Nucleotide-selective amplification and array-based detection for identifying multiple somatic mutations.

In the context of personalized and cost-effective treatment, knowledge of the mutational status of specific genes is advantageous to predict which patients are responsive to therapies. As an alternative to one-by-one detection or massive sequencing, the presented genotyping tool determines multiple polymorphic sequences that vary a single nucleotide. The biosensing method includes an effective enrichment of mutant variants and selective recognition by colorimetric DNA arrays. The proposed approach is the hybridization between sequence-tailored probes and products from PCR with SuperSelective primers to discriminate specific variants in a single locus. A fluorescence scanner, a documental scanner, or a smartphone captured the chip images to obtain spot intensities. Hence, specific recognition patterns identified any single-nucleotide change in the wild-type sequence overcoming qPCR methods and other array-based approaches. Studied mutational analyses applied to human cell lines provided high discrimination factors, the precision was 95%, and the sensitivity was 1% mutant of total DNA. Also, the methods showed a selective genotyping of the KRAS gene from tumorous samples (tissue and liquid biopsy), corroborating results by NGS. The developed technology supported on low-cost robust chips and optical reading provides an attractive pathway toward implementing fast, cheap, reproducible discrimination of oncological patients.

A genosensor for detecting single-point mutations in dendron chips after blocked recombinase polymerase amplification.

A biosensing system was developed to accurately detect a single nucleotide change in the target organism genome, integrating a selective isothermal amplification and a sensitive dendron-mediated DNA hybridization assay in the array format. The novelty arises from the coupling reactions of the dendron and its use as a crosslinker. The allele-specific probes were oligonucleotide-dendron conjugates prepared by fast and clean click-chemistry (thiol-yne reaction) and coupled onto the photo-activated surface of polycarbonate substrates (carbodiimide reaction). The output was forest-array chips with multipoint-site crosslinkers and compatible with current microarray fabrication technologies. The products of blocked recombinase polymerase amplification (blocked RPA), formed at 37 °C, were hybridized with attached probes for specific nucleotide genotyping. The developed approach exhibited sensitive recognition of DNA variants compared to chips based on linear crosslinkers (10-100 fold), showing excellent analytical performances for planar chip and fluidic formats. The methodology was successfully applied to detect the H1047R mutation in the PIK3CA gene (c.3140A > G) from clinical samples of human cancer tissues, the results being consistent with sequencing techniques. The colorimetric biosensing method was reliable, versatile, low cost, sensitive (detection limit genomic DNA: 0.02 ng µL-1), and specific (accuracy >95%).

Magnetic concentration of allele-specific products from recombinase polymerase amplification.

The studied challenge is the specific detection of low-abundant genomic variants that differ by a single nucleotide from the wild type. The combination of blocked recombinase polymerase amplification (RPA) and selective capture by probes immobilised on magnetic-core particles integrated into a flow system is presented. The sensing principle was demonstrated as the effective concentration-detection of the specific generated products was achieved. The analytical performance of resulting assay was successfully compared to PCR-based methods or array formats, providing faster effective detection of the selective products. As proof of concept, the single-nucleotide substitutions of the KRAS gene at codon 12 were studied in chip with parallel microchambers and permanent magnets. The blocked RPA products (generated at 37 °C) from tumour biopsies (extracted DNA 4 ng) provided a specific fluorescent bead-line that depends on the present mutation. The results agree with those reported by next-generation sequencing and provide new opportunities for in vitro diagnostic and personalised medicine.

Blocked recombinase polymerase amplification for mutation analysis of PIK3CA gene.

A blocked recombinase polymerase amplification (blocked-RPA) approach has been developed for the enrichment of mutated templates in heterogeneous specimens as tumor tissues. This isothermal amplification technique opens alternative solutions for meeting the technological demand of physician office laboratories. Herein, the detection of mutations in PIK3CA gene, such as p.E545K, and p.H1047L, is presented. The main element was an oligonucleotide (dideoxycytidine functionalized at 3'-end) which matched with wild-type sequence in the target locus. The amplification was performed operating at 37 °C during 40 min. The results demonstrated that the competition between the upstream primer and the blocker reduced the percentage of amplified wild-type allele, making the detection of the present mutation easier. For mutation discrimination, a fast hybridization assay was performed in microarray format on plastic chip and colorimetric detection. This approach enabled the reliable discrimination of specific mutations against a background of up to 95% wild-type DNA. The applicability of the method, based on the combination of blocked-RPA and low-cost chip hybridization, was successfully proven for the genotyping of various cancer cell lines as well as tumor tissues. The assignations agreed with those provided by next-generation sequencing. Therefore, these investigations would support a personalized approach to patient care based on the molecular signature of human cancers.

