LSSmScarlet, dCyRFP2s, dCyOFP2s and CRISPRed2s, Genetically Encoded Red Fluorescent Proteins with a Large Stokes Shift.

Genetically encoded red fluorescent proteins with a large Stokes shift (LSSRFPs) can be efficiently co-excited with common green FPs both under single- and two-photon microscopy, thus enabling dual-color imaging using a single laser. Recent progress in protein development resulted in a great variety of novel LSSRFPs; however, the selection of the right LSSRFP for a given application is hampered by the lack of a side-by-side comparison of the LSSRFPs' performance. In this study, we employed rational design and random mutagenesis to convert conventional bright RFP mScarlet into LSSRFP, called LSSmScarlet, characterized by excitation/emission maxima at 470/598 nm. In addition, we utilized the previously reported LSSRFPs mCyRFP1, CyOFP1, and mCRISPRed as templates for directed molecular evolution to develop their optimized versions, called dCyRFP2s, dCyOFP2s and CRISPRed2s. We performed a quantitative assessment of the developed LSSRFPs and their precursors in vitro on purified proteins and compared their brightness at 488 nm excitation in the mammalian cells. The monomeric LSSmScarlet protein was successfully utilized for the confocal imaging of the structural proteins in live mammalian cells and multicolor confocal imaging in conjugation with other FPs. LSSmScarlet was successfully applied for dual-color two-photon imaging in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet protein at the resolution of 1.4 Å that revealed a hydrogen bond network supporting excited-state proton transfer (ESPT). Quantum mechanics/molecular mechanics molecular dynamic simulations confirmed the ESPT mechanism of a large Stokes shift. Structure-guided mutagenesis revealed the role of R198 residue in ESPT that allowed us to generate a variant with improved pH stability. Finally, we showed that LSSmScarlet protein is not appropriate for STED microscopy as a consequence of LSSRed-to-Red photoconversion with high-power 775 nm depletion light.

Large-Scale Purification of Photon-Upconversion Nanoparticles by Gel Electrophoresis for Analogue and Digital Bioassays.

The performance of photon-upconversion nanoparticles (UCNPs) as background-free luminescent labels in bioanalytical applications strongly depends on the preparation of well-defined and water-dispersible nanoconjugates. We have exploited the separation power of agarose-gel electrophoresis to purify milligram amounts of homogeneous UCNPs covered with carboxylated silica, biotin, or streptavidin with recovery rates of 30 to 50%. Clusters containing discrete numbers of UCNPs were isolated from the gel and reanalyzed by agarose-gel electrophoresis, single-nanoparticle-upconversion microscopy, and additional complementary methods. The purified nanoconjugates improved conventional (analogue) bioaffinity assays and provided highly monodisperse conjugates for assays that rely on counting individual UCNPs (digital assays).

Bioluminescence imaging: a shining future for cardiac regeneration.

Advances in bioanalytical techniques have become crucial for both basic research and medical practice. One example, bioluminescence imaging (BLI), is based on the application of natural reactants with light-emitting capabilities (photoproteins and luciferases) isolated from a widespread group of organisms. The main challenges in cardiac regeneration remain unresolved, but a vast number of studies have harnessed BLI with the discovery of aequorin and green fluorescent proteins. First described in the luminous hydromedusan Aequorea victoria in the early 1960s, bioluminescent proteins have greatly contributed to the design and initiation of ongoing cell-based clinical trials on cardiovascular diseases. In conjunction with advances in reporter gene technology, BLI provides valuable information about the location and functional status of regenerative cells implanted into numerous animal models of disease. The purpose of this review was to present the great potential of BLI, among other existing imaging modalities, to refine effectiveness and underlying mechanisms of cardiac cell therapy. We recount the first discovery of natural primary compounds with light-emitting capabilities, and follow their applications to bioanalysis. We also illustrate insights and perspectives on BLI to illuminate current efforts in cardiac regeneration, where the future is bright.

Biosynthesis of luminescent quantum dots in an earthworm.

The synthesis of designer solid-state materials by living organisms is an emerging field in bio-nanotechnology. Key examples include the use of engineered viruses as templates for cobalt oxide (Co(3)O(4)) particles, superparamagnetic cobalt-platinum alloy nanowires and gold-cobalt oxide nanowires for photovoltaic and battery-related applications. Here, we show that the earthworm's metal detoxification pathway can be exploited to produce luminescent, water-soluble semiconductor cadmium telluride (CdTe) quantum dots that emit in the green region of the visible spectrum when excited in the ultraviolet region. Standard wild-type Lumbricus rubellus earthworms were exposed to soil spiked with CdCl(2) and Na(2)TeO(3) salts for 11 days. Luminescent quantum dots were isolated from chloragogenous tissues surrounding the gut of the worm, and were successfully used in live-cell imaging. The addition of polyethylene glycol on the surface of the quantum dots allowed for non-targeted, fluid-phase uptake by macrophage cells.

Layer-by-layer assembled Fe3O4@C@CdTe core/shell microspheres as separable luminescent probe for sensitive sensing of Cu2+ ions.

