Sapna K. Deo

Bioluminescence-Based Detection of MicroRNA, miR21 in Breast Cancer Cells

A hybridization assay for the detection of microRNA, miR21 in cancer cells using the bioluminescent enzyme Renilla luciferase (Rluc) as a label, has been developed. MicroRNAs are small RNAs found in plants, animals, and humans that perform key functions in gene silencing and affect early-stage cell development, cell differentiation, and cell death. miRNAs are considered useful early diagnostic and prognostic markers of cancer, candidates for therapeutic intervention, and targets for basic biomedical research. However, methods for highly sensitive and rapid detection of miRNA directly from samples such as cells that can serve as a suitable diagnostics platform are lacking. In that regard, the utilization of the bioluminescent label, Rluc, that offers the advantage of high signal-to-noise ratio, allows for the development of highly sensitive assays for the determination of miRNA in a variety of matrixes. In this paper, we have described the development of a competitive oligonucleotide hybridization assay for the detection of miR21 using the free miR21 and Rluc-labeled miR21 that competes to bind to an immobilized miR21 complementary probe. The miR21 microRNA chosen for this study is of biomedical significance because its levels are elevated in a variety of cancers. Using the optimized assay, a detection limit of 1 fmol was obtained. The assay was employed for the detection of miR21 in human breast adenocarcinoma MCF-7 cells and nontumorigenic epithelial MCF-10A cells. The comparison of miR21 expression level in two cell lines demonstrated higher expression of miR21 in breast cancer cell line MCF-7 compared to the nontumorigenic MCF-10A cells. Further, using the assay developed, the miR21 quantification could be performed directly in cell extracts. The hybridization assay was developed in a microplate format with a total assay time of 1.5 h and without the need for sample PCR amplification. The need for early molecular markers and their detection methods in cancer diagnosis is tremendous. The characteristics of the assay developed in this work show its suitability for early cancer diagnosis based on miRNA as a biomarker.

Fluorescence glucose detection: Advances toward the ideal in vivo biosensor

The importance of glucose monitoring for in vivo as well as for ex vivo applications has driven a vast number of scientific groups to pursue the development of an advanced glucose sensor. Such a sensor must be robust, versatile, and capable of the long-term, accurate and reproducible detection of glucose levels in various testing media. Among the different configurations and signal transduction mechanisms used, fluorescence-based glucose sensors constitute a growing class of glucose sensors represented by an increasing number of significant contributions to the field over the last few years. This manuscript reviews the progress in the development of fluorescence based glucose sensors resulting from the advances in the design of new receptor systems for glucose recognition and the utilization of new fluorescence transduction schemes.

Trends in microRNA detection

MicroRNAs (miRNAs) are short, ~22 nucleotide length RNAs that perform gene regulation. Recently, miRNA has been shown to be linked with the onset of cancer and other diseases based on miRNA expression levels. It is important, therefore, to understand miRNA function as it pertains to disease onset; however, in order to fully understand miRNA’s role in a disease, it is necessary to detect the expression levels of these small molecules. The most widely used miRNA detection method is Northern blotting, which is considered as the standard of miRNA detection methods. This method, however, is time-consuming and has low sensitivity. This has led to an increase in the amount of detection methods available. These detection methods are either solid phase, occurring on a solid support, or solution phase, occurring in solution. While the solid-phase methods are adaptable to high-throughput screening and possess higher sensitivity than Northern blotting, they lack the ability for in vivo use and are often time-consuming. The solution-phase methods are advantageous in that they can be performed in vivo, are very sensitive, and are rapid; however, they cannot be applied in high-throughput settings. Although there are multiple detection methods available, including microarray technology, luminescence-based assays, electrochemical assays, etc., there is still much work to be done regarding miRNA detection. The current gaps of miRNA detection include the ability to perform multiplex, sensitive detection of miRNA with single-nucleotide specificity along with the standardization of these new methods. Current miRNA detection methods, gaps in these methods, miRNA therapeutic options, and the future outlook of miRNA detection are presented here.

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.

