Cloning of the Orange Light‐Producing Luciferase from Photinus scintillans—A New Proposal on how Bioluminescence Color is Determined

Unlike the enchanting yellow‐green flashes of light produced on warm summer evenings by Photinus pyralis, the most common firefly species in North America, the orange lights of Photinus scintillans are infrequently observed. These Photinus species, and likely all bioluminescent beetles, use the same substrates beetle luciferin, ATP and oxygen to produce light. It is the structure of the particular luciferase enzyme that is the key to determining the color of the emitted light. We report here the molecular cloning of the P. scintillans luc gene and the expression and characterization of the corresponding novel recombinant luciferase enzyme. A comparison of the amino acid sequence with that of the highly similar P. pyralis enzyme and subsequent mutagenesis studies revealed that the single conservative amino acid change tyrosine to phenylalanine at position 255 accounted for the entire emission color difference. Additional mutagenesis and crystallographic studies were performed on a H‐bond network, which includes the position 255 residue and five other stringently conserved beetle luciferase residues, that is proximal to the substrate/emitter binding site. The results are interpreted in the context of a speculative proposal that this network is key to the understanding of bioluminescence color determination.     

Gaussianization: An Efficient Multivariate Density Estimation Technique for Statistical Signal Processing

Multivariate density estimation is an important problem that is frequently encountered in statistical learning and signal processing. One of the most popular techniques is Parzen windowing, also referred to as kernel density estimation. Gaussianization is a procedure that allows one to estimate multivariate densities efficiently from the marginal densities of the individual random variables. In this paper, we present an optimal density estimation scheme that combines the desirable properties of Parzen windowing and Gaussianization, using minimum Kullback–Leibler divergence as the optimality criterion for selecting the kernel size in the Parzen windowing step. The utility of the estimate is illustrated in classifier design, independent components analysis, and Prices’ theorem.     

Simulating listener errors in using genetic algorithms for perceptual optimization

The genetic algorithm (GA) was previously suggested for fitting hearing aid or cochlear implant features by using listener's subjective judgment. In the present study, two human factors that might affect the outcome of the GA when used for perceptual optimization were explored with simulations. Listeners with varying sensitivity in discriminating sentences of different intelligibility and with varying error rates in entering their judgment to the GA were simulated. A comparison of the simulation results with the results from human subjects reported by Ba?kent et al. Ear Hear. 28(3) 277-289 (2007) showed that these factors could reduce the performance of the GA considerably.     

Forced Folding of a Disordered Protein Accesses an Alternative Folding Landscape

Intrinsically disordered proteins (IDPs) are involved in diverse cellular functions. Many IDPs can interact with multiple binding partners, resulting in their folding into alternative ligand‐specific functional structures. For such multi‐structural IDPs, a key question is whether these multiple structures are fully encoded in the protein sequence, as is the case in many globular proteins. To answer this question, here we employed a combination of single‐molecule and ensemble techniques to compare ligand‐induced and osmolyte‐forced folding of α‐synuclein. Our results reveal context‐dependent modulation of the protein′s folding landscape, suggesting that the codes for the protein′s native folds are partially encoded in its primary sequence, and are completed only upon interaction with binding partners. Our findings suggest a critical role for cellular interactions in expanding the repertoire of folds and functions available to disordered proteins.     

Thermal Analysis of Micro- and Nano-Lignocellulosic Reinforced Styrene Maleic Anhydride Composite Foams

The aim of this study was to measure the thermal properties of foamed nano/macro filler–reinforced styrene maleic anhydride (SMA) composites. SMA (66%) as a polymer matrix (10% maleic anhydride content) and various fillers including wood flour, starch, α-cellulose, microcrystalline cellulose and cellulose nanofibrils as reinforcing agents (30%) and lubricant (4%) were used to manufacture the composites in a twin-screw extruder. According to the thermogravimetric analysis (TGA) results, thermal degradation of all the foamed composites was found to be lower than that of SMA composites. The storage modulus values were negatively affected with a second time foaming (reprocessing [recycling] the initially processed composites a second time), as were loss modulus and T_g. As a result, second-time-foamed composite modulus values were lower than those of the foamed composites. According to the melt flow index (MFI) results, viscosity of the SMA was found to increase with the addition of fillers.     

On two classes of approximation processes of integral type

The paper aims to study two classes of linear positive operators representing modifications of Picard and Gauss operators. The new operators reproduce both constants and a given exponential function. Approximation properties in polynomial weighted spaces are investigated and the speed of convergence is measured using a certain weighted modulus of smoothness. Also, the asymptotic behavior of the integral operators are established. Finally, aspects on generalized convexity are analyzed.

