Low-cost genotyping method based on allele-specific recombinase polymerase amplification and colorimetric microarray detection

[EN] The costs of current genotyping methods limit their application to personalized therapy. The authors describe an alternative approach for the detection of single-point-polymorphisms using recombinant polymerase amplification as an allele-specific technique. The use of short and chemically modified primers and locked nucleic acids allowed for a selective isothermal amplification of wild-type or mutant variants at 37 °C within 40 min. An amplification chip platform containing 100 wells was manufactured with a 3D printer and using thermoplastic polylactic acid. The platform reduces reagent consumption and allows parallelization. As a proof of concept, the method was applied to the genotyping of four SNPs that are related to the treatment of tobacco addiction. The target polymorphisms included rs4680 (COMT gene), rs1799971 (OPRM1 gene), rs1800497 (ANKK1 gene), and rs16969968 (CHRNA5 gene). The genotype populations can be well discriminated.The authors acknowledge the financial support received from the Generalitat Valenciana (GVA-PROMETEOII/2014/040 project and GRISOLIA/2014/024 PhD grant) and the Spanish Ministry of Economy and Competitiveness (MINECO CTQ2013-45875-R project).Yamanaka, E.; Tortajada-Genaro, LA.; Maquieira, A. (2017). Low-cost genotyping method based on allele-specific recombinase polymerase amplification and colorimetric microarray detection. Microchimica Acta. 184(5):1453-1462. https://doi.org/10.1007/s00604-017-2144-0S145314621845Manolio TA, Chisholm RL, Ozenberger B, Roden DM, Williams MS, Wilson R et al (2013) Implementing genomic medicine in the clinic: the future is here. Genitourin Med 15:258–267Scott SA (2013) Clinical pharmacogenomics: opportunities and challenges at point-of-care. Clin Pharmacol Ther 93:33Limaye N (2013) Pharmacogenomics, Theranostics and personalized medicine-the complexities of clinical trials: challenges in the developing world. Appl Transl Genomics 2:17–21Abul-Husn NS, Owusu Obeng A, Sanderson SC, Gottesman O, Scott SA (2014) Implementation and utilization of genetic testing in personalized medicine. Pharmacogenomics Pers Med 7:227–240Knez K, Spasic D, Janssen KP, Lammertyn J (2014) Emerging technologies for hybridization based single nucleotide polymorphism detection. Analyst 139:353–370Shen W, Tian Y, Ran T, Gao Z (2015) Genotyping and quantification techniques for single-nucleotide polymorphisms. TrAC Trends Anal Chem 69:1–13Milbury CA, Li J, Makrigiorgos GM (2009) PCR-based methods for the enrichment of minority alleles and mutations. Clin Chem 55:632–640Asari M, Watanabe S, Matsubara K, Shiono H, Shimizu K (2009) Single nucleotide polymorphism genotyping by mini-primer allele-specific amplification with universal reporter primers for identification of degraded DNA. Anal Biochem 386:85–90Taira C, Matsuda K, Yamaguchi A, Sueki A, Koeda H, Takagi F, Kobayashi Y, Sugano M, Honda T (2013) Novel high-speed droplet-allele specific-polymerase chain reaction: application in the rapid genotyping of single nucleotide polymorphisms. Clin Chim Acta 424:39–46Tortajada-Genaro LA, Mena S, Niñoles R, Puigmule M, Viladevall L, Maquieira A (2016) Genotyping of single nucleotide polymorphisms related to attention-deficit hyperactivity disorder. Anal Bioanal Chem 408:2339–2345Woolley CF, Hayes MA (2014) Emerging technologies for biomedical analysis. Analyst 139:2277–2288Craw P, Balachandran W (2012) Isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review. Lab Chip 12:2469–2486Zhang L, Zhang Y, Wang C, Feng Q, Fan F, Zhang G, Kang X, Qin X, Sun J, Li Y, Jiang X (2014) Integrated microcapillary for sample-to-answer nucleic acid pretreatment, amplification, and detection. Anal Chem 86:10461–10466Chen F, Zhao Y, Fan C, Zhao Y (2015) Mismatch extension of DNA polymerases and high-accuracy single nucleotide polymorphism