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Background: Despite high survival after bidirectional cavopulmonary anastomosis, a considerable number of patients suffer significant post-operative morbidities related to prolonged length of stay. Methods: A single-center retrospective cohort study of all consecutive patients undergoing a first-time bidirectional cavopulmonary anastomosis from 2006 to 2019. Results: Prolonged length of stay was defined as hospital stay greater than the 75th percentile for our cohort. Of 195 patients who met inclusion criteria, the median post-operative length of stay was 8 days (interquartile range, 4-15 days). Prolonged length of stay was defined as greater than 15 days. In multivariate analysis, greater than mild systemic atrioventricular valve regurgitation (odds ratio 3.7, 95% CI 1.05-13.068, p = 0.04), longer length of stay after the initial palliative procedure (odds ratio 1.028, 95% CI 1.004-1.05, p = 0.02), and pre-operative higher superior vena cava oxygen saturation (odds ratio 0.922, 95% CI 0.85-0.99, p = 0.04) maintained statistical significance as independent risk and protective factors for prolonged length of stay. A one-level increase in the severity of pre-operative systemic atrioventricular valve regurgitation was associated with a multiplicative change in the odds ratio of prolonged length of stay of 5.45 (p = 0.005) independent of the severity of systemic ventricular dysfunction. Conclusion: Pre-operative characteristics with greater than mild systemic atrioventricular valve regurgitation, longer length of stay after the initial palliative procedure, and lower superior vena cava oxygen saturation were associated with prolonged length of stay after a first-time bidirectional cavopulmonary anastomosis.

The concentrations of LSN3154567 in the dog plasma samples.

NAMPT, an enzyme essential for NAD+ biosynthesis, has been extensively studied as an anticancer target for developing potential novel therapeutics. Several NAMPT inhibitors have been discovered, some of which have been subjected to clinical investigations. Yet, the on-target hematological and retinal toxicities have hampered their clinical development. In this study, we report the discovery of a unique NAMPT inhibitor, LSN3154567. This molecule is highly selective and has a potent and broad spectrum of anticancer activity. Its inhibitory activity can be rescued with nicotinic acid (NA) against the cell lines proficient, but not those deficient in NAPRT1, essential for converting NA to NAD+. LSN3154567 also exhibits robust efficacy in multiple tumor models deficient in NAPRT1. Importantly, this molecule when coadministered with NA does not cause observable retinal and hematological toxicities in the rodents, yet still retains robust efficacy. Thus, LSN3154567 has the potential to be further developed clinically into a novel cancer therapeutic. Mol Cancer Ther; 16(12); 2677–88. ©2017 AACR.

Table S1: anti-proliferative activity of LSN3154567 against different cancer cell lines; Table S2: effects of NAM on the anti-proliferative activity of LSN3154567

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.

Coronary artery disease in palliated hypoplastic left heart syndrome is uncommon. Myocardial infarction from a coronary thrombus, serving as a substrate for ventricular arrhythmia in Fontan physiology, is under-reported despite known hypercoagulopathic state. Traditional risk factors for coronary artery occlusion include intracardiac thrombi, hyperlipidemia, and hypertension. Baffle leaks and abnormal ventriculocoronary fistulae found in these patients are contributing factors. We sought to assess and describe coronary artery involvement in this complex patient population. Our research highlights both the need to assess distal coronary vasculature and to thoroughly evaluate hemodynamics and biventricular function with new-onset troponin leak or ventricular arrhythmias.

In forensics, mitochondrial DNA (mtDNA) analysis is foremost applied to rootless hairs often lacking detectable nuclear DNA. Sanger sequencing is the routine mtDNA method in most forensic laboratories, even though interpretation of mixed samples and heteroplasmic sites can be challenging. Individuals may hold cells with low-level heteroplasmy variants below the detection threshold and other cells where this minor variant is the major one. This difference may be interpreted as a mismatch between reference and evidentiary trace samples, such as buccal specimens and rootless hairs. Such mismatches may be solved by Massively Parallel Sequencing (MPS), allowing more sensitive quantitative analysis for mixed positions than Sanger. The mtDNA control region was analysed in buccal reference samples from 26 individuals and 475 corresponding hairs by MPS and compared to Sanger sequencing data generated on the same samples. With MPS, mixed contributions down to 3% were regarded, leading to a substantial increase in the frequency of heteroplasmy. Our results demonstrate that previously reported mismatches between buccal reference and hair shaft samples by Sanger are detected as low-level heteroplasmy by MPS. A detailed overview of buccal and hair heteroplasmy is provided and implications for MPS-based mtDNA analysis in the context of forensic cases are discussed.

