A Fluorogenic Chemogenetic pH Sensor for Imaging Protein Exocytosis.

Fluorescent protein-based pH biosensors enable the tracking of pH changes during protein trafficking and, in particular, exocytosis. The recent development of chemogenetic reporters combining synthetic fluorophores with self-labeling protein tags offers a versatile alternative to fluorescent proteins that combines the diversity of chemical probes and indicators with the selectivity of the genetic encoding. However, this hybrid protein labeling strategy does not avoid common drawbacks of organic fluorophores such as the risk of off-target signal due to unbound molecules. Here, we describe a novel fluorogenic and chemogenetic pH sensor based on a cell-permeable molecular pH indicator called pHluo-Halo-1, whose fluorescence can be locally activated in cells by reaction with HaloTag, ensuring excellent signal selectivity in wash-free imaging experiments. pHluo-Halo-1 was selected out of a series of four fluorogenic molecular rotor structures based on protein chromophore analogues. It displays good pH sensitivity with a pKa of 6.3 well-suited to monitor pH variations during exocytosis and an excellent labeling selectivity in cells. It was applied to follow the secretion of CD63-HaloTag fusion proteins using TIRF microscopy. We anticipate that this strategy based on the combination of a tunable and chemically accessible fluorogenic probe with a well-established protein tag will open new possibilities for the development of versatile alternatives to fluorescent proteins for elucidating the dynamics and regulatory mechanisms of proteins in living cells.

Effect of various light spectra on amino acids and pigment production of Arthrospira platensis using flat-plate photobioreactor.

Today, the use of nutrients derived from natural bioactive compounds application in the food, pharmaceutical, and cosmetic industries is on the increase. This paper aimed to evaluate the amino acids profile (essential and non-essential) and pigments composition (chlorophyll a, carotenoids, and phycocyanin) of Arthrospira platensis (a blue-green microalga) cultivation in a flat-plate photobioreactor under various types of light-emitting diodes (red: 620-680 nm, white: 380-780 nm, yellow: 570-600nm, blue: 445-480 nm). The maximum biomass concentration (604.96 mg L-1) occurred when the red LED was applied for cultivation, and the minimum biomass concentration (279.39 mg L-1) was obtained under blue LED. The sequence of pigments and amino acids concentrations (mg L-1culture volume) was approximately in accordance with the biomass productivity. It means the red light produces the maximum concentration of pigments (chlorophyll a: 5.42, carotenoids: 2.92, phycocyanin: 67.54 mg L-1) and amino acids (essential amino acids: 110.47, nonessential amino acids: 179.10 mg L-1). Nevertheless, when these values were measured in mg per g of dry weight, the utmost contents were observed in microalgal products cultivated under blue LED. These consequences are due to the highest cell productivity and the most extended length of cells that occurred under red and blue LEDs, respectively.

Conformational change and GTPase activity of human tubulin: A comparative study on Alzheimer's disease and healthy brain.

In Alzheimer's disease (AD), the most common form of dementia, microtubules (MTs) play a pivotal role through their highly dynamic structure and instability. They mediate axonal transport that is crucial to synaptic viability. MT assembly, dynamic instability and stabilization are modulated by tau proteins, whose detachment initiates MT disintegration. Albeit extensive research, the role of GTPase activity in molecular mechanism of stability remains controversial. We hypothesized that GTPase activity is altered in AD leading to microtubule dynamic dysfunction and ultimately to neuronal death. In this paper, fresh tubulin was purified by chromatography from normal young adult, normal aged, and Alzheimer's brain tissues. Polymerization pattern, assembly kinetics and dynamics, critical concentration, GTPase activity, interaction with tau, intermolecular geometry, and conformational changes were explored via Förster Resonance Energy Transfer (FRET) and various spectroscopy methods. Results showed slower MT assembly process in samples from the brains of people with AD compared with normal young and aged brains. This observation was characterized by prolonged lag phase and increased critical and inactive concentration of tubulin. In addition, the GTPase activity in samples from AD brains was significantly higher than in both normal young and normal aged samples, concurrent with profound conformational changes and contracted intermolecular MT-tau distances as revealed by FRET. These alterations were partially restored in the presence of a microtubule stabilizer, paclitaxel. We proposed that alterations of both tubulin function and GTPase activity may be involved in the molecular neuropathogenesis of AD, thus providing new avenues for therapeutic approaches.