diagnostics by gold nanoparticle-improved isothermal amplification. Anal Chem 87:8718–8723Li J, Macdonald J (2015) Advances in isothermal amplification: novel strategies inspired by biological processes. Biosens Bioelectron 64:196–211Santiago-Felipe S, Tortajada-Genaro LA, Morais S, Puchades R, Maquieira A (2014) One-pot isothermal DNA amplification–hybridisation and detection by a disc-based method. Sens Actuator B-Chem 204:273–281Santiago-Felipe S, Tortajada-Genaro LA, Puchades R, Maquieira Á (2016) Parallel solid-phase isothermal amplification and detection of multiple DNA targets in microliter-sized wells of a digital versatile disc. Microchim Acta 183:1195–1202Tortajada-Genaro LA, Santiago-Felipe S, Amasia M, Russom A, Maquieira A (2015) Isothermal solid-phase recombinase polymerase amplification on microfluidic digital versatile discs (DVDs). RSCAdv 5:29987–29995Li Z, Liu Y, Wei Q, Liu Y, Liu W, Zhang X, Yu Y (2016) Picoliter well Array Chip-based digital recombinase polymerase amplification for absolute quantification of nucleic acids. PLoS One 11:e0153359Daher RK, Stewart G, Boissinot M, Boudreau DK, Bergeron MG (2015) Influence of sequence mismatches on the specificity of recombinase polymerase amplification technology. Mol Cell Probes 29:116–121Shin Y, Perera AP, Kim KW, Park MK (2013) Real-time, label-free isothermal solid-phase amplification/detection (ISAD) device for rapid detection of genetic alteration in cancers. Lab Chip 13:2106–2114NgePN RCI, Woolley AT (2013) Advances in microfluidic materials, functions, integration, and applications. Chem Rev 113:2550–2583Bhattacharjee N, Urrios A, Kang S, Folch A (2016) The upcoming 3D-printing revolution in microfluidics. Lab Chip 16:1720–1742Waheed S, Cabot JM, Macdonald NP, Lewis T, Guijt RM, Paull B, Breadmore MC (2016) 3D printed microfluidic devices: enablers and barriers. Lab Chip 16:1993–2013Bierut LJ, Madden PA, Breslau N, Johnson EO, Hatsukami D, Pomerleau OF, Swan GE, Rutter J, Bertelsen S, Fox L, Fugman D, Goate AM, Hinrichs AL, Konvicka K, Martin NG, Montgomery GW, Saccone NL, Saccone SF, Wang JC, Chase GA, Rice JP, Ballinger DG (2007) Novel genes identified in a high-density genome wide association study for nicotine dependence. Hum MolGen 16:24–35Carpenter MJ, Jardin BF, Burris JL, Mathew AR, Schnoll RA, Rigotti NA, Cummings KM (2013) Clinical strategies to enhance the efficacy of nicotine replacement therapy for smoking cessation: a review of the literature. Drugs 73:407–426Moody C, Newell H, Viljoen H (2016) FA mathematical model of recombinase polymerase amplification under continuously stirred conditions. Biochem Eng J 112:193–201Dimitrov RA, Zuker M (2004) Prediction of hybridization and melting for double-stranded nucleic acids. Biophys J 87:215–226Zhang C, Xing D (2007) Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends. Nucleic Acids Res 35:4223–4237Liu B, Huang PJJ, Zhang X, Wang F, Pautler R, IpACF LJ (2013) Parts-per-million of polyethylene glycol as a non-interfering blocking agent for homogeneous biosensor development. Anal Chem 85:10045–10050Wu J, Kodzius R, Cao W, Wen W (2014) Extraction, amplification and detection of DNA in microfluidic chip-based assays. Microchim Acta 181:1611–1631Li J, Wang L, Mamon H, Kulke MH, Berbeco R, Makrigiorgos GM (2008) Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nat Med 14:579–584Shen R, Fan JB, Campbell D, Chang W, Chen J, Doucet D, Yeakley J, Bibikova M, Garcia EW, McBride C, Steemers F, Garcia F, Kermani BG, Gunderson K, Oliphant A (2005) High-throughput SNP genotyping on universal bead arrays. Mut Res Fund Mol M 573:70–82David SP, Strong DR, Leventhal AM, Lancaster MA, McGeary JE, Munafò MR, Bergen AW, Swan GE, Benowitz NL, Tyndale RF, Conti DV, Brown RA, Lerman C, Niaura R (2013) Influence of a dopamine pathway additive genetic efficacy score on smoking cessation: results from two randomized clinical trials of bupropion. Addiction 108:2202–221

