Forensic evaluation of two nucleic acid extraction systems and validation of a RT-qPCR protocol for identification of SARS-CoV-2 in post-mortem nasopharyngeal swabs.

The COVID-19 outbreak has represented a challenge for the international scientific community and particularly for forensic sciences. The lack of Coronavirus post-mortem testing led the National Institute of Toxicology and Forensic Sciences (INTCF) from Spain to verify the performance and utility of a quantitative reverse transcription PCR (RT-qPCR) clinical diagnosis protocol for SARS-CoV-2 detection (TaqPath™ COVID-19 CE-IVD RT-PCR Kit), to shed light on the cause of death (COD) in potentially COVID-19 cases in judicial autopsies. Two different RNA extraction methods were also tested (EZ1® DSP Virus Kit on the EZ1® Advanced XL robot versus MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit) regarding extraction efficiency, precision and contamination. RT-qPCR was evaluated for precision, specificity, limit of detection and concordance. Both the automated and the manual RNA extraction procedures showed good efficiency, but the automated virus extraction by bio-robot produced more reproducible results than the manual extraction. The SARS-CoV-2 RT-qPCR assay showed high sensitivity with a detection limit up to 10 copies/reaction and high specificity, as no cross-reactivity was detected between any of the 12 different RNA viruses tested, including three types of coronaviruses (SARS-CoV, NL63 and 229E). Reproducibility and repeatability of the studied method as well as concordance with other SARS-CoV-2 molecular detection protocols were also demonstrated.

The first GHEP-ISFG collaborative exercise on forensic applications of massively parallel sequencing.

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.

Massively parallel sequence data of 31 autosomal STR loci from 496 Spanish individuals revealed concordance with CE-STR technology and enhanced discrimination power.

This study reports Short Tandem Repeat (STR) sequence-based allele data from 496 Spanish individuals across 31 autosomal STR (auSTR) loci included in the Precision ID GlobalFiler™ NGS STR Panel v2: D12S391, D13S317, D8S1179, D21S11, D3S1358, D5S818, D1S1656, D2S1338, vWA, D2S441, D5S2800, D7S820, D16S539, D6S474, D12ATA63, D4S2408, D6S1043, D19S433, D14S1434, CSF1PO, D10S1248, D18S51, D1S1677, D22S1045, D2S1776, D3S4529, FGA, Penta D, Penta E, TH01 and TPOX. The sequence of each allele was aligned to the reference sequence GRCh37 (hg19) and formatted according to the guidance of the International Society for Forensic Genetics. A subset of 221 samples was evaluated for testing concordance with allele calls derived from CE-based analysis using PowerPlex Fusion 6C, and there was 99.95% allele concordance. Twenty-five out of 31 auSTR loci showed an increased number of alleles due to repeat region sequence variation and/or single nucleotide polymorphisms (SNP) residing in the flanking regions. A total of 18 loci showed increased observed heterozygosity due to sequence variation; the loci exhibiting the greatest increase were: D13S317 (12% points), D5S818 (10% points), D8S1179 (7% points), D3S1358 (7% points), and D21S11 (6% points). The combined match probability decreased from 2.022E-24 (length-based data) to 1.042E-27 (sequence-based data) for the 20 CODIS core STR loci. The combined match probability (sequence-based data) for the 31 STR loci studied was 4.777E-40. The combined typical paternity index increased from 1.118E + 12 to 8.179E + 13 using length and sequence-based data, respectively. This Spanish population study performed in the framework of the EU-funded DNASEQEX project is expected to provide STR sequence-based allele frequencies for forensic casework and support implementation of massively parallel sequencing (MPS) technology in forensic laboratories.

Current state-of-art of STR sequencing in forensic genetics.

The current state of validation and implementation strategies of massively parallel sequencing (MPS) technology for the analysis of STR markers for forensic genetics use is described, covering the topics of the current catalog of commercial MPS-STR panels, leading MPS-platforms, and MPS-STR data analysis tools. In addition, the developmental and internal validation studies carried out to date to evaluate reliability, sensitivity, mixture analysis, concordance, and the ability to analyze challenged samples are summarized. The results of various MPS-STR population studies that showed a large number of new STR sequence variants that increase the power of discrimination in several forensically relevant loci are also presented. Finally, various initiatives developed by several international projects and standardization (or guidelines) groups to facilitate application of MPS technology for STR marker analyses are discussed in regard to promoting a standard STR sequence nomenclature, performing population studies to detect sequence variants, and developing a universal system to translate sequence variants into a simple STR nomenclature (numbers and letters) compatible with national STR databases.

Formulation and communication of evaluative forensic science expert opinion-A GHEP-ISFG contribution to the establishment of standards.

Communicating and interpreting genetic evidence in the administration of justice is currently a matter of great concern, due to the theoretical and technical complexity of the evaluative reporting and large difference in expertise between forensic experts and law professionals. A large number of initiatives have been taken trying to bridge this gap, contributing to the education of both parties. Results however have not been very encouraging, as most of these initiatives try to cope globally with the problem, addressing simultaneously theoretical and technical approaches which are in a quite heterogeneous state of development and validation. In consequence, the extension and complexity of the resulting documents disheartens their study by professionals (both jurists and geneticists) and makes a consensus very hard to reach even among the genetic experts' community. Here we propose a 'back-to-basics', example-driven approach, in which a model report for the two most common situations faced by forensic laboratories is presented. We do hope that this strategy will provide a solid basis for a stepwise generalisation.

