Green fluorescent protein based pH indicators for in vivo use: a review

Green fluorescent protein (GFP) and its variants have been used as fluorescent reporters in a variety of applications for monitoring dynamic processes in cells and organisms, including gene expression, protein localization, and intracellular dynamics. GFP fluorescence is stable, species-independent, and can be monitored noninvasively in living cells by fluorescence microscopy, flow cytometry, or macroscopic imaging techniques. Owing to the presence of a phenol group on the chromophore, most GFP variants display pH-sensitive absorption and fluorescence bands. Such behavior has been exploited to genetically engineer encodable pH indicators for studies of pH regulation within specific intracellular compartments that cannot be probed using conventional pH-sensitive dyes. These pH indicators contributed to shedding light on a number of cell functions for which intracellular pH is an important modulator. In this review we discuss the photophysical properties that make GFPs so special as pH indicators for in vivo use and we describe the probes that are utilized most by the scientific community.     

Complementary Approaches to Imaging Subcellular Lipid Architectures in Live Bacteria Using Phosphorescent Iridium Complexes and Raman Spectroscopy

A family of three neutral iridium(III) tetrazolato complexes are investigated as bacterial imaging agents. The complexes offer a facile tuning of the emission colour from green (520 nm) to red (600 nm) in aqueous media, while keeping the excitation wavelength unchanged. The three complexes do not inhibit the bacterial growth of Bacillus Cereus, used as a model in this study, and exhibit extremely fast cellular uptake. After a minute incubation time, the nontoxic complexes show subcellular localisation in spherical structures identified as lipid vacuoles. Confocal Raman imaging has been exploited for the first time on live bacteria, to provide direct and label-free mapping of the lipid-enriched organelles within B. cereus, complementing the use of luminescent probes. Examination of the Raman spectra not only confirmed the presence of lipophilic inclusions in B. cereus but offered additional information about their chemical composition, suggesting that the lipid vacuoles may contain polyhydroxybutyrate (PHB).     

The Experimental Observation of the Intramolecular NO2/CO Interaction in Solution

The weak electrostatic interaction between nitro and carbonyl moieties has been observed by means of variable‐temperature NMR spectroscopy. Its energetic contribution was evaluated to be about 3 kcal mol^?1 by DFT calculations, and confirmed by the measurement of internal energy barriers to the rotation of suitable nitroaryl rings.     

How to Turn an Electron Transfer Protein into a Redox Enzyme for Biosensing

Cytochrome c is a small globular protein whose main physiological role is to shuttle electrons within the mitochondrial electron transport chain. This protein has been widely investigated, especially as a paradigmatic system for understanding the fundamental aspects of biological electron transfer and protein folding. Nevertheless, cytochrome c can also be endowed with a non-native catalytic activity and be immobilized on an electrode surface for the development of third generation biosensors. Here, an overview is offered of the most significant examples of such a functional transformation, carried out by either point mutation(s) or controlled unfolding. The latter can be induced chemically or upon protein immobilization on hydrophobic self-assembled monolayers. We critically discuss the potential held by these systems as core constituents of amperometric biosensors, along with the issues that need to be addressed to optimize their applicability and response.     

Breathing Rhythm Variations during Wash-In Do Not Influence Exhaled Volatile Organic Compound Profile Analyzed by an Electronic Nose

E-noses are innovative tools used for exhaled volatile organic compound (VOC) analysis, which have shown their potential in several diseases. Before obtaining a full validation of these instruments in clinical settings, a number of methodological issues still have to be established. We aimed to assess whether variations in breathing rhythm during wash-in with VOC-filtered air before exhaled air collection reflect changes in the exhaled VOC profile when analyzed by an e-nose (Cyranose 320). We enrolled 20 normal subjects and randomly collected their exhaled breath at three different breathing rhythms during wash-in: (a) normal rhythm (respiratory rate (RR) between 12 and 18/min), (b) fast rhythm (RR > 25/min) and (c) slow rhythm (RR < 10/min). Exhaled breath was collected by a previously validated method (Dragonieri et al., J. Bras. Pneumol. 2016) and analyzed by the e-nose. Using principal component analysis (PCA), no significant variations in the exhaled VOC profile were shown among the three breathing rhythms. Subsequent linear discriminant analysis (LDA) confirmed the above findings, with a cross-validated accuracy of 45% (p = ns). We concluded that the exhaled VOC profile, analyzed by an e-nose, is not influenced by variations in breathing rhythm during wash-in.     

In Situ Observation of Step Evolution on {100} Face of KH2PO4 Crystal in Solution by Atomic Force Microscopy (AFM)

In situ observation of the surface morphology of {100} face of Potassium Dihydrogen Phosphate (KDP) crystals growing in solution was made with the use of Atomic Force Microscope. The slight movement of the steps connected with regeneration of the crystal surface has been detected for very low supersaturation. The height of the elementary steps has been established.     

Engineered Green Fluorescence Proteins for Proteomics and Biomolecular Electronic Applications

The discovery of the photochromic characteristics of engineered green fluorescent proteins (GFPs) allows new proteomics and biomolecular electronic applications. In particular, photoreversibility among two distinct optical states can lead to the realization of a bio-optical high density storage memory. Here we review our recent work on an optically bistable GFP and we report the recent developments of self-assembly methods for spatial immobilization of proteins into well-definite 2D patterns.     

An investigation of the condensation kinetics in poly(ester-amide) and poly(ester-sulphide) preparation

Synthetic processes leading to hydrophilic biodegradable polymers for bio-inspired applications were investigated from a kinetic point of view. In accordance with the reported mechanism of ester aminolysis, polycondensation reactions of α-amino-ω-esters, diesters, and diamines resulted markedly dependent on the basicity of the alkoxide leaving-group, being relatively fast for penthachlorophenate monomers. Furthermore, experimental data concerning the homopolycondensation of penthachlorophenyl α-amino-ω-oligo(ethyleneglycol) succinates of different degree of oligomerization clearly showed the existence of concurrent first and second-order processes, which were attributed to the intramolecular cyclization and intermolecular polycondensation reaction, respectively. In contrast to theoretical predictions based on the collision theory, however, minor incidence of the cyclization reaction was shown by the shortest monomers, thus suggesting a significant kinetic effect due to steric hindrance and solvent-reagent interactions. Analysis of the base-catalyzed Michael-type addition of α,ω-oligo(oxyethylene)dithiols to methyl (meth)acrylate allowed for the optimization of the relevant polymerization process involving hydrophilic diacrylates. Interestingly, very low reaction rates were determined for methacrylic components, supposedly because of steric and electronic factors connected to the presence of the α-methyl group. Minor effects on the reaction rate were also induced by solvent polarity and catalyst nature.