A Deep Learning Image System for Classifying High Oleic Sunflower Seed Varieties.

Sunflower seeds, one of the main oilseeds produced around the world, are widely used in the food industry. Mixtures of seed varieties can occur throughout the supply chain. Intermediaries and the food industry need to identify the varieties to produce high-quality products. Considering that high oleic oilseed varieties are similar, a computer-based system to classify varieties could be useful to the food industry. The objective of our study is to examine the capacity of deep learning (DL) algorithms to classify sunflower seeds. An image acquisition system, with controlled lighting and a Nikon camera in a fixed position, was constructed to take photos of 6000 seeds of six sunflower seed varieties. Images were used to create datasets for training, validation, and testing of the system. A CNN AlexNet model was implemented to perform variety classification, specifically classifying from two to six varieties. The classification model reached an accuracy value of 100% for two classes and 89.5% for the six classes. These values can be considered acceptable, because the varieties classified are very similar, and they can hardly be classified with the naked eye. This result proves that DL algorithms can be useful for classifying high oleic sunflower seeds.

Riboflavin-binding proteins for singlet oxygen production.

miniSOG, developed as the first fully genetically encoded singlet oxygen photosensitiser, has found various applications in cell imaging and functional studies. Yet, miniSOG has suboptimal properties, including a low yield of singlet oxygen generation, which can nevertheless be improved tenfold upon blue light irradiation. In a previous study, we showed that this improvement was due to the photolysis of the miniSOG chromophore, flavin mononucleotide (FMN), into lumichrome, with concomitant removal of the phosphoribityl tail, thereby improving oxygen access to the alloxazine ring. We thus reasoned that a chromophore with a shorter tail would readily improve the photosensitizing properties of miniSOG. In this work, we show that the replacement of FMN by riboflavin (RF), which lacks the bulky phosphate group, significantly improves the singlet oxygen quantum yield (ΦΔ). We then proceeded to mutagenize the residues stabilizing the phosphate group of FMN to alter the chromophore specificity. We identified miniSOG-R57Q as a flavoprotein that selectively binds RF in cellulo, with a modestly improved ΦΔ. Our results show that it is possible to modify the flavin specificity of a given flavoprotein, thus providing a new option to tune its photophysical properties, including those leading to photosensitization. We also determined the structure of miniSOG-Q103L, a mutant with a much increased ΦΔ, which allowed us to postulate the existence of another access channel to FMN for molecular oxygen.

An optogenetic toolbox of LOV-based photosensitizers for light-driven killing of bacteria.

Flavin-binding fluorescent proteins (FPs) are genetically encoded in vivo reporters, which are derived from microbial and plant LOV photoreceptors. In this study, we comparatively analyzed ROS formation and light-driven antimicrobial efficacy of eleven LOV-based FPs. In particular, we determined singlet oxygen (1O2) quantum yields and superoxide photosensitization activities via spectroscopic assays and performed cell toxicity experiments in E. coli. Besides miniSOG and SOPP, which have been engineered to generate 1O2, all of the other tested flavoproteins were able to produce singlet oxygen and/or hydrogen peroxide but exhibited remarkable differences in ROS selectivity and yield. Accordingly, most LOV-FPs are potent photosensitizers, which can be used for light-controlled killing of bacteria. Furthermore, the two variants Pp2FbFP and DsFbFP M49I, exhibiting preferential photosensitization of singlet oxygen or singlet oxygen and superoxide, respectively, were shown to be new tools for studying specific ROS-induced cell signaling processes. The tested LOV-FPs thus further expand the toolbox of optogenetic sensitizers usable for a broad spectrum of microbiological and biomedical applications.

Correction: Cationic phthalocyanine dendrimers as potential antimicrobial photosensitisers.

Correction for 'Cationic phthalocyanine dendrimers as potential antimicrobial photosensitisers' by Rubén Ruiz-González et al., Org. Biomol. Chem., 2017, 15, 9008-9017.

Influence of foliar riboflavin applications to vineyard on grape amino acid content.

