Direct detection and quantification of microRNAs

The recent discovery of the potent regulatory nature of microRNAs (miRNAs), a relatively new class of approximately 22 nucleotide RNAs, has made them a primary focus in today’s biochemical and medical research. The relationship between miRNA expression patterns and the onset of cancer, as well as other diseases, has glimpsed the potential of miRNAs as disease biomarkers or drug targets, making them a primary research focus. Their promising future in medicine is hinged upon improving our scientific understanding of their intricate regulatory mechanisms. In the realm of analytical chemistry, the main challenge associated with miRNA is its detection. Their extremely small size and low cellular concentration poses many challenges for achieving reliable results. Current reviews in this area have focused on adaptations to microarray, PCR, and Northern blotting procedures to make them suitable for miRNA detection. While these are extremely powerful methods and accepted as the current standards, they are typically very laborious, semi-quantitative, and often require expensive imaging equipment and/or radioactive/toxic labels. This review aims to highlight emerging techniques in miRNA detection and quantification that exhibit superior flexibility and adaptability as well as matched or increased sensitivity in comparison to the current standards. Specifically, this review will cover colorimetric, fluorescence, bioluminescence, enzyme, and electrochemical based methods, which drastically reduce procedural complexity and overall expense of operation thereby increasing the accessibility of this field of research. The methods are presented and discussed as to their improvements over current standard methods as well as their potential complications preventing acceptance as standard procedures. These new methods have addressed the many of the problems associated with miRNA detection through the employment of enzyme-based signal amplification, enhanced hybridization conditions using PNA capture probes, highly sensitive and flexible forms of spectroscopy, and extremely responsive electrocatalytic nanosystems, among other approaches

Aequorin and Obelin Mutants with Differing Wavelengths and Bioluminescence

The invention relates to aequorin and obelin mutants whose emission is shifted with respect to wild type. The shift in emission is accomplished using a combination of mutations of amino acids within aequorin or obelin that affect bioluminescence; use of different types of chromophores, i.e., coelenterazines with variable emission characteristics; and modifications of the photoprotein with fluorophores that will allow for emission of light at longer wavelengths as a result of energy transfer. Additionally, an assay employing aequorin mutants to test for HIV-1 protease inhibitors is disclosed

Methods and Kit for Determination of Prostacyclin in Plasma

A solid-phase immunoassay for 6-keto-Prostaglandin F1α, the stable hydrolysis product of prostacyclin (Prostaglandin I2) is disclosed. Prostacyclin, a potent vasodilator with anti-platelet and anti-proliferative properties is an effective treatment for primary pulmonary hypertension and pulmonary arterial hypertension associated with scleroderma and scleroderma-like syndrome. Levels of 6-keto-Prostaglandin F1α can be directly correlated with levels of prostacyclin. Therefore, 6-keto-Prostaglandin F1α has become the indicator of choice to measure prostacyclin levels. The single step immunoassay for 6-keto-Prostaglandin F1α uses the bioluminescent protein, aequorin as a label. Analyte-label conjugates were constructed by linking the carboxyl group of 6-keto-Prostaglandin F1α and lysine residues of aequorin by chemical conjugation methods. The binding properties of 6-keto-Prostaglandin F1α towards its antibody and the bioluminescent properties of aequorin are retained in the conjugate. The concentration of 6-keto-Prostaglandin F1α after extraction from plasma shows good correlation with the concentration of 6-keto-Prostaglandin F1α obtained without prior extraction of the same plasma sample. The assay allows the measurement of 6-keto-Prostaglandin F1α directly in plasma without any pre-treatment of the samples, which results in a much simpler method with a faster assay time

Stimuli-Responsive Hydrogel Microdomes Integrated with Genetically Engineered Proteins for High-Throughput Screening of Pharmaceuticals

A hydrogel microdome that can swell in response to a stimuli or target molecule is formed by polymerizing a mixture comprising a monomer capable of forming a hydrogel with a biopolymer. An array of hydrogel microdomes can be formed on a substrate by microspotting the mixture and polymerizing. The array can be used for high-throughput screening of analytes as well as for use as an actuator and biosensor using the swelling property of the hydrogel