Measuring empathy feelings in football through media value / Valor mediático en el fútbol y grado de afinidad de los fans

This paper applies media value appraisals to measure the degree of identification that players and teams inspire in football supporters. Using a large data set of media value scores in season 2014/15, we rank the most popular football players and clubs worldwide; as well as the teams’ managers with the greatest media value exposure. Then, building upon individual ratings, we determine the hierarchy of the 'Big Five' European domestic leagues, according to their comparative media value status

En este artículo se emplean indicadores de valor mediático en el fútbol para estimar el grado de identificación que jugadores y clubs inspiran entre los aficionados. A partir de amplias bases de datos del valor mediático en la temporada 2014/15, se elaboran los rankings mundiales de jugadores y clubs, así como el ranking de los entrenadores con mayor visibilidad en los medios. Además, agregando registros individuales, puede determinarse la jerarquía de estatus mediático de las cinco grandes ligas domésticas de Europa

[The use, care and policy of complementary and integrative practices in primary health care]. / Uso, cuidado e política das práticas integrativas e complementares na Atenção Primária à Saúde.

The use of Complementary and Integrative Practices (CIP) is on the increase and its institutionalization in Primary Health Care (PHC) is a challenge. This article discusses the use, care, and policies of CIP at international and national levels found in the indexed literature. A review of the literature in PubMed/Medline and the Virtual Health Library was conducted using the key search words "Homeopathy", "Acupuncture", "Herbal Medicine", "Body Practices", "Primary Health Care" and other related terms in English, Spanish and Portuguese between 2002 and 2011. The use in the literature of CIP for the treatment of specific diseases from a biomedical perspective was observed, as well as evaluations of its use for the treatment of specific diseases focused on the reaction of the users and professionals and the analysis of the political, economic and social viability of CIP in health services. The conclusion drawn is that what is predominant in the literature is the quest for the scientific validation of CIP and a biomedical methodological bias in the designs of the studies, which does not contribute to clarifying the potential care of CIP in PHC.

Uso, cuidado e política das práticas integrativas e complementares na Atenção Primária à Saúde / The use, care and policy of complementary and integrative practices in primary health care

ResumoO uso das Práticas Integrativas e Complementares (PIC) é crescente e sua institucionalização na Atenção Primária à Saúde (APS) um desafio. Este artigo discute o uso, o cuidado e as políticas das PIC na esfera internacional e nacional, por meio da literatura indexada. Foi realizada revisão da literatura no PubMed/Medline e na Biblioteca Virtual da Saúde com os unitermos "Homeopatia", "Acupuntura", "Fitoterapia", "Práticas Corporais", "Atenção Primária à Saúde" e correlatos, nas línguas inglesa, espanhola e portuguesa, entre 2002 e 2011. Observou-se na literatura avaliações do uso das PIC para o tratamento de patologias específicas, com perspectiva biomédica; avaliações do seu uso para o tratamento de patologias específicas, porém focadas nos sentidos atribuídos pelos usuários e profissionais; análise da viabilidade política, econômica e social das PIC nos serviços de saúde. Conclui-se que predomina na literatura a busca pela validação científica das PIC e um viés metodológico biomédico no desenho dos estudos, o qual não contribui para esclarecer o potencial de cuidado das PIC na APS.

AbstractThe use of Complementary and Integrative Practices (CIP) is on the increase and its institutionalization in Primary Health Care (PHC) is a challenge. This article discusses the use, care, and policies of CIP at international and national levels found in the indexed literature. A review of the literature in PubMed/Medline and the Virtual Health Library was conducted using the key search words "Homeopathy", "Acupuncture", "Herbal Medicine", "Body Practices", "Primary Health Care" and other related terms in English, Spanish and Portuguese between 2002 and 2011. The use in the literature of CIP for the treatment of specific diseases from a biomedical perspective was observed, as well as evaluations of its use for the treatment of specific diseases focused on the reaction of the users and professionals and the analysis of the political, economic and social viability of CIP in health services. The conclusion drawn is that what is predominant in the literature is the quest for the scientific validation of CIP and a biomedical methodological bias in the designs of the studies, which does not contribute to clarifying the potential care of CIP in PHC.

Insulin-receptor substrate-2 (irs-2) is required for maintaining glucokinase and glucokinase regulatory protein expression in mouse liver.

