Amplification-free profiling of microRNA-122 biomarker in DILI patient serums, using the luminex MAGPIX system.

Dynamic chemical labelling is a single-base specific method to enable detection and quantification of micro-Ribonucleic Acids in biological fluids without extraction and pre-amplification. In this study, dynamic chemical labelling was combined with the Luminex MAGPIX system to profile levels of microRNA-122 biomarker in serum from patients with Drug-Induced Liver Injury.

O-Alkyl Hydroxamates Display Potent and Selective Antileishmanial Activity.

Leishmania (L.) infantum causes visceral, cutaneous, and mucosal leishmaniasis in humans and canine leishmaniasis in dogs. Herein, we describe that O-alkyl hydroxamate derivatives displayed potent and selective in vitro activity against the amastigote stage of L. infantum while no activity was observed against promastigotes. Compound 5 showed potent in vivo activity against L. infantum. Moreover, the combination of compound 5 supported on gold nanoparticles and meglumine antimoniate was also effective in vivo and improved the activity of these compounds compared to that of the individual treatment. Docking studies showed that compound 5 did not reach highly conserved pocket C and established interactions with the semiconserved residues V44, A45, R242, and E243 in pocket A of LiSIR2rp1. The surface space determined by these four amino acids is not conserved in human sirtuins. Compound 5 represents a new class of selective ligands with antileishmanial activity.

PCR-free and chemistry-based technology for miR-21 rapid detection directly from tumour cells.

miRNAs are well known for being implicated in a myriad of biological situations, including those related to serious diseases. Amongst miRNAs, miRNA-21 has the spotlight as it is reported to be up-regulated in multiple severe pathological conditions, being its quantification a key point in medicine. To date, most of the techniques for miRNA quantification have shown to be less effective than expected; thus, we herein present a novel, rapid, cost-effective, robust and PCR-free approach, based on dynamic chemistry, for the identification and quantification of miRNA directly from tumour cells using both FACS and a fluorescent microplate. This dynamic chemistry novel application involves bead based reagents and allows quantifying the number of miR-21 molecules presented in MDA-MB-468 and H1975 tumour cells.

New Platform for the Direct Profiling of microRNAs in Biofluids.

Circulating microRNAs have been identified as potential biomarkers for early detection, prognosis, and prediction of several diseases. Their use in clinical diagnostics has been limited by the lack of suitable detection techniques. Most of the current technologies suffer from requiring complex protocols, not yet able to deliver robust and cost-effective assays in the field of clinical diagnostics. In this work, we report the development of a breakthrough platform for profiling circulating microRNAs. The platform comprises a novel silicon photomultiplier-based reader in conjunction with a chemical-based method for nucleic acid detection. Accurate microRNAs profiling without extraction, pre-amplification, or pre-labeling of the target is now achievable. We designed and synthesized a set of reagents that combined the chemical-based method with a chemiluminescent reaction. The signals generated were read out using a novel, compact silicon photomultiplier-based reader. The platform sensitivity was determined by measuring known concentrations of hsa-miR-21-5p spike-ins. The limit of detection was calculated as 4.7 pmol/L. The platform was also successfully used to directly detect hsa-miR-21-5p in eight non-small cell lung cancer plasma samples. Levels of plasma hsa-miR-21-5p expression were also measured via TaqMan RT-qPCR. The successful integration of the unique chemical-based method for nucleic acid detection with the novel silicon photomultiplier-based reader created an innovative product (ODG platform) with diagnostic utility, for the direct qualitative and quantitative analysis of microRNA biomarkers in biological fluids.

A colorimetric strategy based on dynamic chemistry for direct detection of Trypanosomatid species.

