery occlusion. Study groups were control, and those treated with the alpha1-adrenoceptor-subtype blockers WB4101 (0.5 mg/kg i.v.) or chloroethylclonidine (1.9 mg/kg i.v.). For the first set of experiments all animals were in sinus rhythm and heart rate was slower in the chloroethylclonidine-pretreated animals than the WB4101-treated group (P < 0.05). During occlusion, ventricular tachycardia and ventricular fibrillation incidence did not differ among control, WB4101 or chloroethylclonidine (3 dogs with ventricular fibrillation in each group and 0, 2 and 3 dogs respectively with ventricular tachycardia), but ventricular premature depolarizations were significantly reduced by both interventions, and nonsustained ventricular tachycardia was suppressed by WB4101. In a second set of experiments, animals were atrially paced at a cycle length of 300 ms, and divided into control, WB4101-treated or chloroethylclonidine-treated, as above. Here, 9/10 chloroethylclonidine-treated animals developed ventricular tachycardia and fibrillation during occlusion, whereas only 4/10 controls and 4/10 WB4101-treated animals did so (P < 0.05). In conclusion, during sinus rhythm, both types of alpha1-adrenoceptor subtype blockade significantly suppressed ventricular premature depolarizations and neither affected ventricular tachycardia and fibrillation. In contrast, when heart rate was held constant, chloroethylclonidine clearly enhanced the occurrence of ventricular fibrillation during occlusion. These results suggest the alpha1-adrenoceptor subtype blocked by chloroethylclonidine, but not that blocked by WB4101, is capable of increasing the incidence of lethal arrhythmias that occur at rapid atrial rates during ischemia.

The synthesis of racemic 3-aminobicyclo[3.3.0]octane-1,3-dicarboxylic acids (2 and 3) which are conformationally constrained ACPD analogues, has been achieved in seven steps starting from the readily available Weiss diketone (4). Partial reduction of of 4 to 5, followed by phenyl ring oxidation with RuCl3/NaIO4, gave the bicyclic ketoacid 6 which, after Bucherer-Bergs reaction and fractional crystallization, afforded spirohydantoins 7 and 8 in a 2/1 ratio. Both isomers were hydrolyzed to amino acids 2 and 3. Optical resolution of racemic 7 was performed by crystallization of the corresponding (−)-(S)-brucine diasteromeric salts and, after decomposition and hydrolysis, (+)-(1S ∗ , 3R ∗ , 5R ∗ ) and (−)-(1R ∗ , 3S ∗ , 5S ∗ )- 3-aminobicyclo[3,3,0]octane-1,3-dicarboxylic acids (2a and 2b) were obtained to be biologically compared with (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD). Due to the solubility profile of hydantoins 7 and amino acids 2, the enantiomeric purity was measured in the dimethyl derivative 9, being determined by chiral-HPLC.

Mitochondrial DNA hypervariable regions I and II were sequenced from 403 unrelated Argentine individuals. The aim of this study was to create a population database as well as to identify the population diversity for this genetic marker by classifying it into haplogroups. The sequence polymorphisms of the HVI and HVII regions were determined by PCR and direct sequencing. The haplotypes found were checked by phylogenetic haplogroup analysis to decrease haplotype assignation errors and to avoid artificial recombination. We found 78 different haplogroups in this set of samples. A high percentage of haplotypes (53%) belong to European haplogroups due to the large flow of European immigrants from colonial times. However, we also observed a high percentage of haplotypes that belong to Amerindian haplogroups (39%), which were conserved through the female Amerindian population contribution. Furthermore, we found a small group of haplotypes with Sub-Saharan African origin (3.5%) due to the slave trade at the beginning of Argentina's colonization. The sequences found showed that this set of samples has an abundant haplogroup diversity because of the European and Amerindian ethnic group contribution.