Nanoparticles from Microalgae and Their Biomedical Applications.

Over the years, microalgae have been a source of useful compounds mainly used as food and dietary supplements. Recently, microalgae have been used as a source of metabolites that can participate in the synthesis of several nanoparticles through inexpensive and environmentally friendly routes alternative to chemical synthesis. Notably, the occurrence of global health threats focused attention on the microalgae application in the medicinal field. In this review, we report the influence of secondary metabolites from marine and freshwater microalgae and cyanobacteria on the synthesis of nanoparticles that were applied as therapeutics. In addition, the use of isolated compounds on the surface of nanoparticles to combat diseases has also been addressed. Although studies have proven the beneficial effect of high-value bioproducts on microalgae and their potential in medicine, there is still room for understanding their exact role in the human body and translating lab-based research into clinical trials.

Structural and mechanistic insights into the interaction of the circadian transcription factor BMAL1 with the KIX domain of the CREB-binding protein.

The mammalian CLOCK:BMAL1 transcription factor complex and its coactivators CREB-binding protein (CBP)/p300 and mixed-lineage leukemia 1 (MLL1) critically regulate circadian transcription and chromatin modification. Circadian oscillations are regulated by interactions of BMAL1's C-terminal transactivation domain (TAD) with the KIX domain of CBP/p300 (activating) and with the clock protein CRY1 (repressing) as well as by the BMAL1 G-region preceding the TAD. Circadian acetylation of Lys537 within the G-region enhances repressive BMAL1-TAD-CRY1 interactions. Here, we characterized the interaction of the CBP-KIX domain with BMAL1 proteins, including the BMAL1-TAD, parts of the G-region, and Lys537 Tethering the small compound 1-10 in the MLL-binding pocket of the CBP-KIX domain weakened BMAL1 binding, and MLL1-bound KIX did not form a ternary complex with BMAL1, indicating that the MLL-binding pocket is important for KIX-BMAL1 interactions. Small-angle X-ray scattering (SAXS) models of BMAL1 and BMAL1:KIX complexes revealed that the N-terminal BMAL1 G-region including Lys537 forms elongated extensions emerging from the bulkier BMAL1-TAD:KIX core complex. Fitting high-resolution KIX domain structures into the SAXS-derived envelopes suggested that the G-region emerges near the MLL-binding pocket, further supporting a role of this pocket in BMAL1 binding. Additionally, mutations in the second CREB-pKID/c-Myb-binding pocket of the KIX domain moderately impacted BMAL1 binding. The BMAL1(K537Q) mutation mimicking Lys537 acetylation, however, did not affect the KIX-binding affinity, in contrast to its enhancing effect on CRY1 binding. Our results significantly advance the mechanistic understanding of the protein interaction networks controlling CLOCK:BMAL1- and CBP-dependent gene regulation in the mammalian circadian clock.

Extensive Characterization of the Human Salivary Basic Proline-Rich Protein Family by Top-Down Mass Spectrometry.

Human basic proline-rich proteins and basic glycosylated proline-rich proteins, encoded by the polymorphic PRB1-4 genes and expressed only in parotid glands, are the most complex family of adult salivary proteins. The family includes 11 parent peptides/proteins and more than 6 parent glycosylated proteins, but a high number of proteoforms with rather similar structures derive from polymorphisms and post-translational modifications. 55 new components of the family were characterized by top-down liquid chromatography-mass spectrometry and tandem-mass platforms, bringing the total number of proteoforms to 109. The new components comprise the three variants P-H S1 → A, P-Ko P36 → S, and P-Ko A41 → S and several of their naturally occurring proteolytic fragments. The paper represents an updated reference for the peptides included in the heterogeneous family of proteins encoded by PRB1/PRB4. MS data are available via ProteomeXchange with the identifier PXD009813.

Crystal structure of 3-hydroxybenzoate 6-hydroxylase uncovers lipid-assisted flavoprotein strategy for regioselective aromatic hydroxylation.

3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is a dimeric flavoprotein that catalyzes the NADH- and oxygen-dependent para-hydroxylation of 3-hydroxybenzoate to 2,5-dihydroxybenzoate. In this study, we report the crystal structure of 3HB6H as expressed in Escherichia coli. The overall fold of 3HB6H is similar to that of p-hydroxybenzoate hydroxylase and other flavoprotein aromatic hydroxylases. Unexpectedly, a lipid ligand is bound to each 3HB6H monomer. Mass spectral analysis identified the ligand as a mixture of phosphatidylglycerol and phosphatidylethanolamine. The fatty acid chains occupy hydrophobic channels that deeply penetrate into the interior of the substrate-binding domain of each subunit, whereas the hydrophilic part is exposed on the protein surface, connecting the dimerization domains via a few interactions. Most remarkably, the terminal part of a phospholipid acyl chain is directly involved in the substrate-binding site. Co-crystallized chloride ion and the crystal structure of the H213S variant with bound 3-hydroxybenzoate provide hints about oxygen activation and substrate hydroxylation. Essential roles are played by His-213 in catalysis and Tyr-105 in substrate binding. This phospholipid-assisted strategy to control regioselective aromatic hydroxylation is of relevance for optimization of flavin-dependent biocatalysts.