     

Acanthifoliosides, minor steroidal saponins from the Caribbean sponge Pandaros acanthifolium

Seven novel steroid glycosides, acanthifoliosides A-F (1-6), and the methyl ester of 6 (7), were isolated from the marine sponge Pandaros acanthifolium as minor components. Acanthifoliosides are characterized by a rare C-15 and C-16 oxidized D ring, which was previously found in saponins produced by starfishes. Very uncommon is the presence of additional sugar residues at C-15 or C-16. Their structures were determined on the basis of extensive spectroscopic analyses, including two-dimensional NMR and HRESIMS data. The absolute configurations of the aglycones were assigned by comparison between experimental and TDDFT calculated CD spectra of 1, whereas the absolute configurations of the monosaccharide units were determined by chiral GC analyses of the acid methanolysates. Some of the acanthifoliosides exhibit moderate antiprotozoal activity but to a lesser extent than the most potent pandarosides.     

Fast and stable photochromic oxazines for fluorescence switching

The stringent limitations imposed by diffraction on the spatial resolution of fluorescence microscopes demand the identification of viable strategies to switch fluorescence under optical control. In this context, the photoinduced and reversible transformations of photochromic compounds are particularly valuable. In fact, these molecules can be engineered to regulate the emission intensities of complementary fluorophores in response to optical stimulations. On the basis of this general design logic, we assembled a functional molecular construct consisting of a borondipyrromethene fluorophore and a nitrospiropyran photochrome and demonstrated that the emission of the former can be modulated with the interconversion of the latter. This fluorophore-photochrome dyad, however, has a slow switching speed and poor fatigue resistance. To improve both parameters, we developed a new family of photochromic switches based on the photoinduced opening and thermal closing of an oxazine ring. These compounds switch back and forth between ring-closed and -open isomers on nanosecond-microsecond timescales and tolerate thousands of switching cycles with no sign of degradation. In addition, the attachment of appropriate chromophoric fragments to their switchable oxazine ring can be exploited to either deactivate or activate fluorescence reversibly in response to illumination with a pair of exciting beams. Specifically, we assembled three dyads, each based on either a borondipyrromethene or a coumarin fluorophore and an oxazine photochrome, and modulated their fluorescence in a few microseconds with outstanding fatigue resistance. The unique photochemical and photophysical properties of our fluorophore-photochrome dyads can facilitate the development of switchable fluorophores for superresolution imaging and, ultimately, provide valuable molecular probes for the visualization of biological samples on the nanometer level.     

Solvatochromic rotaxane molecular shuttles

A strongly fluorescent bistable rotaxane is described in which the relative position of the macrocyclic ring with respect to a solvatochromic fluorophore gives a strong response in the spectral domain.     

Quantitative and sensitive detection of rare mutations using droplet-based microfluidics

Somatic mutations within tumoral DNA can be used as highly specific biomarkers to distinguish cancer cells from their normal counterparts. These DNA biomarkers are potentially useful for the diagnosis, prognosis, treatment and follow-up of patients. In order to have the required sensitivity and specificity to detect rare tumoral DNA in stool, blood, lymph and other patient samples, a simple, sensitive and quantitative procedure to measure the ratio of mutant to wild-type genes is required. However, techniques such as dual probe TaqMan(?) assays and pyrosequencing, while quantitative, cannot detect less than ～1% mutant genes in a background of non-mutated DNA from normal cells. Here we describe a procedure allowing the highly sensitive detection of mutated DNA in a quantitative manner within complex mixtures of DNA. The method is based on using a droplet-based microfluidic system to perform digital PCR in millions of picolitre droplets. Genomic DNA (gDNA) is compartmentalized in droplets at a concentration of less than one genome equivalent per droplet together with two TaqMan(?) probes, one specific for the mutant and the other for the wild-type DNA, which generate green and red fluorescent signals, respectively. After thermocycling, the ratio of mutant to wild-type genes is determined by counting the ratio of green to red droplets. We demonstrate the accurate and sensitive quantification of mutated KRAS oncogene in gDNA. The technique enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines and the precise quantification of a mutated KRAS gene in the presence of a 200,000-fold excess of unmutated KRAS genes. The sensitivity is only limited by the number of droplets analyzed. Furthermore, by one-to-one fusion of drops containing gDNA with any one of seven different types of droplets, each containing a TaqMan(?) probe specific for a different KRAS mutation, or wild-type KRAS, and an optical code, it was possible to screen the six common mutations in KRAS codon 12 in parallel in a single experiment.