Naked Eye Y Amelogenin Gene Fragment Detection Using DNAzymes on a Paper-based Device

Nowadays, there are strong efforts in developing new technology for rapid detection of specific DNA sequences for environmental monitoring, forensic analysis and rapid biomedical diagnosis applications. That is where microfluidic paper-based analytical devices are positioned as suitable platforms for the development of point of care analytical devices, due to their simple fabrication protocols, ease of use and low cost. Herein, a methodology for in situ single strand DNA detection by using a colorimetric assay based on the formation of a DNAzyme within a paper substrate was developed. A DNAzyme that could only be formed in the presence of a specific sequence of the Y human amelogenin gene was designed. The performance of the DNAzyme was followed colorimetrically first in solution and then in paper substrates. The reaction was found to be specific to the Y fragment selected as analyte. The DNAzyme reaction on paper enabled the unequivocal colorimetric identification of the Y single strand DNA fragment both qualitatively, with the naked eye (143 ng), and quantitatively by image analysis (45.7 ng). As a proof of concept, a microfluidic paper-based device, pre-loaded with all DNAzyme reagents, was characterized and implemented for the simultaneous detection of X and Y single strand DNA fragments.This work was supported by Gobierno de España, Ministerio de Economía y Competitividad, with Grant No. BIO2016-80417-P; funding from Basque Government (Grant: IT1271-19) and European Union funds: DNASURF (H2020-MSCA-RISE-778001). E.A.-H. acknowledges funding from the Basque Government, Department of Education, for predoctoral fellowship 2016

Beta-Glucans Improve Growth, Viability and Colonization of Probiotic Microorganisms

Probiotics, prebiotics and synbiotics are frequently-used components for the elaboration of functional food. Currently, most of the commercialized probiotics are limited to a few strains of the genera Bifidobacteria, Lactobacillus and Streptococcus, most of which produce exopolysaccharides (EPS). This suggests that the beneficial properties of these microorganisms may be related to the biological activities of these biopolymers. In this work we report that a 2-substituted-(1,3)-β-d-glucan of non-dairy bacterial origin has a prebiotic effect on three probiotic strains. Moreover, the presence of this β-d-glucan potentiates in vitro adhesion of the probiotic Lactobacillus plantarum WCFS1 to human intestinal epithelial cells

Biodegradation of ochratoxin A by Pediococcus parvulus isolated from Douro wines

Lactic acid bacteria (LAB) are a promising solution to reduce exposure to dietary mycotoxins because of the unique mycotoxin decontaminating characteristic of some LAB. Ochratoxin A (OTA) is one of the most prominent mycotoxins found in agricultural commodities. The present work reports on the ability of Pediococcus parvulus strains that were isolated from Douro wines that spontaneously underwent malolactic fermentation to detoxify OTA. These strains were identified and characterised using a polyphasic approach that employed both phenotypic and genotypic methods. When cultivated on OTA-supplemented MRS media, OTA was biodegraded into OTα by certain P. parvulus strains. The presence of OTα was confirmed using LC-MS/MS. The conversion of OTA into OTα indicates that the OTA amide bond was hydrolysed by a putative peptidase. The rate of OTA biodegradation was found to be dependent on the inoculum size and on the incubation temperature. Adsorption assays with dead P. parvulus cells showed that approximately 1.3% ± 1.0 of the OTA was adsorbed onto cells wall, which excludes this mechanism in the elimination of OTA by strains that degrades OTA. Under optimum conditions, 50% and 90% of OTA was degraded in 6 and 19 h, respectively. Other LAB strains that belonged to different species were tested but did not degrade OTA. OTA biodegradation by P. parvulus UTAD 473 was observed in grape must. Because some P. parvulus strains have relevant probiotic properties, the strains that were identified could be particularly relevant to food and feed applications to counteract the toxic effects of OTA.This work was funded by FEDER funds through the "Programa Operacional Factores de Competitividade - COMPETE" and by national funds through "Fundacao para a Ciencia e a Tecnologia - FCT", Ref. FCOMP-01-0124-FEDER-028029 and PTDC/AGR-TEC/3900/2012, respectively. The authors also thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013 and the Project "BioInd - Biotechnology and Bioengineering for improved Industrial and Agro-Food processes, REF. NORTE-07-0124-FEDER-000028" co-funded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER. Luis Abrunhosa was supported by the grant SFRH/BPD/43922/2008 from FCT