High-Entropy Diborides-Silicon Carbide Composites by Reactive and Non-Reactive Spark Plasma Sintering: A Comparative Study.

The reactive spark plasma sintering (R-SPS) method was compared in this work with the two-step SHS-SPS route, based on the combination of the self-propagating high-temperature synthesis (SHS) with the SPS process, for the fabrication of dense (Hf0.2Mo0.2Ti0.2Ta0.2Nb0.2)B2-SiC and (Hf0.2Mo0.2Ti0.2Ta0.2Zr0.2)B2-SiC ceramics. A multiphase and inhomogeneous product, containing various borides, was obtained at 2000 °C/20 min by R-SPS from transition metals, B4C, and Si. In contrast, if the same precursors were first reacted by SHS and then processed by SPS under the optimized condition of 1800 °C/20 min, the desired ceramics were successfully attained. The resulting sintered samples possessed relative densities above 97% and displayed uniform microstructures with residual oxide content <2.4 wt.%. The presence of SiC made the sintering temperature milder, i.e., 150 °C below that needed by the corresponding additive-free system. The fracture toughness was also markedly improved, particularly when considering the Nb-containing system processed at 1800 °C/20 min, whereas the fracture toughness progressively decreased (from 7.35 to 5.36 MPa m1/2) as the SPS conditions became more severe. SiC addition was found to inhibit the volatilization of metal oxides like MoO3 formed during oxidation experiments, thus avoiding mass loss in the ceramics. The benefits above also likely took advantage of the fact that the two composite constituents were synthesized in parallel, according to the SHS-SPS approach, rather than being produced separately and combined subsequently, so that strong interfaces between them were formed.

Correction: Pakhomova et al. High-Entropy Diborides-Silicon Carbide Composites by Reactive and Non-Reactive Spark Plasma Sintering: A Comparative Study. Materials 2024, 17, 718.

In the original publication [...].

Microalgae-derived Co3O4 nanomaterials for catalytic CO oxidation.

Efficient carbon monoxide oxidation is important to reduce its impacts on both human health and the environment. Following a sustainable synthesis route toward new catalysts, nanosized Co3O4 was synthesized based on extracts of microalgae: Spirulina platensis, Chlorella vulgaris, and Haematococcus pluvialis. Using the metabolites in the extract and applying different calcination temperatures (450, 650, 800 °C) led to Co3O4 catalysts with distinctly different properties. The obtained Co3O4 nanomaterials exhibited octahedral, nanosheet, and spherical morphologies with structural defects and surface segregation of phosphorous and potassium, originating from the extracts. The presence of P and K in the oxide nanostructures significantly improved their catalytic CO oxidation activity. When normalized by the specific surface area, the microalgae-derived catalysts exceeded a commercial benchmark catalyst. In situ studies revealed differences in oxygen mobility and carbonate formation during the reaction. The obtained insights may facilitate the development of new synthesis strategies for manufacturing highly active Co3O4 nanocatalysts.

Optimization of Brilliant Blue R photocatalytic degradation by silver nanoparticles synthesized using Chlorella vulgaris.

Synthesis of silver nanoparticles (Ag NPs) using microalgae is gaining recognition for its environmentally friendly and cost-effective nature while maintaining high activity of NPs. In the present study, Ag NPs were synthesized using a methanolic extract of Chlorella vulgaris and subjected to calcination. The X-ray diffraction (XRD) analysis showed a crystalline nature of the products with Ag2O and Ag phases with an average crystalline size of 16.07 nm before calcination and an Ag phase with 24.61 nm crystalline size after calcination. Fourier transform infrared spectroscopy (FTIR) revealed the capping functional groups on Ag NPs, while scanning electron microscopy (SEM) displayed their irregular morphology and agglomeration after calcination. The organic coating was examined by energy-dispersive X-ray spectroscopy (EDX) and thermogravimetric (TGA) analyses, confirming the involvement of the metabolites. The UV-Vis analysis showed a difference in optical properties due to calcination. Synthesized Ag NPs were applied for the photodegradation of hazardous dye Brilliant Blue R in visible light. Different values of light intensity, catalyst dose, initial dye concentration, and pH were tested to identify the optimal set of operating conditions. The highest degradation efficiency of 90.6% with an apparent rate constant of 0.04402 min-1 was achieved after 90 min of irradiation in the highest tested catalyst dosage.

Recombinant glutathione transferases from flufenacet-resistant black-grass (Alopecurus myosuroides Huds.) form different flufenacet metabolites and differ in their interaction with pre- and post-emergence herbicides.