Nitrogen is an important element for grapevine and winemaking, which affects plant development, grape juice fermentation and has a potential effect in modulating wine quality. The aim was to study the influence of foliar applications of riboflavin (vitamin B2) to vineyard on grape nitrogen composition. This vitamin has a reported capacity to protect different plant species, but its application to favor grape and grape juice quality had not previously been studied. This work reports the oenological properties and the effect on amino acid concentration of grape juices obtained from grapes treated with riboflavin at two different doses compared to control. Results showed that probable alcohol, malic acid, color intensity and hue had significant differences when the riboflavin treatments were applied. Most of the amino acids presented the highest concentrations when the lowest riboflavin dose was used. These results are promising in terms of fermentation development and grape juice nitrogen composition.

Cationic phthalocyanine dendrimers as potential antimicrobial photosensitisers.

In the present study we describe the synthesis, photophysical properties and the photoinactivation performance against representative microorganisms of two families of cationic dendrimeric phthalocyanines as potential photosensitisers. Four charged dendrimeric compounds varying in their degree of ionicity (4 or 8 positive charges) and the coordinating metal (zinc or ruthenium) are compared and assessed as potential photosensitising agents in terms of their antimicrobial activity.

Acid- and hydrogen-bonding-induced switching between 22-π and 18-π electron conjugations in 2-aminothiazolo[4,5-c]porphycenes.

2-Aminothiazolo[4,5-c]porphycenes are a novel class of 22-π electron aromatic porphycene derivatives prepared by click reaction of porphycene isothiocyanates with primary and secondary amines with high potential as near-infrared theranostic labels. Herein, the optical and photophysical properties of 2-aminothiazolo[4,5-c]porphycenes have been studied, revealing a strong dependence on hydrogen bond donor solvents and acids. High hydrogen bond donor solvents and acids shift the absorption and fluorescence emission of 2-aminothiazolo[4,5-c]porphycenes to the blue due to a contraction of their aromatic system from 22-π to 18-π electrons. Finally, the aromatic shift has been successfully used to measure the pH using 2-aminothiazoloporphycene-labelled gold nanoclusters, paving the way for the use of these compounds as near infrared pH-sensitive probes.

NanoSOSG: A Nanostructured Fluorescent Probe for the Detection of Intracellular Singlet Oxygen.

A biocompatible fluorescent nanoprobe for singlet oxygen (1 O2 ) detection in biological systems was designed, synthesized, and characterized, that circumvents many of the limitations of the molecular probe Singlet Oxygen Sensor Green® (SOSG). This widely used commercial singlet oxygen probe was covalently linked to a polyacrylamide nanoparticle core using different architectures to optimize the response to 1 O2 . In contrast to its molecular counterpart, the optimum SOSG-based nanoprobe, which we call NanoSOSG, is readily internalized by E. coli cells and does not interact with bovine serum albumin. Furthermore, the spectral characteristics do not change inside cells, and the probe responds to intracellularly generated 1 O2 with an increase in fluorescence.

Photodynamic Synergistic Effect of Pheophorbide a and Doxorubicin in Combined Treatment against Tumoral Cells.

A combination of therapies to treat cancer malignancies is at the forefront of research with the aim to reduce drug doses (ultimately side effects) and diminish the possibility of resistance emergence given the multitarget strategy. With this goal in mind, in the present study, we report the combination between the chemotherapeutic drug doxorubicin (DOXO) and the photosensitizing agent pheophorbide a (PhA) to inactivate HeLa cells. Photophysical studies revealed that DOXO can quench the excited states of PhA, detracting from its photosensitizing ability. DOXO can itself photosensitize the production of singlet oxygen; however, this is largely suppressed when bound to DNA. Photodynamic treatments of cells incubated with DOXO and PhA led to different outcomes depending on the concentrations and administration protocols, ranging from antagonistic to synergic for the same concentrations. Taken together, the results indicate that an appropriate combination of DOXO with PhA and red light may produce improved cytotoxicity with a smaller dose of the chemotherapeutic drug, as a result of the different subcellular localization, targets and mode of action of the two agents.

Precision Optogenetic Tool for Selective Single- and Multiple-Cell Ablation in a Live Animal Model System.