Bacterias lácticas de sidra natural: implicación en alteraciones y potencial prebiótico de cepas productoras de (1,3)(1,2)-ß-D-glucanos

Esta investigación estudia una población de niños que nacieron prematuros con muy bajo peso (<1500gr) a la edad de 2 años (edad corregida) comparando su nivel de desarrollo psicomotor y las características del vínculo madre-niño, con una muestra equivalente de niños nacidos sanos a término. Los objetivos son: valorar las diferencias en el tipo de vínculo o de apego madre-hijo y comparar los índices de desarrollo de ambas poblaciones. Finalmente, evaluar la existencia de posibles asociaciones entre los índices de desarrollo y el tipo de vínculo y estudiar las influencias de los antecedentes médicos, y las características personales y familiares. Es un estudio observacional de cohortes retrospectivas. La muestra de prematuros incluye los nacidos en el Hospital de Cruces (Bizkaia) durante 2 años y que están en el programa de seguimiento del Servicio de Neonatología de dicho centro (N=90) y se comparan con 96 niños que nacieron sanos a término con edad y características sociodemográficas similares. Los prematuros estudiados presentaron una inmadurez y riesgo biológico de grado medio-severo: EG media 29,98 sem, PN 1.159,76 gr, y 57% más de 1 semana de hospitalización en UCI. No se encuentran diferencias significativas entre los subgrupos de alto riesgo (al menos 2 de estás 3 características: PN <1000gr, EG<29sem, más de 7 dias en UCI) y bajo riesgo. Hay diferencias significativas en los índices de desarrollo, más bajos en los prematuros, pero las puntuaciones de ambos están dentro de la normalidad. Las madres de prematuros presentan más estrés durante el primer año de su hijo (61%) comparando con el grupo control (39%). No hay diferencias entre las madres de ambas poblaciones en el modelo representacional de apego: el porcentaje de modelo vincular inseguro es similar. Los niños prematuros presentan más problemas de alimentación y más trastornos de comportamiento externalizado que el grupo de no prematuros a los 2 años de edad.Esta tesis ha sido financiada por una beca predoctoral de formación de investigadores del Departamento de Agricultura, Pesca y Alimentación del Gobierno Vasco y se ha realizado en la Unidad de Microbiología del departamento de Química Aplicada de la Facultad de Ciencias Químicas de la Universidad de País Vasco/Euskal Herriko Unibertsitatea (UPV/EHU) en Donostia.Peer Reviewe

Unconventional Application of Conventional Enzymatic Substrate: First Fluorogenic Immunoassay Based on Enzymatic Formation of Quantum Dots

In this study, a simple fluorogenic immunoassay based on in situ formation of semiconductor quantum dots (QDs) is described. We discovered that alkaline phosphatase (ALP), the enzyme broadly used in enzyme-linked immuno-sorbent assay (ELISA), is able to trigger formation of fluorescent CdS QDs. ALP-catalyzed hydrolysis of <i>p</i>-nitrophenyl phosphate (pNPP) leads to the formation of <i>p</i>-nitrophenol and inorganic phosphate. The latter stabilizes CdS QDs produced in situ through interaction of Cd²⁺ with S^2– ions. So, the specific interaction of analyte (antibody) with ALP-labeled antibody can be detected through formation of CdS QDs, monitored by recording emission spectra at λ_ex = 290 nm. The fluorescence intensity showed to be dependent on the concentration of target antibody. This method allowed us to detect as low as 0.4 ng mL^–1 of analyte antibody with a linear range up to 10 ng mL^–1. The sensitivity of this novel assay showed to be 1 order of magnitude better than that of the standard method based on colorimetric <i>p</i>-nitrophenyl phosphate assay