Meconio y exposición prenatal a neurotóxicos

6 pages, 1 figures, 2 tables.[ESP] Introducción. La ubicuidad con la que se encuentran la mayoría de las substancias neurotóxicas en el medio ambiente implica a los pediatras en la necesidad de desarrollar métodos para medir la magnitud de la exposición durante los periodos vulnerables del desarrollo. Una forma útil de abordar este problema consiste en analizar muestras biológicas que acumulen las substancias neurotóxicas o sus metabolitos durante el periodo fetal.Método. Revisión bibliográfica sistemática de los últimos 20 años obtenida principalmente de Medline; Science Citation Index y Embase sobre los estudios con meconio como matriz de exposición prenatal a substancias neurotóxicas. El perfil de búsqueda utilizado fue: meconium, prenatal exposure, biological markers, matrices, environmental pollutants, nervous system poisonings, neurotoxicity sindromes. Hemos seleccionado los trabajos más importantes y de sus referencias se han obtenido los más relevantes de los años previos a la búsqueda.Resultados. Tradicionalmente, los esfuerzos para determinar la exposición fetal se han centrado en el análisis de sangre de cordón u orina de la madre o el neonato. El meconio es fácilmente disponible, es inerte, acumula los neurotóxicos y/o sus metabolitos desde la semana 12 de gestación donde quedan “fosilizados” hasta el nacimiento. Puede constituir un instrumento muy importante para investigar la exposición fetal a los distintos contaminantes ambientales y en particular a neurotóxicos.Conclusiones. Las exposiciones fetales a los distintos neurotóxicos estudiados a través de sangre materna, de cordón, pelo, uña, placenta y orina parecen ser menos predictivas sobre los efectos neurológicos que las mediciones de los mismos realizadas en meconio. Son necesarios más estudios en este campo.Implementar y desarrollar la medida en meconio de una amplia gama de sustancias neurotóxicas ayudará en la práctica pediátrica a una intervención e identificación temprana mostrando las exposiciones que puedan provocar daño y facilitando el desarrollo de medidas preventivas y rehabilitadoras.[ENG] Brackground. The environmental ubiquity of most neurotoxicants implies the pediatricians in the development of methods for exposure measurement during the vulnerable periods of development. The analysis of biological samples able to accumulate the neurotoxicant substances or its metabolites during the fetal period is a useful approach to fulfil this objective.Material and methods. A systematic literature review of the last 20 years in Medline, Science Citation Index and Embase on the studies with meconium like womb of prenatal exposure to neurotoxicants has been undertaken. The search profile was: “meconium”, “prenatal exposure”, “biological markers”, “matrices”, “environmental pollutants”, “nervous system poisonings”, “neurotoxicity sindromes”. We selected the most relevant articles and retrieved more from their references.Results. Traditionally, the efforts to determine the fetal exposure have been centered in the analysis of cord blood, urinates of the mother or of the neonato. Meconium is easily available, inert, accumulates the neurotoxicants and/or its metabolitos from week 12 of gestation where they are "fossilized" until the birth. It can constitute a very important instrument for the investigation of the fetal exposure to the different environmental pollutants and in particular to neurotoxicants.Conclusions. Foetal exposure to different neurotoxicants monitored from maternal blood, cord blood, hair, fingernail, placenta and urinates seem to be less predictive for neurological effects than meconium. However, more studies are needed to confirm this hypothesis.Implementation and measurement in meconium of a wide range of neurotoxic substances will be of help in the pediatric practice for intervention and early identification as it will reveal harmful exposures and facilitate the implementation of preventive measures.Los autores quieren expresar su agradecimiento a los miembros de la red de Investigación Colaborativa INMA, y en especial a Amparo Quiles Latorre, Elena Romero Aliaga y Sandra Pérez Aliaga, por su apoyo y colaboración en la realización del trabajo de campo; al equipo de enfermería de la 7ª, 8ª y 9ª de la maternidad del Hospital Materno-Infantil Universitario La Fe y a los recién nacidos y sus felices padres que con su colaboración y entusiasmo hacen posible llevar a término estos estudios.Peer reviewe

