NGS y Metagenómica

The Next Generation Sequencing (NGS) allows to sequence the whole genome of an organism, compared to Maxam and Gilbert and Sanger sequencing that only allow to sequence, hardly, a single gene. Removing the separation of DNA fragments by electrophoresis, and the development of techniques that let the parallelization (analysing simultaneously several DNA fragments) have been crucial for the improvements of this process. The new companies in this ambit, Roche and Illumina, bet for different protocols to achieve these goals. Illumina bets for the sequencing by synthesis (SBS), requiring the library preparation and the use of adapters. Likewise, Illumina has replaced Roche because its lower rate of misincorporation, making it ideal for studies of genetic variability, transcriptomic, epigenomic, and metagenomic, in which this study will focus. However, it is noteworthy that the last progress in sequencing is carried out by the third generation sequencing, using nanotechnology to design small sequencers that sequence the whole genome of an organism quickly and inexpensively. Moreover, they provide more reliable data than current systems because they sequence a single molecule, solving the problem of synchronisation. In this way, PacBio and Nanopore allow a great progress in diagnostic and personalized medicine. Metagenomics provide to make a qualitative and quantitative analysis of the various species present in a sample. The main advantage of this technique is the no necessary isolation and growth of the species, allowing the analysis of nonculturable species. The Illumina protocol studies the variable regions of the 16S rRNA gene, which contains variable and not variables regions providing a phylogenetic classification. Therefore, metagenomics is a topic of interest to know the biodiversity of complex ecosystems and to study the microbiome of patients given the high involvement with certain microbial profiles on the condition of certain metabolic diseases.

Positive biocompatibility of nanocrystalline glass-like carbon thin films

The interest in carbon nanomaterials with high transparency and electrical conductivity has grown within the last decade in view of a wide variety of applications, including biocompatible sensors, diagnostic devices and bioelectronic implants. The aim of this work is to test the biocompatibility of particular nanometer-thin nanocrystalline glass-like carbon films (NGLC), a disordered structure of graphene flakes joined by carbon matrix (Romero et al., 2016). We used a cell line (SN4741) from substantia nigra dopaminergic cells derived from transgenic mouse embryo cells (Son et al., 1999). Some cells were cultured on top of NGLC films (5, 20 and 80 nm) and other with NGLC nanoflakes (approx. 5-10 mm2) in increasing concentrations: 1, 5, 10, 20 and 50 μg/ml, during 24 h, 3 days and 7 days. Cells growing in normal conditions were defined under culture with DMEM supplemented with 10% FCS, Glucose (0,6%), penicillin-streptomycin (50U/ml) and L-glutamine (2mM) at 5%CO2 humidified atmosphere. Nanoflakes were resuspended in DMEM at the stock concentration (2 g/l). The experiments were conducted in 96 well plates (Corning) using 2500 cells per well. For MTT analysis, the manufacturer recommendations were followed (Roche, MTT kit assay): a positive control with a 10% Triton X-100 treatments (15 minutes) and a negative control without neither Triton X-100 nor NGLC. As apoptosis/necrosis assay we used LIVE/DEAD® Viability/Cytotoxicity Assay Kit (Invitrogen). In a separate experiment, cells were cultured on top of the NGLC films for 7 days. Primary antibodies: anti-synaptophysin (SYP, clone SY38, Chemicon) and goat anti-GIRK2 (G-protein-regulated inward-rectifier potassium channel 2 protein) (Abcom) following protocol for immunofluorescence. WB for proteins detection performed with a polyclonal anti-rabbit proliferating cell nuclear antigen (PCNA). Results demonstrated the biocompatibility with different concentration of NGLC varying the degree of survival from a low concentration (1 mg/ml) in the first 24 h to high concentrations (20-50 g/ml) after 7 days as it is corroborated by the PCNA analysis. Cells cultured on top of the film showed after 7 days axonal-like alignment and edge orientation as well as net-like images. Neuronal functionality was demonstrated to a certain extent through the analysis of coexistence between SYP and GIRK2. In conclusion, this nanomaterial could offer a powerful platform for biomedical applications such as neural tissue engineering