Enzymatic Product-Mediated Stabilization of CdS Quantum Dots Produced <i>In Situ</i>: Application for Detection of Reduced Glutathione, NADPH, and Glutathione Reductase Activity

Glutathione is the most abundant nonprotein molecule in the cell and plays an important role in many biological processes, including the maintenance of intracellular redox states, detoxification, and metabolism. Furthermore, glutathione levels have been linked to several human diseases, such as AIDS, Alzheimer disease, alcoholic liver disease, cardiovascular disease, diabetes mellitus, and cancer. A novel concept in bioanalysis is introduced and applied to the highly sensitive and inexpensive detection of reduced glutathione (GSH), over its oxidized form (GSSG), and glutathione reductase (GR) in human serum. This new fluorogenic bioanalytical system is based on the GSH-mediated stabilization of growing CdS nanoparticles. The sensitivity of this new assay is 5 pM of GR, which is 3 orders of magnitude better than other fluorogenic methods previously reported

Naturally occurring 2-substituted (1,3)-β-D-glucan producing Lactobacillus suebicus and Pediococcus parvulus strains with potential utility in the production of functional foods

35 p.-4 fig.-2 fig. supl.We have isolated three lactic acid bacteria (Lactobacillus suebicus CUPV221, Pediococcus parvulus CUPV1 and P. parvulus CUPV22) that produced high levels of 2-substituted (1,3)-β-d-glucans which increased the viscosity of the growth media. The (1,3)-β-d-glucan consisted of two main molecular species, with masses of approximately 107 and 104 Da, whose proportions varied among the strains. The three strains survived exposure to saliva and simulated gastric conditions at pH 5, with P. parvulus CUPV22 surviving at pH 3.1, and L. suebicus CUPV221 surviving at pH 1.8. All strains were resistant to pancreatin and bile salts. P. parvulus CUPV22 exhibited the highest adhesion (10.5%) to Caco-2 cells, which decreased to 1.2% after washing the cells. Finally, P. parvulus CUPV22 and L. suebicus CUPV221 induced the production of inflammation-related cytokines by polarized macrophages, and interestingly, L. suebicus stimulated the production of cytokine IL-10. These results indicate that the three strains have potential utility for the production of functional foodsThis work was supported by the Ministerio de Ciencia e Innovación (Projects AGL2006-11932 and AGL2009-12998, Subprojects 01 and 02), the Universidad del País Vasco (UPV/EHU) (EHU08/37) and the Diputación Foral de Gipuzkoa, Programa Red Gipuzkoana de Ciencia, Tecnología e Innovación (co-financed by the European Union). Gaizka Garai-Ibabe acknowledges the Gobierno Vasco (Dpto. Agricultura, Pesca y Alimentación) for the pre-doctoral fellowshipPeer reviewe

Peroxidase-Mimicking DNAzyme Modulated Growth of CdS Nanocrystalline Structures in Situ through Redox Reaction: Application to Development of Genosensors and Aptasensors

This work demonstrates the use of the peroxidase-mimicking DNAzyme (peroxidase-DNAzyme) as general and inexpensive platform for development of fluorogenic assays that do not require organic fluorophores. The system is based on the affinity interaction between the peroxidase-DNAzyme bearing hairpin sequence and the analyte (DNA or low molecular weight molecule), which changes the folding of the hairpin structure and consequently the activity of peroxidase-DNAzyme. Hence, in the presence of the analyte the peroxidase-DNAzyme structure is disrupted and does not catalyze the aerobic oxidation of l-cysteine to cystine. Thus, l-cysteine is not removed from the system and the fluorescence of the assay increases due to the in situ formation of fluorescent CdS nanocrystals. The capability of the system as a platform for fluorogenic assays was demonstrated through designing model geno- and aptasensor for the detection of a tumor marker DNA and a low molecular weight analyte, adenosine 5′triphosphate (ATP), respectively