Consumer electronics devices for DNA genotyping based on loop-mediated isothermal amplification and array hybridisation

[EN] Consumer electronic technologies offer practical performances to develop compact biosensing systems intended for the point-of-care testing of DNA biomarkers. Herein a discrimination method for detecting single nucleotide polymorphisms, based on isothermal amplification and on-chip hybridisation, was developed and integrated into user-friendly optical devices: e.g., USB digital microscope, flatbed scanner, smartphone and DVD drive. In order to adequately identify a single base change, loop-mediated isothermal amplification (LAMP) was employed, with high yields (8 orders) within 45 min. Subsequently, products were directly hybridised to the allele-specific probes attached to plastic chips in an array format. After colorimetric staining, four consumer electronic techniques were compared. Sensitive precise measurements were taken (high signal-to-noise ratios, 10-mu m image resolution, 99% scan-to-scan reproducibility). These features confirmed their potential as analytical tools, are a competitive alternative to fluorescence scanners, and incorporate additional advantages, such as user-friendly interface and connectivity for telemedicine needs. The analytical performances of the integrated platform (assay and reader) in the human samples were also excellent, with a low detection limit (100 genomic DNA copies), and reproducible (< 15%) and cheap assays (< 10 (sic)/test). The correct genotyping of a genetic biomarker (single-nucleotide polymorphism located in the GRIK4 gene) was achieved as the assigned genotypes agreed with those determined by using sequencing. The portability, favourable discriminating and read-out capabilities reveal that the implementation of mass-produced low-cost devices into minimal-specialised clinical laboratories is closer to becoming a reality.The authors acknowledge the financial support received from the Generalitat Valenciana, Spain (GVA-PROMETEOII/2014/040 Project and GRISOLIA/2014/024 Ph.D. grant) and the Spanish Ministry of Economy and Competitiveness, Spain (MINECO CTQ2016-75749-R project) by U. E. FEDER funds. The authors also thank J. Carrascosa for supporting the DVD reader measurements.Tortajada-Genaro, LA.; Yamanaka, ES.; Maquieira Catala, A. (2019). Consumer electronics devices for DNA genotyping based on loop-mediated isothermal amplification and array hybridisation. Talanta. 198:424-431. https://doi.org/10.1016/j.talanta.2019.01.124S42443119