Insulin receptor substrate (IRS) proteins play important roles in hepatic nutrient homeostasis. Since glucokinase (GK) and glucokinase regulatory protein (GKRP) function as key glucose sensors, we have investigated the expression of GK and GKRP in liver of Irs-2 deficient mice and Irs2(-/-) mice where Irs2 was reintroduced specifically into pancreatic ß-cells [RIP-Irs-2/IRS-2(-/-)]. We observed that liver GK activity was significantly lower (p<0.0001) in IRS-2(-/-) mice. However, in RIP-Irs-2/IRS-2(-/-) mice, GK activity was similar to the values observed in wild-type animals. GK activity in hypothalamus was not altered in IRS-2(-/-) mice. GK and GKRP mRNA levels in liver of IRS-2(-/-) were significantly lower, whereas in RIP-Irs-2/IRS-2(-/-) mice, both GK and GKRP mRNAs levels were comparable to wild-type animals. At the protein level, the liver content of GK was reduced in IRS-2(-/-) mice as compared with controls, although GKRP levels were similar between these experimental models. Both GK and GKRP levels were lower in RIP-Irs-2/IRS-2(-/-) mice. These results suggest that IRS-2 signalling is important for maintaining the activity of liver GK. Moreover, the differences between liver and brain GK may be explained by the fact that expression of hepatic, but not brain, GK is controlled by insulin. GK activity was restored by the ß-cell compensation in the RIP-Irs-2/IRS-2 mice. Interestingly, GK and GKRP protein expression remained low in RIP-Irs-2/IRS-2(-/-) mice, perhaps reflecting different mRNA half-lives or alterations in the process of translation and post-translational regulation.

Clinical evaluation of Tiny® 2.5- and 3.0-mm narrow-diameter implants as definitive implants in different clinical situations: a retrospective cohort study.

AIM: The aim of the present study was to assess retrospectively the survival of narrow-diameter implants (2.5 and 3 mm in diameter) in patients with insufficient bone ridge thickness for placement of standard-diameter implants. MATERIAL AND METHODS: Fifty-one patients with 89 inserted narrow-diameter implants (2.5 and 3.0 mm) were included. Patients were treated with one or more narrow-diameter implants between June 2004 and December 2005. The observation period for all included implants was at least 3 years after implant loading. Outcome measures were implant survival, complications and marginal bone level changes evaluated on panoramic radiographs. RESULTS: The mean follow-up period for all implants was 48 months. Only one implant was lost, yielding survival rates of 98.9% and 98.0% for the implant- and subject-based analyses, respectively. Six complications were reported. Twenty-four months after implant insertion, mean bone loss was 1.26 mm (SD 0.51). CONCLUSIONS: Narrow-diameter implants can be successfully used to treat narrow bone ridges up to 3 years after loading.

A multicentre placebo-controlled randomised clinical trial of antibiotic prophylaxis for placement of single dental implants.

PURPOSE: To compare the efficacy and safety of 2 g amoxicillin orally with identical placebo tablets 1 hour before implant placement when placing single implants in bone types II and III. MATERIAL AND METHODS: 12 private dental clinics in Spain agreed to participate in this trial. A total of 105 patients were recruited. Patients were randomised for consumption orally of 2g amoxicillin or identical placebo tablets. Only patients needing single implants were included. Outcome measures were post-operative infections, adverse events and implant failures. Characteristics of the saprophytic flora were also studied in all patients. Patients were seen 3 days, 10 days, 1 month and 3 months postoperatively. RESULTS: A total of 105 patients (n = 52 in the amoxicillin group and n = 53 in the placebo group) were evaluated and none were excluded from the study at 3 months. Six post-operative infections occurred and two implants were lost in each group. There were no statistically significant differences for postoperative infection, adverse events, implant failures and the characteristics of saprophytic flora between groups. The use of amoxicillin did not either alter or modify the characteristics of the saprophytic flora nor provoke remarkable side effects. CONCLUSIONS: Antibiotic prophylaxis may not be needed when placing single implants in patients with bone types II and III.

Metacarpal sexual determination in a Spanish population.

Anthropologists and forensic pathologist determine the sex of skeletons by analyzing quantitative and qualitative characters in the bone remains. Generally, the skull and os coxae are the elements most used, but they are not always preserved. In such cases, the investigator needs to have available other techniques based on different remains. The aim of the present work is to develop and describe discriminating functions for sex determination in a recent Spanish population using metacarpal morphology. A sample of bones corresponding to a contemporary Spanish population deposited at the Complutense University of Madrid (UCM) was analyzed. This sample comprised 697 metacarpals, corresponding to 79 adult individuals (37 men and 42 women). These allowed us to obtain 120 unifactorial discriminant functions. We selected the 10 equations, one for each metacarpal from both hands, that provided the best sexual discrimination. The correct sex classification rank progressed from 81%, for right (R) metacarpals IV and V, to 91%, for left (L) metacarpal II. The results suggest that metacarpals are structures that can be used for sex determination in paleoanthropological and forensic identifications.

Population data for 16 Y-chromosome STRs in four populations from Pyrenees (Spain).

Population frequencies for the eight Y-STR loci included in the "minimal haplotype" from Y-STR Haplotype Reference Database (YHRD) plus other eight Y-STRs (DYS434, DYS435, DYS436, DYS437, DYS438, DYS439, GATA H4 and GATA A10) were obtained for a sample of 133 males from four main geographical areas in the Pyrenees (Spain): Vall D'Aran (Lérida), Cerdanya (Gerona), Alt Urgell (Lérida) and Jacetania (Huesca). Haplotype diversities were estimated in the four populations.