Photoproteins in Bioanalysis

Preface. List of Contributors. 1 The Photoproteins (Osamu Shimomura). 1.1 Discovery of Photoprotein. 1.2 Various Types of Photoproteins Presently Known. 1.2.1 Radiolarian (Protozoa) Photoproteins. 1.2.2 Coelenterate Photoproteins. 1.2.3 Ctenophore Photoproteins. 1.2.4 Pholasin (Pholas Luciferin). 1.2.5 Chaetopterus Photoprotein. 1.2.6 Polynoidin. 1.2.7 Symplectin. 1.2.8 Luminodesmus Photoprotein. 1.2.9 Ophiopsila Photoprotein. 1.3 Basic Strategy of Extracting and Purifying Photoproteins. 1.4 The Photoprotein Aequorin. 1.4.1 Extraction and Purifi cation of Aequorin. 1.4.1.1 Hydrophobic Interaction Chromatography. 1.4.2 Properties of Aequorin. 1.4.2.1 Stability. 1.4.2.2 Freeze-drying. 1.4.3 Specifi city to Ca 2+ . 1.4.4 Luminescence of Aequorin by Substances Other Than Divalent Cations. 1.4.5 Mechanism of Aequorin Luminescence and Regeneration of Aequorin. 1.4.5.1 Structure of Aequorin. 1.4.5.2 Luminescence Reaction. 1.4.5.3 Regeneration. 1.4.6 Inhibitors of Aequorin Luminescence. 1.4.7 Recombinant Aequorin. 1.4.8 Semi-synthetic Aequorins. 1.4.8.1 e-Aequorins. References. 2 Luminous Marine Organisms (Steven H.D. Haddock). 2.1 Introduction. 2.1.1 Non-luminous Taxa. 2.1.2 Luminous Taxa. 2.2 Taxonomic Distribution of Bioluminescence. 2.2.1 Bacterial Luminescence. 2.2.2 Dinofl agellate Luciferin. 2.2.3 Cypridina (Vargula) Luciferin. 2.2.4 Coelenterazine. 2.2.5 Other Luciferins: Known and Unknown. 2.3 Functions. 2.3.1 Startle or Distract. 2.3.2 Burglar Alarm. 2.3.3 Counterillumination. 2.3.4 Mating Displays. 2.3.5 Prey Attraction. References. 3 Beetle Luciferases: Colorful Lights on Biological Processes and Diseases (Vadim R. Viviani and Yoshihiro Ohmiya). 3.1 Introduction. 3.2 Beetle Luciferases. 3.3 Bioanalytical Assays of ATP. 3.3.1 Biomass Estimation and Microbiological Contamination. 3.3.2 Cytotoxicity and Cell Viability Tests. 3.3.3 Enzymatic Assays. 3.4 Luciferases as Reporter Genes. 3.4.1 Dual and Multiple Reporter Assays. 3.5 Biophotonic Imaging in Animals: A Living Light on Diseases. 3.5.1 Pathogen Infection in Living Models. 3.5.2 Drug Screening. 3.5.3 Tumor Proliferation and Regression Studies. 3.5.4 Gene Delivery and Gene Therapy. 3.5.5 Luciferase as Biomarkers for Cell Traffi cking Studies. 3.5.6 Immunoassays. 3.6 Biophotonic Imaging in Plants. 3.7 Biosensors: Sensing the Environment. 3.8 Novel Luciferases: Different Colors for Different Occasions. References. 4 Split Luciferase Systems for Detecting Protein-Protein Interactions in Mammalian Cells Based on Protein Splicing and Protein Complementation (Yoshio Umezawa). 4.1 Introduction. 4.2 Protein Splicing-based Split Firefl y Luciferase System [23]. 4.2.1 Split Luciferase Works as a Probe for Protein Interaction. 4.3 Split Renilla Luciferase Complementation System [33]. 4.3.1 Time Course of the Interaction Between Y941 and SH2n. 4.3.2 Location of the Interaction Between Y941 and SH2n. References. 5 Photoproteins in Nucleic Acid Analysis (Theodore K. Christopoulos, Penelope C. Ioannou, and Monique Verhaegen). 5.1 Hybridization Assays. 5.2 Quantitative Polymerase Chain Reaction. 5.3 Genotyping of Single-nucleotide Polymorphisms. 5.4 Conjugation of Aequorin to Oligodeoxynucleotide Probes. 5.5 Development of New Recombinant Bioluminescent Reporters. 5.6 Signal Amplifi cation by in Vitro Expression of DNA Reporters Encoding Bioluminescent Proteins. 5.7 Conclusions. References. 6 Bioluminescence Resonance Energy Transfer in Bioanalysis (Suresh Shrestha and Sapna K. Deo). 6.1 Introduction. 6.2 BRET Principle, Effi ciency, and Instrumentation. 6.3 Comparison of BRET and FRET. 6.4 Examples of BRET Donor-Acceptor Pairs. 