Systems and Methods for Diagnosis and Monitoring of Bacteria-Related Conditions

The presently-disclosed subject matter provides systems, methods, and kits for diagnosing and/or monitoring a bacteria-related condition of interest in a subject by providing a cell sensing system, each system containing a reporter molecule capable of detecting binding of a quorum sensing molecule and capable of generating a detectable signal

Potential Impacts of PCBs on Sediment Microbiomes in a Tropical Marine Environment

Within the tropical marine study site of Guánica Bay, Puerto Rico, polychlorinated biphenyls (PCBs) are subjected to coastal and oceanic currents coupled with marine microbial and geochemical processes. To evaluate these processes a hydrodynamic model was developed to simulate the transport of PCBs within nearshore and offshore marine areas of Guánica Bay. Material transport and circulation information from the model were matched with measurements from samples collected from within the bay. These samples, consisting of both intertidal and submerged sediments, were analyzed for physical characteristics (organic carbon, grain size, and mineralogy), microbial characteristics (target bacteria levels and microbial community analyses), presence of PCBs, and PCB-degrading enzymes. Results show that the bay geometry and bathymetry limit the mixing of the extremely high levels of PCBs observed in the eastern portion of the bay. Bay bottom sediments showed the highest levels of PCBs and these sediments were characterized by high organic carbon content and finer grain size. Detectable levels of PCBs were also observed within sediments found along the shore. Microbes from the bay bottom sediments showed a greater relative abundance of microbes from the Chloroflexi, phylum with close phylogenetic associations with known anaerobic PCB-degrading organisms. Based on quantitative PCR measurement of the biphenyl dioxygenase gene, the intertidal sediments showed the greatest potential for aerobic PCB degradation. These results elucidate particular mechanisms of PCB’s fate and transport in coastal, tropical marine environments

Comparative Study of the Performance of Two Different Luciferases for the Analysis of Fumonisin B1 in Wheat Samples

The development of two different immunoassays for the determination of fumonisin B1 in wheat samples is reported. A previously described mimopeptide for fumonisin B1 (FB1) was used to produce fusion proteins in combination with two different luciferases: Gaussia luciferase (GLuc) and NanoLuc luciferase (NLuc). The production, expression and the development of two immunoassays based on these fusion proteins (A2- GLuc and A2-NLuc) is detailed. The assay showing the best performance, A2-NLuc, with a limit of detection of 0.61 ngmL 1 and a dynamic range from 1.9 to 95 ngmL 1 , was employed for the analysis of spiked wheat samples, a reference matrix material, as well as naturally contaminated wheat samples. The recoveries obtained in the spiked samples were acceptable, between 81.5 and 109%, with relative standard deviations lower than 14%. The analysis of naturally contaminated wheat was validated by a liquid chromatography coupled to tandem mass detection method

A Targeted and Adjuvanted Nanocarrier Lowers the Effective Dose of Liposomal Amphotericin B and Enhances Adaptive Immunity in Murine Cutaneous Leishmaniasis

Background: Amphotericin B (AmB), the most effective drug against leishmaniasis, has serious toxicity. As Leishmania species are obligate intracellular parasites of antigen presenting cells (APC), an immunopotentiating APC-specific AmB nanocarrier would be ideally suited to reduce the drug dosage and regimen requirements in leishmaniasis treatment. Here, we report a nanocarrier that results in effective treatment shortening of cutaneous leishmaniasis in a mouse model, while also enhancing L. major specific T-cell immune responses in the infected host. Methods: We used a Pan-DR-binding epitope (PADRE)-derivatized-dendrimer (PDD), complexed with liposomal amphotericin B (LAmB) in an L. major mouse model and analyzed the therapeutic efficacy of low-dose PDD/ LAmB vs full dose LAmB. Results: PDD was shown to escort LAmB to APCs in vivo, enhanced the drug efficacy by 83% and drug APC targeting by 10-fold and significantly reduced parasite burden and toxicity. Fortuitously, the PDD immunopotentiating effect significantly enhanced parasite-specific T-cell responses in immunocompetent infected mice. Conclusions: PDD reduced the effective dose and toxicity of LAmB and resulted in elicitation of strong parasite specific T-cell responses. A reduced effective therapeutic dose was achieved by selective LAmB delivery to APC, bypassing bystander cells, reducing toxicity and inducing antiparasite immunity