Human platelet-rich plasma improves the nesting and differentiation of human chondrocytes cultured in stabilized porous chitosan scaffolds.

The clinical management of large-size cartilage lesions is difficult due to the limited regenerative ability of the cartilage. Different biomaterials have been used to develop tissue engineering substitutes for cartilage repair, including chitosan alone or in combination with growth factors to improve its chondrogenic properties. The main objective of this investigation was to evaluate the benefits of combining activated platelet-rich plasma with a stabilized porous chitosan scaffold for cartilage regeneration. To achieve this purpose, stabilized porous chitosan scaffolds were prepared using freeze gelation and combined with activated platelet-rich plasma. Human primary articular chondrocytes were isolated and cultured in stabilized porous chitosan scaffolds with and without combination to activated platelet-rich plasma. Scanning electron microscopy was used for the morphological characterization of the resulting scaffolds. Cell counts were performed in hematoxylin and eosin-stained sections, and type I and II collagen expression was evaluated using immunohistochemistry. Significant increase in cell number in activated platelet-rich plasma/stabilized porous chitosan was found compared with stabilized porous chitosan scaffolds. Chondrocytes grown on stabilized porous chitosan expressed high levels of type I collagen but type II was not detectable, whereas cells grown on activated platelet rich plasma/stabilized porous chitosan scaffolds expressed high levels of type II collagen and type I was almost undetectable. In summary, activated platelet-rich plasma increases nesting and induces the differentiation of chondrocytes cultured on stabilized porous chitosan scaffolds.

Vitamin D Receptor Activation Reduces Angiotensin-II-Induced Dissecting Abdominal Aortic Aneurysm in Apolipoprotein E-Knockout Mice.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a vascular disorder characterized by chronic inflammation of the aortic wall. Low concentrations of vitamin D3 are associated with AAA development; however, the potential direct effect of vitamin D3 on AAA remains unknown. This study evaluates the effect of oral treatment with the vitamin D3 receptor (VDR) ligand, calcitriol, on dissecting AAA induced by angiotensin-II (Ang-II) infusion in apoE(-/-) mice. APPROACH AND RESULTS: Oral treatment with calcitriol reduced Ang-II-induced dissecting AAA formation in apoE(-/-) mice, which was unrelated to systolic blood pressure or plasma cholesterol concentrations. Immunohistochemistry and reverse-transcription polymerase chain reaction analysis demonstrated a significant increase in macrophage infiltration, neovessel formation, matrix metalloproteinase-2 and matrix metalloproteinase-9, chemokine (CCL2 [(C-C motif) ligand 2], CCL5 [(C-C motif) ligand 5], and CXCL1 [(C-X-C motif) ligand 1]) and vascular endothelial growth factor expression in suprarenal aortic walls of apoE(-/-) mice infused with Ang-II, and all were significantly reduced by cotreatment with calcitriol. Phosphorylation of extracellular signal-regulated kinases 1/2, p38 mitogen-activated protein kinase, and nuclear factor-κB was also decreased in the suprarenal aortas of apoE(-/-) mice cotreated with calcitriol. These effects were accompanied by a marked increase in VDR-retinoid X receptor (RXR) interaction in the aortas of calcitriol-treated mice. In vitro, VDR activation by calcitriol in human endothelial cells inhibited Ang-II-induced leukocyte-endothelial cell interactions, morphogenesis, and production of endothelial proinflammatory and angiogenic chemokines through VDR-RXR interactions, and knockdown of VDR or RXR abolished the inhibitory effects of calcitriol. CONCLUSIONS: VDR activation reduces dissecting AAA formation induced by Ang-II in apoE(-/-) mice and may constitute a novel therapeutic strategy to prevent AAA progression.