Cocaína, violencia y género desde el punto de vista de los profesionales

Objetivo: El presente estudio pretende conocer los comportamientos violentos de hombres y mujeres consumidores de cocaína desde la perspectiva de los profesionales del área de drogodependencias y la violencia.Metodología: Se utilizó metodología cualitativa, concretamente se realizaron entrevistas semiestructuradas a una muestra representativa de profesionales de Unidades de Conductas Adictivas (UCA) y de Centros Mujer 24 Horas (CM24H) de la Comunitat Valenciana. El análisis de los datos, a excepción de las preguntas cerradas que se analizaron estadísticamente, se realizó siguiendo la Grounded Theory, lo que implica un método de comparaciones constantes que intenta generar teoría a partir de los datos empíricos y ofrece la posibilidad de comprensión del fenómeno desde el interior del mismo.Resultados: En opinión de los profesionales los hombres manifiestan más violencia de tipo físico que las mujeres, siendo más probable que éstas manifiesten su violencia verbalmente. Las conductas violentas suelen ir dirigidas hacia la pareja en el caso de los hombres y en el caso de las mujeres hacia sus hijos. Los hombres cocainómanos tienen más redes de apoyo en el proceso de tratamiento, normalmente reciben soporte de su pareja, no ocurriendo así en el caso de las mujeres cocainómanas que normalmente comienzan el tratamiento por voluntad propia y son apoyadas por algún miembro de su familia, que normalmente suele ser una mujer. Es habitual la existencia de violencia durante el proceso de tratamiento, pues las mujeres que se encuentran incluidas en estos programas de tratamiento suelen ser víctimas de violencia.Discusión: La relación entre consumo de cocaína y violencia es muy compleja, en este estudio se indica que las conductas violentas entre consumidores de cocaína pueden variar según género. Es importante continuar la tarea investigadora para profundizar en estas diferencias y mejorar así la calidad de los programas asistenciales dirigidos a esta población

El emprendimiento como propuesta de cambio y herramienta clave para acercar la formación universitaria a la realidad social del mercado laboral

La discusión relativa a si la formación universitaria prepara para la realidad del mercado laboral, siempre ha despertado gran interés en nuestra sociedad y es una necesidad sin resolver en la actualidad. A pesar de los cambios que ha traído consigo la convergencia europea a la Universidad española, la formación de nuestros estudiantes sigue careciendo de las herramientas que les demanda la sociedad del siglo XXI y el mundo empresarial. El espíritu emprendedor transmitido por los docentes en las aulas, respondería a esa necesidad y ayudaría a ese acercamiento al mundo laboral de los estudiantes universitarios que tanto se demanda. Pero la capacidad emprendedora ¿es enseñable? ¿Se puede evaluar? La revisión de la literatura más reciente es ambigua respecto a qué evaluar (actitud, valor, competencia, habilidad, rasgo de personalidad, intención), y eso dificulta el diseño de planes efectivos de desarrollo del emprendimiento entre nuestro alumnado. El trabajo que presentamos se posiciona razonadamente en el aspecto a medir y plantea una propuesta de innovación dirigida a fomentar en los alumnos universitarios las competencias emprendedoras, y junto a ellas, la inteligencia emocional, la felicidad y la creatividad como elementos facilitadores del rendimiento académico y herramientas clave para su incorporación al mercado laboral.The discussion on the potential role of Higher education to prepare for the labour market has always given a great interest in our society and it is an unresolved need today. Despite of the changes promoted by the European convergence process to the Spanish universities, the student training still lacks the skills demanded by the 21st century society and the business world. The entrepreneurial spirit transmitted by the teachers in the classrooms would respond to this social necessity and would help to introduce university students to the working world that is in a high demand nowadays. But is the entrepreneurial capacity teachable? Can it be evaluated? The review of the most recent literature is ambiguous about its evaluation (attitude, value, competence, ability, personality trait, intention), and this difficulties an effective design about entrepreneurship development plans among our students. The work we present is reasonably positioned in the aspect to be measured and offers a proposal for innovation aimed to encourage the entrepreneurial competences in the university students, and taken together, the emotional intelligence, happiness and creativity as facilitator elements for a higher academic performance and as key tools for their incorporation into the labour market

Adult diagnosis of Swyer-James-MacLeod syndrome: a case report

<p>Abstract</p> <p>Introduction</p> <p>Swyer-James-MacLeod syndrome or unilateral hyperlucent lung syndrome is a rare entity associated with postinfectious bronchiolitis obliterans occurring in childhood. It is characterized by hypoplasia and/or agenesis of the pulmonary arteries resulting in pulmonary parenchyma hypoperfusion.</p> <p>Case presentation</p> <p>Here we report the case of a 53-year-old Caucasian woman with Swyer-James-MacLeod syndrome found in the differential diagnosis workup for a new onset of heart failure, secondary to pulmonary arterial hypertension complicated by a patent ductus arteriosus.</p> <p>Conclusion</p> <p>Typically, this disorder is diagnosed in childhood after evaluation for recurrent respiratory infections, but sometimes an indolent course means diagnosis is not made until adulthood.</p