A novel multifunctional microsphere with a fluorescent CdTe quantum dots (QDs) shell and a magnetic core (Fe(3)O(4)) has been successfully developed and prepared by a combination of the hydrothermal method and layer-by-layer (LBL) self-assembly technique. The resulting fluorescent Fe(3)O(4)@C@CdTe core/shell microspheres are utilized as a chemosensor for ultrasensitive Cu(2+) ion detection. The fluorescence of the obtained chemosensor could be quenched effectively by Cu(2+) ions. The quenching mechanism was studied and the results showed the existence of both static and dynamic quenching processes. However, static quenching is the more prominent of the two. The modified Stern-Volmer equation showed a good linear response (R(2) = 0.9957) in the range 1-10 µM with a quenching constant (K(sv)) of 4.9 × 10(4) M(-1). Most importantly, magnetic measurements showed that the Fe(3)O(4)@C@CdTe core/shell microspheres were superparamagnetic and they could be separated and collected easily using a commercial magnet in 10 s. These results obtained not only provide a way to solve the embarrassments in practical sensing applications of QDs, but also enable the fabrication of other multifunctional nanostructure-based hybrid nanomaterials.

LC-MS and microscale NMR analysis of luciferin-related compounds from the bioluminescent earthworm Fridericia heliota.

This paper presents the main results of RP-HPLC-MS and microscale NMR analysis performed on Accompanying similar to Luciferin (AsLn(x)), compounds present in extracts of the bioluminescent earthworm Fridericia heliota that display similarities with Fridericia's luciferin, the substrate of the bioluminescent reaction. Three isomers of AsLn were discovered, AsLn(1), AsLn(2) and AsLn(3), all of which present a molecular weight of 529 Da. Their UV-Vis absorption spectra show maxima at 235 nm for AsLn(1), 238 and 295 nm for AsLn(2) and 241 and 295nm for AsLn(3). MS(n) fragmentation patterns suggest the existence of carboxylic acid and hydroxyl moieties, and possibly chemical groups found in other luciferins like pterin or benzothiazole. The major isomer, AsLn(2), presents an aromatic ring and alkene and alkyl moieties. These luciferin-like compounds can be used as models that could give further insights into the structure of this newly discovered luciferin.

Rapid, continuous purification of proteins in a microfluidic device using genetically-engineered partition tags.

High-throughput screening assays of native and recombinant proteins are increasingly crucial in life science research, including fields such as drug screening and enzyme engineering. These assays are typically highly parallel, and require minute amounts of purified protein per assay. To address this need, we have developed a rapid, automated microscale process for isolating specific proteins from sub-microlitre volumes of E. Coli cell lysate. Recombinant proteins are genetically tagged to drive partitioning into the PEG-rich phase of a flowing aqueous two-phase system, which removes approximately 85% of contaminating proteins, as well as unwanted nucleic acids and cell debris, on a simple microfluidic device. Inclusion of the genetic tag roughly triples recovery of the autofluorescent protein AcGFP1, and also significantly improves recovery of the enzyme glutathione S-transferase (GST), from nearly zero recovery for the wild-type enzyme, up to 40% with genetic tagging. The extraction process operates continuously, with only a single step from cell lysate to purified protein, and does not require expensive affinity reagents or troublesome chromatographic steps. The two-phase system is mild and does not disrupt protein function, as evidenced by recovery of active enzymes and functional fluorescent protein from our microfluidic process. The microfluidic aqueous two-phase extraction forms the core component of an integrated lab-on-a-chip device comprising cell culture, lysis, purification and analysis on a single device.

Purification and partial spectral characterization of a novel luciferin from the luminous enchytraeid Fridericia heliota.

A homogeneous luciferin preparation has been obtained from the luminous soil enchytraeid Fridericia heliota, which has an ATP-dependent luminescent system. A procedure for luciferin purification without losing fractions of active luciferase has been developed. The luciferin specific activity is 4000 times increased; its UV absorption spectrum maximum is 294 nm with a local minimum at 262 nm. The luciferin of the enchytraeid F. heliota is significantly different from firefly luciferin, whose luminescent reaction also requires ATP, and it also appears to have no similarities to other known luciferins.

Study on the thermal stability of green fluorescent protein (GFP) in glucose parenteral formulations.

Large volume parenteral solutions (LVPS) that are widely used in the healthcare system must be processed by moist-heat treatment to an assured sterility level in which the efficacy is measured by a bioindicator (BI) that provides fast, accurate and reliable results. This study evaluated the thermal stability of green fluorescent protein (GFP) into glucose-based LVPS (1.5-50%) solutions to determine its utility as a BI for thermal processes. GFP, expressed by Escherichia coli, isolated/purified by TPP/HIC, was diluted in buffered (each 10mM: Tris-EDTA, pH 8; phosphate, pH 6 and 7; acetate, pH 5) and in water for injection (WFI; pH 6.70+/-0.40) glucose solutions (1.5-50%) and exposed to constant temperatures from 80 degrees C to 95 degrees C. The thermal stability was expressed in decimal reduction time (D-value, time required to reduce 90% of the GFP fluorescence intensity). At 95 degrees C, the D-values for GFP in 1.5-50% glucose were: (i) 1.63+/-0.23 min (pH 5); (ii) 2.64+/-0.26 min (WFI); (iii) 2.50+/-0.18 min (pH 6); (iv) 3.24+/-0.28 min (pH 7); (v) 2.89+/-0.44 min (pH 8). By the convenient measure of fluorescence intensity and its thermal stability, GFP has the potential as a BI to assay the efficacy of moist-heat processing of LVPS at temperatures < or =100 degrees C.