Leishmaniasis and Chagas disease are endemic in many countries, and re-emerging in the developed countries. A rapid and accurate diagnosis is important for early treatment for reducing the duration of infection as well as for preventing further potential health complications. In this work, we have developed a novel colorimetric molecular assay that integrates nucleic acid analysis by dynamic chemistry (ChemNAT) with reverse dot-blot hybridization in an array format for a rapid and easy discrimination of Leishmania major and Trypanosoma cruzi. The assay consists of a singleplex PCR step that amplifies a highly homologous DNA sequence which encodes for the RNA component of the large ribosome subunit. The amplicons of the two different parasites differ between them by single nucleotide variations, known as "Single Nucleotide Fingerprint" (SNF) markers. The SNF markers can be easily identified by naked eye using a novel micro Spin-Tube device "Spin-Tube", as each of them creates a specific spot pattern. Moreover, the direct use of ribosomal RNA without requiring the PCR pre-amplification step is also feasible, further increasing the simplicity of the assay. The molecular assay delivers sensitivity capable of identifying up to 8.7 copies per µL with single mismatch specificity. The Spin-Tube thus represents an innovative solution providing benefits in terms of time, cost, and simplicity, all of which are crucial for the diagnosis of infectious disease in developing countries.

A PCR-free technology to detect and quantify microRNAs directly from human plasma.

A novel sensitive, specific and rapid method for the detection and quantification of microRNAs without requiring extraction from their biological sources is now available using a novel chemical based, PCR-free technology for nucleic acid testing. In this study, we both demonstrate how this method can be used to profile miR-451a, an important miRNA in erythropoiesis, and compare with the gold standard RT-qPCR.

Identification of Trypanosomatids by detecting Single Nucleotide Fingerprints using DNA analysis by dynamic chemistry with MALDI-ToF.

Protozoan parasites of the Trypanosomatidae family can cause devastating diseases in humans and animals, such as Human African Trypanosomiasis or Sleeping Sickness, Chagas disease and Leishmaniasis. Currently, there are molecular assays for detecting parasitic infections and their post-treatment monitoring based on nucleic acid amplification, but there are still certain limitations which limit the development of assays that can detect and discriminate between parasite infections with a single test. Here, we present the development of a novel molecular assay for the rapid identification of Trypanosomatids, integrating DNA analysis by dynamic chemistry in conjunction with Matrix-Assisted Laser Desorption Ionization - Time-of-Flight Mass Spectrometry (MALDI-ToF). Differentiation of Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp. is now possible using a single reaction tube, and enables rapid identification of Trypanosomatids. The test is based on a singleplex PCR, using a specific primer pair that amplifies a 155 base pair segment of the 28S ribosomal RNA gene, within a conserved homology region of Trypanosomatidae species. Amplified fragments are analysed by dynamic chemistry using two abasic PNA probes and the four reactive nucleobases - containing an aldehyde functional group - with MALDI-ToF to identify unique molecular patterns created by each specie due to their single base differences (Single Nucleotide Fingerprint 'SNF') in this highly homologous region. This novel assay offers the possibility to expand routine diagnostic testing for Trypanosomatids, and monitoring of therapeutic responses to these infectious diseases.

Novel bead-based platform for direct detection of unlabelled nucleic acids through Single Nucleobase Labelling.

Over the last decade, circulating microRNAs have received attention as diagnostic and prognostic biomarkers. In particular, microRNA122 has been demonstrated to be an early and more sensitive indicator of drug-induced liver injury than the widely used biomarkers such as alanine aminotransferase and aspartate aminotransferase. Recently, microRNA122 has been used in vitro to assess the cellular toxicity of new drugs and as a biomarker for the development of a rapid test for drug overdose/liver damage. In this proof-of-concept study, we report a PCR-free and label-free detection method that has a limit of detection (3 standard deviations) of 15 fmoles of microRNA122, by integrating a dynamic chemical approach for "Single Nucleobase Labelling" with a bead-based platform (Luminex®) thereby, in principle, demonstrating the exciting prospect of rapid and accurate profiling of any microRNAs related to diseases and toxicology.