Biology subjects

Lopez Diaz, M.

Publications and source records attributed to Lopez Diaz, M..

3 recordsLinked to original sources

bioRxiv2022

Application of RT-LAMP-CRISPR-Cas13a technology to the detection of OXA-48 producing Klebsiella pneumoniae

Carbapenem-resistant pathogens have been recognized as a health concern because of their ability to cause severe infections and because they are difficult to detect in laboratories. Researchers are making great efforts to develop a diagnostic technique with high levels of sensitivity and specificity, as accurate, early diagnosis is required to prevent the spread of these microorganisms and improve the prognosis of patients. In this context, CRISPR-Cas systems are proposed as promising tools for the development of diagnostic techniques due to their high specificity: Cas13 endonuclease discriminates single nucleotide changes and displays collateral activity against single-stranded RNA molecules. This technology is usually combined with isothermal pre-amplification reactions in order to increase the sensitivity of diagnosis. We have developed an RT-LAMP-CRISPR-Cas13a-based assay for the detection of Klebsiella pneumoniae OXA-48 producer strains in clinical samples without the need for RNA extraction. The assay exhibited 100 % specificity, sensitivity, positive predictive value and negative predictive value.

microbiology↗

bioRxiv2017

Novel methods of isolation and amplification of progenitor cells applied to avian primordial germ cells

BackgroundThe progenitor cells in adult tissues are scarce and have a great regenerative potential. In this study novel methods were used to improve the isolation and culture of the chicken primordial germ cells (PGCs) from stage X and HH 8-9 embryos. The cellular size and external glycoprotein envelope were the two criteria studied and used.\n\nResultsPGCs were segregated with high efficiency and purity, from stage X and HH 8-9 gross cell suspensions through cell strainers with 10 m of pore size. In embryos in toto, WGA Alexa 594 (affinity for N-acetylglucosamine) and Con A Alexa 488 (binding D-mannosyl) were used to characterize external polysaccharides of the PGCs. The PGCs in stage X embryos (zone pellucida), have predominately Nacetylglucosamine and later on, in HH 8-9 embryos (cephalic zone), -D mannosyl residues, in a specific manner. In coated plates with the appropriate lectin and in alkaline conditions, isolated cells from stage X and HH 8-9 embryos formed numerous clumped PGC-LCs with spherical shape \"germspheres\". In all isolates from single embryo, immunohistochemistry confirmed that they were PGCs and revealed that the \"germspheres\" were formed by hundreds of positive cells to VASA and SSEA-1. N-acethyl D+glucosamine supplementation to the culture media greatly enhances the amplification of isolated PGC-LCs.\n\nConclusionsThese gentle and quick strategies with high yields of PGCs can be potentially useful for many progenitor cells in Regenerative Medicine.

developmental biology↗

bioRxiv2016

Effective Cell Immunoablation in Undisrupted Developing Avian Embryos

In birds the construction of germline chimeras by grafting exogenous primordial germ cells (PGCs) during embryonic development is feasible since they migrate to the gonads through the blood. Up to date, the efficiencies are highly variable, in part dependent on the destruction of endogenous PGCs in the recipient embryo. We show an almost complete ablation of the endogenous PGCs in stage X embryos using a baby rabbit serum (BRS), with previous cellular signaling by specific antibodies (SSEA1). The application of the treatments, either on epiblast or subgerminaly, produced the reduction of the PGCs in the embryos in a dose dependent manner. No malformations or damages were detected in the treated embryos. However, subgerminal injection of this cocktail produced a massive cellular destruction in all embryos. Therefore, sequential application is a selective and effective method to produce receptor embryos. Nevertheless, it can also be highly destructive if the mixture is applied locally, this could be useful in the treatment of malignancies.\n\nSUMMARY STATEMENTAn immunosurgery procedure is described that yields an almost complete ablation of primordial germ cells in early developing chick embryos, thus increasing the expected rates of chimerism when foreign PGCs are grafted onto these embryos

developmental biology↗