Computationally designed peptides for zika virus detection: An incremental construction approach

Herein, and in contrast to current production of anti-Zika virus antibodies, we propose a semi-combinatorial virtual strategy to select short peptides as biomimetic antibodies/binding agents for the detection of intact Zika virus (ZIKV) particles. The virtual approach was based on generating different docking cycles of tetra, penta, hexa, and heptapeptide libraries by maximizing the discrimination between the amino acid motif in the ZIKV and dengue virus (DENV) envelope protein glycosylation site. Eight peptides, two for each length (tetra, penta, hexa, and heptapeptide) were then synthesized and tested vs. intact ZIKV particles by using a direct enzyme linked immunosorbent assay (ELISA). As a reference, we employed a well-established anti-ZIKV antibody, the antibody 4G2. Three peptide-based assays had good detection limits with dynamic range starting from 105 copies/mL of intact ZIKV particles; this was one order magnitude lower than the other peptides or antibodies. These three peptides showed slight cross-reactivity against the three serotypes of DENV (DENV-1,-2, and-3) at a concentration of 106 copies/mL of intact virus particles, but the discrimination between the DENV and ZIKV was lost when the coating concentration was increased to 107 copies/mL of the virus. The sensitivity of the peptides was tested in the presence of two biological matrices, serum and urine diluted 1:10 and 1:1, respectively. The detection limits decreased about one order of magnitude for ZIKV detection in serum or urine, albeit still having for two of the three peptides tested a distinct analytical signal starting from 106 copies/mL, the concentration of ZIKV in acute infection

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

Monitoring neural activity with bioluminescence during natural behavior

Existing techniques for monitoring neural activity in awake, freely behaving vertebrates are invasive and difficult to target to genetically identified neurons. We used bioluminescence to non-invasively monitor the activity of genetically specified neurons in freely behaving zebrafish. Transgenic fish with the Ca^(2+)-sensitive photoprotein green fluorescent protein (GFP)-Aequorin in most neurons generated large and fast bioluminescent signals that were related to neural activity, neuroluminescence, which could be recorded continuously for many days. To test the limits of this technique, we specifically targeted GFP-Aequorin to the hypocretin-positive neurons of the hypothalamus. We found that neuroluminescence generated by this group of ~20 neurons was associated with periods of increased locomotor activity and identified two classes of neural activity corresponding to distinct swim latencies. Our neuroluminescence assay can report, with high temporal resolution and sensitivity, the activity of small subsets of neurons during unrestrained behavior

Photoproteins as luminescent labels in binding assays

Certain marine organisms produce calcium-activated photoproteins that allow them to emit light for a variety of purposes, such as defense, feeding, breeding, etc. Even though there are many bioluminescent organisms in nature, only a few photoproteins have been isolated and characterized. The mechanism of emission of light in the blue region is the result of an internal chemical reaction. Because there is no need for excitation through external irradiation for the emission of bioluminescence, the signal produced has virtually no background. This allows for the detection of the proteins at extremely low levels, making these photoproteins attractive labels for analytical applications. In that regard, the use of certain photoproteins, namely, aequorin, obelin, and the green fluorescent protein as labels in the design and development of binding assays for biomolecules has been reviewed. In addition, a related fluorescent photoprotein, the green fluorescent protein (GFP), has been recently employed in bioanalysis. The use of GFP in binding assays is also discussed in this review

Luminescent proteins from Aequorea victoria: applications in drug discovery and in high throughput analysis

Recent progress in generating a vast number of drug targets through genomics and large compound libraries through combinatorial chemistry have stimulated advancements in drug discovery through the development of new high throughput screening (HTS) methods. Automation and HTS techniques are also highly desired in fields such as clinical diagnostics. Luminescence-based assays have emerged as an alternative to radiolabel-based assays in HTS as they approach the sensitivity of radioactive detection along with ease of operation, which makes them amenable to miniaturization. Luminescent proteins provide the advantage of reduced reagent and operating costs because they can be produced in unlimited amounts through the use of genetic engineering tools. In that regard, the use of two naturally occurring and recombinantly produced luminescent proteins from the jellyfish Aequorea victoria, namely, aequorin and the green fluorescent protein (GFP), has attracted attention in a number of analytical applications in diverse research areas. Aequorin is naturally bioluminescent and has therefore, virtually no associated background signal, which allows its detection down to attomole levels. GFP has become the reporter of choice in a variety of applications given that it is an autofluorescent protein that does not require addition of any co-factors for fluorescence emission. Furthermore, the generation of various mutants of GFP with differing luminescent and spectral properties has spurred additional interest in this protein. In this review, we focus on the use of aequorin and GFP in the development of highly sensitive assays that find applications in drug discovery and in high throughput analysis

Bioluminescence resonance energy transfer from aequorin to a fluorophore: an artificial jellyfish for applications in multianalyte detection.

In nature, the green light emission observed in the jellyfish Aequorea victoria is a result of a non-radiative energy transfer from the excited-state aequorin to the green fluorescent protein. In this work, we have modified the photoprotein aequorin by attaching selected fluorophores at a unique site on the protein. This will allow for in vitro transfer of bioluminescent energy from aequorin to the fluorophore thus creating an \u2018\u2018artificial jellyfish\u2019\u2019. The fluorophores are selected such that the excitation spectrum of the fluorophore overlaps with the emission spectrum of aequorin. By modifying aequorin with different fluorophores, bioluminescent labels with different emission maxima are produced, which will allow for the simultaneous detection of multiple analytes. By examining the X-ray crystal structure of the protein, four different sites for introduction of the unique cysteine residue were evaluated. Two fluorophores with differing emission maxima were attached individually to the mutants through the sulfhydryl group of the cysteine molecule. Two of the fluorophore-labeled mutants showed a peak corresponding to fluorophore emission thus indicating resonance energy transfer from aequorin to the fluorophore

Genetically engineered obelin as a bioluminescent label in an assay for a peptide

The marine polyp Obelia longissima produces a protein, obelin, which emits light in a calcium-dependent manner. This photoprotein consists of a stable complex of its apoprotein, a chromophore, and oxygen. In the presence of calcium ions, the protein undergoes a change in conformation that allows it to catalyze the oxidation of the chromophore, coelenterazine, to coelenteramide with the release of light and CO2. Photoproteins are attractive as labels in analytical applications because the bioluminescent signal that they produce is the result of a chemical reaction and, therefore, has virtually no background. Thus, bioluminescence allows for extremely sensitive detection. In that regard, the feasibility of using obelin as a label has been explored with the development of a competitive immunoassay for the determination of a small peptide analyte. To attach the obelin label in a controlled manner to the octapeptide, a fusion protein was produced using recombinant DNA techniques. The protein consisted of the C-terminus of the peptide fused to the N-terminus of obelin. The octapeptide-obelin fusion protein retained the bioluminescence properties of the native protein, and was subsequently used to generate dose-response curves for the free octapeptide

Chemically Tunable Lensing of Stimuli‐Responsive Hydrogel Microdomes

Chemically tunable hydrogel microlenses have been developed from acrylamide hydrogels based on covalently bound hinge‐motion binding proteins, like calmodulin, and low affinity ligands, i.e. phenothiazine. Initially the protein binds to phenothiazine forming chemical crosslinks within the hydrogel, which are released in the presence of the higher affinity ligand chlorpromazine resulting in the swelling of the microlenses