One of the main goals of the Spanish and Portuguese-Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG) is to promote and contribute to the development and dissemination of scientific knowledge in the field of forensic genetics. The GHEP-ISFG supports several Working Commissions which develop different scientific activities. One of them, the Working Commission on "Massively Parallel Sequencing (MPS): Forensic Applications", organized its first collaborative exercise on forensic applications of MPS technology in 2019. The aim of this exercise was to assess the concordance between the MPS results and those obtained with conventional technologies (capillary electrophoresis and Sanger sequencing), as well as to compare the results obtained within the different MPS platforms and/or the different kits/panels and analysis software packages (commercial and open-access) available on the market. The seven participating laboratories analyzed some samples of the annual GHEP-ISFG proficiency test (EIADN No. 27 (2019)), using Ion Torrent™ or MiSeq FGx® platforms. Six of them sent autosomal STR sequence data, five laboratories performed MPS analysis of individual identification SNPs, four laboratories reported MPS data of Y-chromosomal STRs, and X-chromosomal STRs, three laboratories performed MPS analysis of ancestry informative SNPs and phenotype informative SNPs, two labs performed MPS analysis of the mitochondrial DNA control region, and only one lab produced MPS data of lineage informative SNPs. Autosomal STR sequencing results were highly concordant to the consensus obtained by capillary electrophoresis in the EIADN No. 27 (2019) exercise. Furthermore, in general, a high level of concordance was observed between the results of the participating laboratories, regardless of the platform used. The main discordances were due to errors during the analysis process or from sequence data obtained with low depth of coverage. In this paper we highlight some issues that still arise, such as standardization of the nomenclature for STRs analyzed by sequencing with MPS, the universal uptake of a nomenclature framework by the analysis software, and well established validation and accreditation of the new MPS platforms for use in routine forensic case-work.

In both criminal cases and civil cases there is an increasing demand for the analysis of DNA mixtures involving relationships. The goal might be, for example, to identify the contributors to a DNA mixture where the donors may be related, or to infer the relationship between individuals based on a DNA mixture. This paper applies a recent approach to modelling and computation for DNA mixtures involving contributors with arbitrarily complex relationships to two real cases from the Spanish Forensic Police., Comment: 12 pages, 11 tables; new version has additional analysis of first case study, and appendix about methodology

The Piscine Stream Community Estimation System (PiSCES) provides users with a hypothesized fish community for any stream reach in the conterminous United States using information obtained from Nature Serve, the US Geological Survey (USGS), StreamCat, and the Peterson Field Guide to Freshwater Fishes of North America for over 1000 native and non-native freshwater fish species. PiSCES can filter HUC8-based fish assemblages based on species-specific occurrence models; create a community abundance/biomass distribution by relating relative abundance to mean body weight of each species; and allow users to query its database to see ancillary characteristics of each species (e.g., habitat preferences and maximum size). Future efforts will aim to improve the accuracy of the species distribution database and refine/augment increase the occurrence models. The PiSCES tool is accessible at the EPA's Quantitative Environmental Domain (QED) website at https://qed.epacdx.net/pisces/

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.

NAMPT, an enzyme essential for NAD+ biosynthesis, has been extensively studied as an anticancer target for developing potential novel therapeutics. Several NAMPT inhibitors have been discovered, some of which have been subjected to clinical investigations. Yet, the on-target hematological and retinal toxicities have hampered their clinical development. In this study, we report the discovery of a unique NAMPT inhibitor, LSN3154567. This molecule is highly selective and has a potent and broad spectrum of anticancer activity. Its inhibitory activity can be rescued with nicotinic acid (NA) against the cell lines proficient, but not those deficient in NAPRT1, essential for converting NA to NAD+. LSN3154567 also exhibits robust efficacy in multiple tumor models deficient in NAPRT1. Importantly, this molecule when coadministered with NA does not cause observable retinal and hematological toxicities in the rodents, yet still retains robust efficacy. Thus, LSN3154567 has the potential to be further developed clinically into a novel cancer therapeutic. Mol Cancer Ther; 16(12); 2677–88. ©2017 AACR.