6.5 Applications of BRET in Bioanalysis. 6.5.1 Homogeneous Assays. 6.5.2 Protein-Protein Interactions and High-throughput Screening. 6.6 Conclusions. References. 7 Photoproteins as in Vivo Indicators of Biological Function (Rajesh Shinde, Hui Zhao, and Christopher H. Contag). 7.1 Overview. 7.2 Probes Used for in Vivo Bioluminescence Imaging. 7.3 Probes Used for in Vivo Fluorescence Imaging. 7.4 Detection Technologies. 7.5 Current Applications. 7.5.1 Oncology. 7.5.2 Infectious Disease. 7.5.3 Bacterial Infections. 7.5.4 Viral Infections. 7.5.5 Viral-mediated Gene Transfer. 7.5.6 Cell Biology. 7.5.7 Stem Cell Biology. 7.6 Protease Sensors. 7.7 Conclusions. References. 8 Photoproteins as Reporters in Whole-cell Sensing (Jessika Feliciano, Patrizia Pasini, Sapna K. Deo, and Sylvia Daunert). 8.1 Introduction. 8.1.1 Biosensors Using Intact Cells. 8.1.2 Reporter Genes in Genetically Engineered Whole-cell Sensors. 8.2 The Luciferases. 8.2.1 Bacterial Luciferases. 8.2.1.1 luxAB Bioreporters. 8.2.1.2 luxCDABE Bioreporters. 8.2.1.3 Naturally Luminescent Bioreporters. 8.2.2 Eukaryotic Luciferases. 8.2.2.1 Firefl y Luciferase. 8.2.2.2 Sea Pansy Luciferase. 8.3 Aequorin. 8.4 Fluorescent Proteins. 8.4.1 Green Fluorescent Protein. 8.4.2 Red Fluorescent Protein. 8.5 Multiplexing. 8.6 Applications. 8.6.1 Stress Factors and Genotoxicants. 8.6.2 Environmental Pollutants. 8.6.3 Quorum-sensing Signaling Molecules. 8.6.4 Antibiotics. 8.7 Technological Advances. References. 9 Luminescent Proteins in Binding Assays (Aldo Roda, Massimo Guardigli, Elisa Michelini, Mara Mirasoli, and Patrizia Pasini). 9.1 Introduction. 9.2 Protein-Protein and Protein-Ligand Interaction Assays. 9.2.1 FRET and BRET Techniques. 9.2.2 FRET and BRET Applications. 9.2.3 Other Detection Principles. 9.3 Antibody-based Binding Assays. 9.3.1 Chemical Conjugation. 9.3.2 Gene Fusion. 9.3.3 Dual-analyte Assays. 9.3.4 Expression Immunoassays. 9.3.5 BRET-based Immunoassays. 9.4 Biotin-Avidin Binding Assays. 9.5 Nucleic Acid Hybridization Assays. 9.6 Other Binding Assays. 9.7 Concluding Remarks. References. 10 Luminescent Proteins: Applications in Microfl uidics and Miniaturized Analytical Systems (Emre Dikici, Laura Rowe, Elizabeth A. Moschou, Anna Rothert, Sapna K. Deo, and Sylvia Daunert). 10.1 Miniaturization and Microfl uidics. 10.2 Photoproteins and Applications in Miniaturized Detection Systems. 10.2.1 Green Fluorescent Protein. 10.2.1.1 GFP in Miniaturized Microfl uidic-based Assays. 10.2.2 Luciferase. 10.2.2.1 Luciferase in Miniaturized Microfl uidic-based Assays. 10.2.3 Aequorin. 10.2.3.1 Aequorin in Miniaturized Microfl uidic-based Assays. 10.3 Future Perspectives. References. 11 Advances in Instrumentation for Detecting Low-level Bioluminescence and Fluorescence (Eric Karplus). 11.1 Introduction. 11.2 Low Light Levels. 11.3 Methods of Coupling the Signal to the Detector. 11.3.1 Proximity Focusing. 11.3.2 Microscope Objectives. 11.3.3 Macro Lenses. 11.3.4 Fiber Optics. 11.4 Evaluating the Performance of an Optical System. 11.4.1 Numerical Aperture. 11.4.2 Transmission Effi ciency. 11.4.3 Magnifi cation. 11.5 Detector Technologies. 11.6 Selecting the Right Detector. 11.7 Detector Sensitivity. 11.8 Detector Noise. 11.9 Statistics of Photon Counting. 11.10 Summary. References. 12 Photoproteins and Instrumentation: Their Availability and Applications in Bioanalysis (Leslie Doleman, Stephanie Bachas-Daunert, Logan Davies, Sapna K. Deo, and Sylvia Daunert). Aequorin. Obelin. Luciferases. Aequorea and Anthozoa Fluorescent Proteins. Coelenteraziness. Luminometers. Fluorometers. Portable Luminometers. Disclaimer. Subject Index.