One-pot isothermal DNA amplification Hybridisation and detection by a disc-based method

[EN] An integrated sensor comprising isothermal DNA amplification and in situ detection is presented. The method principle is based on recombinase polymerase amplification (RPA) and detection in the microarray format by compact disc technology as a high-throughput sensing platform. Primers were immobilised on the polycarbonate surface of digital versatile discs (DVD) and, after hemi-nested amplification, multiplexing identification of each tethered product was achieved by optical scanning with a 650 nm-laser of the DVD drive. The efficiency of one-pot hybridisation/elongation/detection depended strongly on probedensity and other factors such as the concentration of the unbound primers present in solution. The optimised conditions provided equivalent amplification factors (7.3 x 10(8) -8.9 x 10(8) fold) to those obtained by conventional reactions performed in vials. The proposed method was applied to Salmonella detection (generic by hns and oriC genes, and specific for subspecies I by STM4507 gene). A triplex assay was satisfactorily compared to the non-integrated protocols. Food and vaccine samples were analysed in a shorter time with less handling. The results indicate that the multiplex DVD assay is a simple, competitive, isothermal, portable system that is particularly useful for microbiological routine analysis. (C) 2014 Elsevier B.V. All rights reserved.This research has been funded through Projects GVA-PROMETEO/2010/008 (Generalitat Valenciana) and CTQ/2013/ 45875-R (MINECO). The Spanish Ministry of Education and Science provided S.S.F. with a grant for her PhD studies.Santiago Felipe, S.; Tortajada-Genaro, LA.; Morais, S.; Puchades, R.; Maquieira Catala, Á. (2014). One-pot isothermal DNA amplification Hybridisation and detection by a disc-based method. Sensors and Actuators B: Chemical. 204:273-281. https://doi.org/10.1016/j.snb.2014.07.073S27328120

HEX17(Neumifil) : an intranasal respiratory biotherapeutic with broad-acting antiviral activity

Funding: This research has been funded by Pneumagen Ltd.Broad-acting antiviral strategies to prevent respiratory tract infections are urgently required. Emerging or re-emerging viral diseases caused by new or genetic variants of viruses such as influenza viruses (IFVs), respiratory syncytial viruses (RSVs), human rhinoviruses (HRVs), parainfluenza viruses (PIVs) or coronaviruses (CoVs), pose a severe threat to human health, particularly in the very young or old, or in those with pre-existing respiratory conditions such as asthma or chronic obstructive pulmonary disease (COPD). Although vaccines remain a key component in controlling and preventing viral infections, they are unable to provide broad-spectrum protection against recurring seasonal infections or newly emerging threats. HEX17 (aka Neumifil), is a first-in-class protein-based antiviral prophylactic for respiratory viral infections. HEX17 consists of a hexavalent carbohydrate-binding module (CBM) with high affinity to sialic acids, which are typically present on terminating branches of glycans on viral cellular receptors. This allows HEX17 to block virus engagement of host receptors and inhibit infection of a wide range of viral pathogens and their variants with reduced risk of antiviral resistance. As described herein, HEX17 has demonstrated broad-spectrum efficacy against respiratory viral pathogens including IFV, RSV, CoV and HRV in multiple in vivo and in vitro studies. In addition, HEX17 can be easily administered via an intranasal spray and is currently undergoing clinical trials.Peer reviewe

Hepatitis A virus vaccine escape variants and potential new serotype emergence.

Six hepatitis A virus antigenic variants that likely escaped the protective effect of available vaccines were isolated, mostly from men who have sex with men. The need to complete the proper vaccination schedules is critical, particularly in the immunocompromised population, to prevent the emergence of vaccine-escaping variants