Cell ablation is a strategy to study cell lineage and function during development. Optogenetic methods are an important cell-ablation approach, and we have previously developed a mini singlet oxygen generator (miniSOG) tool that works in the living Caenorhabditis elegans. Here, we use directed evolution to generate miniSOG2, an improved tool for cell ablation via photogenerated reactive oxygen species. We apply miniSOG2 to a far more complex model animal system, Drosophila melanogaster, and demonstrate that it can be used to kill a single neuron in a Drosophila larva. In addition, miniSOG2 is able to photoablate a small group of cells in one of the larval wing imaginal discs, resulting in an adult with one incomplete and one normal wing. We expect miniSOG2 to be a useful optogenetic tool for precision cell ablation at a desired developmental time point in live animals, thus opening a new window into cell origin, fate and function, tissue regeneration, and developmental biology.

Correction: Assessing the potential of photosensitizing flavoproteins as tags for correlative microscopy.

Correction for 'Assessing the potential of photosensitizing flavoproteins as tags for correlative microscopy' by Alberto Rodríguez-Pulido et al., Chem. Commun., 2016, 52, 8405-8408.

Rational Design of a GFP-Based Fluorogenic Caspase Reporter for Imaging Apoptosis In Vivo.

Fluorescence resonance energy transfer-based executioner caspase reporters using GFP are important tools for imaging apoptosis. While these reporters are useful for imaging apoptosis in cultured cells, their in vivo application has been handicapped by poor signal to noise. Here, we report the design and characterization of a GFP-based fluorogenic protease reporter, dubbed ZipGFP. ZipGFP-based TEV protease reporter increased fluorescence 10-fold after activation by protease. A ZipGFP-based executioner caspase reporter visualized apoptosis in live zebrafish embryos with spatiotemporal resolution. Thus, the ZipGFP-based caspase reporter may be useful for monitoring apoptosis during animal development and for designing reporters of proteases beyond the executioner caspases.

Assessing the potential of photosensitizing flavoproteins as tags for correlative microscopy.

Photosensitizing flavoproteins have great potential as tags for correlative light and electron microscopy (CLEM). We examine the photostability of miniSOG mutants and their ability to photo-oxidize diaminobenzidine, both key aspects for CLEM. Our experiments reveal a complex relation between these parameters and the production of different reactive oxygen species.

Anthracene-based fluorescent nanoprobes for singlet oxygen detection in biological media.

We have developed a novel singlet oxygen nanoprobe based on 9,10-anthracenedipropionic acid covalently bound to mesoporous silica nanoparticles. The nanoparticle protects the probe from interactions with proteins, which detract from its ability to detect singlet oxygen. In vitro studies show that the nanoprobe is internalized by cells and is distributed throughout the cytoplasm, thus being capable of detecting intracellularly-generated singlet oxygen.

Rational design of a monomeric and photostable far-red fluorescent protein for fluorescence imaging in vivo.

Fluorescent proteins (FPs) are powerful tools for cell and molecular biology. Here based on structural analysis, a blue-shifted mutant of a recently engineered monomeric infrared fluorescent protein (mIFP) has been rationally designed. This variant, named iBlueberry, bears a single mutation that shifts both excitation and emission spectra by approximately 40 nm. Furthermore, iBlueberry is four times more photostable than mIFP, rendering it more advantageous for imaging protein dynamics. By tagging iBlueberry to centrin, it has been demonstrated that the fusion protein labels the centrosome in the developing zebrafish embryo. Together with GFP-labeled nucleus and tdTomato-labeled plasma membrane, time-lapse imaging to visualize the dynamics of centrosomes in radial glia neural progenitors in the intact zebrafish brain has been demonstrated. It is further shown that iBlueberry can be used together with mIFP in two-color protein labeling in living cells and in two-color tumor labeling in mice.

A Comparative Study on Two Cationic Porphycenes: Photophysical and Antimicrobial Photoinactivation Evaluation.

Over the last decades, the number of pathogenic multi-resistant microorganisms has grown dramatically, which has stimulated the search for novel strategies to combat antimicrobial resistance. Antimicrobial photodynamic therapy (aPDT) is one of the promising alternatives to conventional treatments based on antibiotics. Here, we present a comparative study of two aryl tricationic porphycenes where photoinactivation efficiency against model pathogenic microorganisms is correlated to the photophysical behavior of the porphycene derivatives. Moreover, the extent of photosensitizer cell binding to bacteria has been assessed by flow cytometry in experiments with, or without, removing the unbound porphycene from the incubation medium. Results show that the peripheral substituent change do not significantly affect the overall behavior for both tricationic compounds neither in terms of photokilling efficiency, nor in terms of binding.