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

LY2812223 [(1R,2S,4R,5R,6R)-2-amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid] was identified via structure-activity studies arising from the potent metabotropic glutamate mGlu2/3 receptor agonist LY354740 [(+)-2-aminobicyclo[3.1.0] hexane-2,6-dicarboxylic acid] as an mGlu2-preferring agonist. This pharmacology was determined using stably transfected cells containing either the human mGlu2 or mGlu3 receptor. We extended the pharmacological evaluation of LY2812223 to native brain tissues derived from relevant species used for preclinical drug development as well as human postmortem brain tissue. This analysis was conducted to ensure pharmacological translation from animals to human subjects in subsequent clinical studies. A guanosine 5′-O-(3-[35S]thio)triphosphate (GTPγS) functional binding assay, a method for measuring Gi-coupled signaling that is inherent to the group 2 mGlu receptors, was used to evaluate LY2812223 pharmacology of native mGlu receptors in mouse, rat, nonhuman primate, and human cortical brain tissue samples. In native tissue membranes, LY2812223 unexpectedly acted as a partial agonist across all species tested. Activity of LY2812223 was lost in cortical membranes collected from mGlu2 knockout mice, but not those from mGlu3 knockout mice, providing additional support for mGlu2-preferring activity. Other signal transduction assays were used for comparison with the GTP binding assay (cAMP, calcium mobilization, and dynamic mass redistribution). In ectopic cell line–based assays, LY2812223 displayed near maximal agonist responses at the mGlu2 receptor across all assay formats, while it showed no functional agonist activity at the mGlu3 receptor except in the cAMP assay. In native brain slices or membranes that express both mGlu2 and mGlu3 receptors, LY2812223 displayed unexpected partial agonist activity, which may suggest a functional interplay between these receptor subtypes in the brain.

Small variations between haplotypes detected in different tissues from the same individual have been previously described. These differences complicate the interpretation of mtDNA results in real forensic casework. mtDNA haplotypes detected in hair strands collected at the crime scene have to be frequently compared with haplotypes of reference samples (buccal swabs) from victims or suspects. Nucleotide position 16093 is a well-known hot spot where differences can accumulate between different tissues of the same individual. Intra individual variation was also detected at positions 16182 and 16183 in haplotypes showing an uninterrupted HV1 poly-C stretch (with 16189C). In order to better characterize the type of variation in these positions between buccal cells and hair strands from the same individual, we have performed Sanger sequencing in 25–28 hair strands (411 in total) from 15 individuals showing either an uninterrupted HV1 polyC-stretch (16189C) or 16093C/Y in their buccal cells. The results have been evaluated by also taking into account our previous results published in [ 19 ]. We have found that no variation among hair strands was detected in individuals showing T16093 in buccal cells, while variation in hair strands (T16093, 16093C and 16093Y) were detected in individuals showing 16093C or 16093Y in buccal cells. Regarding nucleotide positions 16182 and 16183 in combination with an uninterrupted polyC-stretch, no variation was detected in hairs from individuals showing A16182 16183C in their buccal cells. In contrast, individuals A16182 A16183 showed hair strands with A16182 16183 M and A16182 16183C. And finally, individuals with 16182C 16183C showed some variation in a small amount of their hair strands (some hairs with 16182 M 16183C). These results can be relevant for forensic practitioners when comparing reference samples with hair strands, which is the type of sample most tested by using mtDNA analysis in forensic casework.

Forensic genetics brings together all the genetic knowledge necessary to solve specific legal problems. In recent decades, new techniques have shown the potential of DNA as a profiling system. These advances have arrived hand in hand with other improvements for the communication of test results, with the introduction of statistical evaluation. In the collective imagination, nourished by TV series such as CSI, forensic evidence is presented as one hundred percent certain, when reality is different. However, statistical analysis has allowed us to turn from handcrafted forensic medicine, based on intuition and experience, to tests based on evidence and data, where uncertainty is quantified in probabilistic terms.

Identification of orthosteric mGlu(2/3) receptor agonists capable of discriminating between individual mGlu2 and mGlu3 subtypes has been highly challenging owing to the glutamate-site sequence homology between these proteins. Herein we detail the preparation and characterization of a series of molecules related to (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate 1 (LY354740) bearing C4-thiotriazole substituents. On the basis of second messenger responses in cells expressing other recombinant human mGlu2/3 subtypes, a number of high potency and efficacy mGlu2 receptor agonists exhibiting low potency mGlu3 partial agonist/antagonist activity were identified. From this, (1R,2S,4R,5R,6R)-2-amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 14a (LY2812223) was further characterized. Cocrystallization of 14a with the amino terminal domains of hmGlu2 and hmGlu3 combined with site-directed mutation studies has clarified the underlying molecular basis of this unique pharmacology. Evaluation of 14a in a rat model responsive to mGlu2 receptor activation coupled with a measure of central drug disposition provides evidence that this molecule engages and activates central mGlu2 receptors in vivo.