MicroRNA Detection: Current Technology and Research Strategies

The relatively new field of microRNA (miR) has experienced rapid growth in methodology associated with its detection and bioanalysis as well as with its role in -omics research, clinical diagnostics, and new therapeutic strategies. The breadth of this area of research and the seemingly exponential increase in number of publications on the subject can present scientists new to the field with a daunting amount of information to evaluate. This review aims to provide a collective overview of miR detection methods by relating conventional, established techniques [such as quantitative reverse transcription polymerase chain reaction (RT-qPCR), microarray, and Northern blotting (NB)] and relatively recent advancements [such as next-generation sequencing (NGS), highly sensitive biosensors, and computational prediction of microRNA/targets] to common miR research strategies. This should guide interested readers toward a more focused study of miR research and the surrounding technology.

A rapid, sensitive, and selective bioluminescence resonance energy transfer (BRET)-based nucleic acid sensing system.

Here we report the design of a bioluminescence resonance energy transfer (BRET)-based sensing system that could detect nucleic acid target in 5 min with high sensitivity and selectivity. The sensing system is based on adjacent binding of oligonucleotide probes labeled with Renilla luciferase (Rluc) and quantum dot (Qd) on the nucleic acid target. Here Rluc, a bioluminescent protein that generates light by a chemical reaction, is employed as an energy donor, and a quantum dot is used as an energy acceptor. Bioluminescence emission of Rluc overlaps with the Qd absorption whereas the emission of Qd is shifted from the emission of Rluc allowing for monitoring of BRET. In the presence of target, the labeled probes bind adjacently in a head-to-head fashion leading to BRET from Rluc to Qd upon addition of a substrate coelenterazine. The sensing system could detect target nucleic acid in buffer as well as in Escherichia coli cellular matrix in 5 min with a detection limit of 0.54 pmol. The ability to detect target nucleic acid rapidly in a cellular matrix with high sensitivity will prove highly beneficial in biomedical and environmental applications.

Aequorin variants with improved bioluminescence properties

The photoprotein aequorin has been widely used as a bioluminescent label in immunoassays, for the determination of calcium concentrations in vivo, and as a reporter in cellular imaging. It is composed of apoaequorin (189 amino acid residues), the imidazopyrazine chromophore coelenterazine and molecular oxygen. The emission characteristics of aequorin can be changed by rational design of the protein to introduce mutations in its structure, as well as by substituting different coelenterazine analogues to yield semi-synthetic aequorins. Variants of aequorin were created by mutating residues His16, Met19, Tyr82, Trp86, Trp108, Phe113 and Tyr132. Forty-two aequorin mutants were prepared and combined with 10 different coelenterazine analogues in a search for proteins with different emission wavelengths, altered decay kinetics and improved stability. This spectral tuning strategy resulted in semi-synthetic photoprotein mutants with significantly altered bioluminescent properties.

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.

Neurotransmitters: The Critical Modulators Regulating Gut–Brain Axis

Neurotransmitters, including catecholamines and serotonin, play a crucial role in maintaining homeostasis in the human body. Studies on these neurotransmitters mainly revolved around their role in the “fight or flight” response, transmitting signals across a chemical synapse and modulating blood flow throughout the body. However, recent research has demonstrated that neurotransmitters can play a significant role in the gastrointestinal (GI) physiology. Norepinephrine (NE), epinephrine (E), dopamine (DA), and serotonin have recently been a topic of interest because of their roles in the gut physiology and their potential roles in GI and central nervous system pathophysiology. These neurotransmitters are able to regulate and control not only blood flow, but also affect gut motility, nutrient absorption, GI innate immune system, and the microbiome. Furthermore, in pathological states, such as inflammatory bowel disease (IBD) and Parkinsons disease, the levels of these neurotransmitters are dysregulated, therefore causing a variety of GI symptoms. Research in this field has shown that exogenous manipulation of catecholamine serum concentrations can help in decreasing symptomology and/or disease progression. In this review article, we discuss the current state‐of‐the‐art research and literature regarding the role of neurotransmitters in regulation of normal GI physiology, their impact on several disease processes, and novel work focused on the use of exogenous hormones and/or psychotropic medications to improve disease symptomology. J. Cell. Physiol. 232: 2359–2372, 2017.