Synthesis, photophysical characterization, and photoinduced antibacterial activity of methylene blue-loaded amino- and mannose-targeted mesoporous silica nanoparticles.

Over the last 20 years, the number of pathogenic multi-resistant microorganisms has grown steadily, which has stimulated the search for new strategies to combat antimicrobial resistance. Antimicrobial photodynamic therapy (aPDT), also called photodynamic inactivation, is emerging as a promising alternative to treatments based on conventional antibiotics. We have explored the effectiveness of methylene blue-loaded targeted mesoporous silica nanoparticles (MSNP) in the photodynamic inactivation of two Gram negative bacteria, namely Escherichia coli and Pseudomonas aeruginosa. For E. coli, nanoparticle association clearly reduced the dark toxicity of MB while preserving its photoinactivation activity. For P. aeruginosa, a remarkable difference was observed between amino- and mannose-decorated nanoparticles. The details of singlet oxygen production in the nanoparticles have been characterized, revealing the presence of two populations of this cytotoxic species. Strong quenching of singlet oxygen within the nanoparticles is observed.

Singlet oxygen photosensitisation by the fluorescent protein Pp2FbFP L30M, a novel derivative of Pseudomonas putida flavin-binding Pp2FbFP.

Flavin-binding fluorescent proteins (FbFPs) are a class of fluorescent reporters that have been increasingly used as reporters in the study of cellular structures and dynamics. Flavin's intrinsic high singlet oxygen ((1)O2) quantum yield (ΦΔ = 0.51) provides a basis for the development of new FbFP mutants capable of photosensitising (1)O2 for mechanistic and therapeutic applications, as recently exemplified by the FbFP miniSOG. In the present work we report an investigation on the (1)O2 photoproduction by Pp2FbFP L30M, a novel derivative of Pseudomonas putida Pp2FbFP. Direct detection of (1)O2 through its phosphorescence at 1275 nm yielded the value ΦΔ = 0.09 ± 0.01, which is the highest (1)O2 quantum yield reported to date for any FP and is approximately 3-fold higher than the ΦΔ for miniSOG. Unlike miniSOG, transient absorption measurements revealed the existence of two independent triplet states each with a different ability to sensitise (1)O2.

An SVM-based classifier for estimating the state of various rotating components in agro-industrial machinery with a vibration signal acquired from a single point on the machine chassis.

The goal of this article is to assess the feasibility of estimating the state of various rotating components in agro-industrial machinery by employing just one vibration signal acquired from a single point on the machine chassis. To do so, a Support Vector Machine (SVM)-based system is employed. Experimental tests evaluated this system by acquiring vibration data from a single point of an agricultural harvester, while varying several of its working conditions. The whole process included two major steps. Initially, the vibration data were preprocessed through twelve feature extraction algorithms, after which the Exhaustive Search method selected the most suitable features. Secondly, the SVM-based system accuracy was evaluated by using Leave-One-Out cross-validation, with the selected features as the input data. The results of this study provide evidence that (i) accurate estimation of the status of various rotating components in agro-industrial machinery is possible by processing the vibration signal acquired from a single point on the machine structure; (ii) the vibration signal can be acquired with a uniaxial accelerometer, the orientation of which does not significantly affect the classification accuracy; and, (iii) when using an SVM classifier, an 85% mean cross-validation accuracy can be reached, which only requires a maximum of seven features as its input, and no significant improvements are noted between the use of either nonlinear or linear kernels.

Synthesis, photophysical studies and ¹O2 generation of carboxylate-terminated zinc phthalocyanine dendrimers.

Highly water-soluble dendrimers have been prepared consisting of a central zinc phthalocyanine moiety and dendritic wedges with terminal carboxylate groups. The biggest polyelectrolyte comprises 32 negative charges at the dendrimer surface. The photophysical studies reveal a strong correlation between the degree of dendritic environment, the extent of aggregation, and the ability to generate singlet oxygen in aqueous media. Compared to dendrimers having an axial derivatization the functionalization on the outer rim also significantly improves the phthalocyanine's ability to photosensitize singlet oxygen.