As part of our ongoing research to identify novel agents acting at metabotropic glutamate 2 (mGlu2) and 3 (mGlu3) receptors, we have previously reported the identification of the C4α-methyl analog of mGlu2/3 receptor agonist 1 (LY354740). This molecule, 1S,2S,4R,5R,6S-2-amino-4-methylbicyclo[3.1.0]hexane-2,6-dicarboxylate 2 (LY541850), exhibited an unexpected mGlu2 agonist/mGlu3 antagonist pharmacological profile, whereas the C4β-methyl diastereomer (3) possessed dual mGlu2/3 receptor agonist activity. We have now further explored this structure–activity relationship through the preparation of cyclic and acyclic C4-disubstituted analogs of 1, leading to the identification of C4-spirocyclopropane 5 (LY2934747), a novel, potent, and systemically bioavailable mGlu2/3 receptor agonist which exhibits both antipsychotic and analgesic properties in vivo. In addition, through the combined use of protein–ligand X-ray crystallography employing recombinant human mGlu2/3 receptor amino terminal domains, molecular mod...

Acarapisosis is a disease of the adult honey bee Apis mellifera L., caused by the tracheal mite Acarapis woodi (Rennie), that affects the prothoracic tracheas of worker honey bees. Although it is not usually considered a real problem for honey bee colonies in southern Europe (mainly Spain and Greece), where the majority of professional beekeepers are located in Europe, recent works have reported the constant presence of this mite in this area, making it a potential cofactor for colony losses. In this study, we developed a specific PCR diagnostic tool that improves the techniques used so far and allowed us to confirm the presence of this parasite in Spain, urging the need to monitor its prevalence and implications in the health of the colonies. Indeed, in a total of 635 apiaries analysed, the prevalence of A. woodi in 2010 was 8.3 and 4 % in 2011. The mite is present in bee colonies over time and should not be underestimated as a possible cofactor in the collapse of bee colonies. Additionally, some positive samples were cloned so a genetic analysis on the diversity within A. woodi isolates was also approached. This allowed us to identify different genetic variants within an isolate, even when they were present at low frequencies. And this genetic analysis revealed the existence of a different clade of Acarapis sequences that could represent a new species or subspecies, although more research is required to verify the identity of this novel lineage at genetic and morphological level.

The microorganisms Ascosphaera apis, Paenibacillus larvae and Melissococcus plutonius are the three most important pathogens that affect honeybee brood. The aim of the present study was to evaluate the prevalence of these pathogens in honeybee colonies and to elucidate their role in the honeybee colony losses in Spain. In order to get it, a multiplex polymerase chain reaction (PCR) assay was developed to simultaneously amplify the16S ribosomal ribonucleic acid (rRNA) gene of P. larvae and M. plutonius, and the 5.8S rRNA gene of A. apis. The multiplex PCR assay provides a quick and specific tool that successfully detected the three infectious pathogens (P. larvae, M. plutonius and A. apis) in brood and adult honeybee samples without the need for microbiological culture. This technique was then used to evaluate the prevalence of these pathogens in Spanish honeybee colonies in 2006 and 2007, revealing our results a low prevalence of these pathogens in most of the geographic areas studied.

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.

Watershed-scale rainfall-runoff models are used for environmental management and regulatory modeling applications, but their effectiveness is limited by predictive uncertainties associated with model input data. This study evaluated the effect of temporal and spatial rainfall resolution on the predictive performance of Hydrological Simulation Program—Fortran (HSPF) using manual and automatic calibration procedures. Furthermore, the effect of automatic parameter estimation on the physical significance of calibrated parameter values was evaluated. Temporal resolutions examined included 15 min, 30 min, 1 h, and 2 h, and spatial resolution effects evaluated included the effect of a spatially averaged network of four rain gauges and Next-Generation Radar (NEXRAD) for selected rain events. Model efficiencies ranged from 0.31 to 0.86 when individual rain gauges (RG71, RG73, RG74, and RG77) were used one at a time. Model efficiency improved and ranged from 0.86 to 0.94 when a spatially averaged network of four ra...

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 determined nutrient export from the Yaquina and Alsea Rivers as part of a larger program for evaluating nutrient sources to coastal waters. The Yaquina and Alsea data indicated that one river typically contained twice the amount of dissolved nitrate-N, although temperature, conductivity and the concentrations of other nutrients were similar. We developed a nitrate export model using multiple linear regression (MLR) to analyze the discriminating variables that included nutrient concentrations and hardwood cover containing approximately 90% red alder (Alnus rubra), a nitrogen-fixing tree species. Using data from the Coastal Landscape Analysis and Modeling Study (CLAMS), hardwood cover was found to be most prevalent in the upper (gaged) Yaquina watershed. Estimated nitrate export was 2.02 Mg km-2 y-1 in the Yaquina and 1.24 Mg km-2 y-1 in the Alsea for 2006. However, the annual nitrate-N exported from the entire Alsea basin (1560 Mg) was slightly greater than that of the Yaquina basin (1320 Mg) s...