Protein complex prediction: A survey.

Protein complexes are one of the most important functional units for deriving biological processes within the cell. Experimental methods have provided valuable data to infer protein complexes. However, these methods have inherent limitations. Considering these limitations, many computational methods have been proposed to predict protein complexes, in the last decade. Almost all of these in-silico methods predict protein complexes from the ever-increasing protein-protein interaction (PPI) data. These computational approaches usually use the PPI data in the format of a huge protein-protein interaction network (PPIN) as input and output various sub-networks of the given PPIN as the predicted protein complexes. Some of these methods have already reached a promising efficiency in protein complex detection. Nonetheless, there are challenges in prediction of other types of protein complexes, specially sparse and small ones. New methods should further incorporate the knowledge of biological properties of proteins to improve the performance. Additionally, there are several challenges that should be considered more effectively in designing the new complex prediction algorithms in the future. This article not only reviews the history of computational protein complex prediction but also provides new insight for improvement of new methodologies. In this article, most important computational methods for protein complex prediction are evaluated and compared. In addition, some of the challenges in the reconstruction of the protein complexes are discussed. Finally, various tools for protein complex prediction and PPIN analysis as well as the current high-throughput databases are reviewed.

Thermodynamics and kinetic analysis of carbon nanofibers as nanozymes.

PURPOSE: Evaluation of structural features, thermodynamics and kinetic properties of carbon nanofibers (CNFs) as artificial nanoscale enzymes (nanozyme). METHODS: Synthesis of CNFs was done using chemical vapor deposition, and transmission electron microscopy (TEM), field emission scanning electron microscopy (FE-SEM) and energy-dispersive x-ray spectroscopy (EDX) were used to provide information on the morphology, elemental monitoring and impurity assay of the CNFs. The thermal features of the CNFs were evaluated using differential thermal analysis (DTA), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) derivative and TGA. The calculated thermo-physical parameters were melting temperature (Tm), weight loss maximum temperature (Tmax ) and enthalpy of fusion (ΔHfusion ). Catalytic activity was assayed by a 4-aminoantypyrine (4-AAP)-H2O2 coupled colorimetric system by UV-visible spectroscopy. RESULTS: FE-SEM and TEM analysis demonstrated parallel graphitic layers and uniformity of atomic orientation and morphology. The EDX spectra approved carbon element as major signal and presence of partial Ti as impurities of CNFs during CVD process. The DTA thermogram showed the endothermic process had a maximum temperature of 82.27°C at -15.48 mV and that thermal decomposition occurred at about 200°C. The TGA-differential gravimetric analysis thermogram showed that Tmax was 700°C. The DSC heat flow curve showed a melting temperature (Tm) of 254.52°C, ΔHfusion of 3.84 J^.g-1, area under the curve of 58.58 mJ and Te (onset) and Tf (end set) temperatures of 246.60°C and 285.67°C, respectively. The peroxidase activity of the CNFs obeyed the Michaelis-Menten equation with a double-reciprocal curve and the calculated Km, Kcat and Vmax kinetic parameters. CONCLUSION: CNFs as peroxidase nanozymes are intrinsically strong and stable nanocatalysts under difficult thermal conditions. The peroxidase activity was demonstrated, making these CNFs candidates for analytical tools under extreme conditions.

Catalase and its mysteries.

Catalase is one of the firsts in every realm of biological sciences. At the same time it also has a number of unusual features. It has one of the highest turnover numbers of all enzymes. It is essential for neutralizing the noxious hydrogen peroxide both in the nature and the various industries such as dairy, textile and pharmaceutics. It also has the merit of being one of the first protein crystals to be isolated. Ironically its three-dimensional structure was discerned some forty years later. However through the times this senile enzyme has continued to intrigue the scientists by surprising facts and phenomena, such as peculiar interweaving of subunits and remarkable thermal stability. It is also known for suicide inactivation by its own substrate. Catalase is known to be implicated in various medical scenarios and its levels have served as a marker in that capacity. It has even been incorporated into several pharmaceuticals. This review strives to clarify these perspectives. It also draws attention to the biophysical contributions offered by thermodynamics and kinetics in these discoveries. The ultimate aim of this review, however, is to state that the venerable catalase will continue to bewilder us with its mysteries well into the twenty-first century.