BACKGROUND: Black-grass (Alopecurus myosuroides Huds.) has become a problematic weed in cereals in Europe. Besides resistance to post-emergent herbicides becoming increasingly widespread, enhanced metabolism of inhibitors of the synthesis of very-long-chain fatty acids (VLCFAs), such as flufenacet, is evolving. Yet, cross-resistance patterns and evolution of this resistance remains poorly understood. RESULTS: The cDNA sequences of five glutathione transferases (GSTs) upregulated in flufenacet resistant black-grass were identified and used for recombinant protein expression. Moderate to slow detoxification of flufenacet was verified for all candidate GSTs expressed in E. coli, and the most active protein produced flufenacet-alcohol instead of a glutathione conjugate, in the presence of reduced glutathione (GSH). Moreover, cross-resistance to other VLCFA-inhibitors e.g., acetochlor and pyroxasulfone and the ACCase inhibitor fenoxaprop was verified in vitro. Various other herbicides of different modes of action including VLCFA-inhibitors were not detoxified by the candidate GSTs. CONCLUSIONS: As several in planta upregulated GSTs detoxified flufenacet in vitro, the shift in sensitivity observed in black-grass populations, is likely a result of an additive effect. The polygenic character and the relatively low turnover rate of the individual GSTs may explain the slow evolution of flufenacet resistance. In addition, flufenacet resistance was accompanied by cross-resistance with some, but not all, herbicides of the same mode of action, and furthermore to the ACCase inhibitor fenoxaprop-ethyl. Hence, not only the rotation of herbicide modes of action, but also of individual active ingredients is important for resistance management. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Snapshots of enzymatic Baeyer-Villiger catalysis: oxygen activation and intermediate stabilization.

Baeyer-Villiger monooxygenases catalyze the oxidation of carbonylic substrates to ester or lactone products using NADPH as electron donor and molecular oxygen as oxidative reactant. Using protein engineering, kinetics, microspectrophotometry, crystallography, and intermediate analogs, we have captured several snapshots along the catalytic cycle which highlight key features in enzyme catalysis. After acting as electron donor, the enzyme-bound NADP(H) forms an H-bond with the flavin cofactor. This interaction is critical for stabilizing the oxygen-activating flavin-peroxide intermediate that results from the reaction of the reduced cofactor with oxygen. An essential active-site arginine acts as anchoring element for proper binding of the ketone substrate. Its positively charged guanidinium group can enhance the propensity of the substrate to undergo a nucleophilic attack by the flavin-peroxide intermediate. Furthermore, the arginine side chain, together with the NADP(+) ribose group, forms the niche that hosts the negatively charged Criegee intermediate that is generated upon reaction of the substrate with the flavin-peroxide. The fascinating ability of Baeyer-Villiger monooxygenases to catalyze a complex multistep catalytic reaction originates from concerted action of this Arg-NADP(H) pair and the flavin subsequently to promote flavin reduction, oxygen activation, tetrahedral intermediate formation, and product synthesis and release. The emerging picture is that these enzymes are mainly oxygen-activating and "Criegee-stabilizing" catalysts that act on any chemically suitable substrate that can diffuse into the active site, emphasizing their potential value as toolboxes for biocatalytic applications.

Joint functions of protein residues and NADP(H) in oxygen activation by flavin-containing monooxygenase.

The reactivity of flavoenzymes with dioxygen is at the heart of a number of biochemical reactions with far reaching implications for cell physiology and pathology. Flavin-containing monooxygenases are an attractive model system to study flavin-mediated oxygenation. In these enzymes, the NADP(H) cofactor is essential for stabilizing the flavin intermediate, which activates dioxygen and makes it ready to react with the substrate undergoing oxygenation. Our studies combine site-directed mutagenesis with the usage of NADP(+) analogues to dissect the specific roles of the cofactors and surrounding protein matrix. The highlight of this "double-engineering" approach is that subtle alterations in the hydrogen bonding and stereochemical environment can drastically alter the efficiency and outcome of the reaction with oxygen. This is illustrated by the seemingly marginal replacement of an Asn to Ser in the oxygen-reacting site, which inactivates the enzyme by effectively converting it into an oxidase. These data rationalize the effect of mutations that cause enzyme deficiency in patients affected by the fish odor syndrome. A crucial role of NADP(+) in the oxygenation reaction is to shield the reacting flavin N5 atom by H-bond interactions. A Tyr residue functions as backdoor that stabilizes this crucial binding conformation of the nicotinamide cofactor. A general concept emerging from this analysis is that the two alternative pathways of flavoprotein-oxygen reactivity (oxidation versus monooxygenation) are predicted to have very similar activation barriers. The necessity of fine tuning the hydrogen-bonding, electrostatics, and accessibility of the flavin will represent a challenge for the design and development of oxidases and monoxygenases for biotechnological applications.

Revealing the moonlighting role of NADP in the structure of a flavin-containing monooxygenase.

Flavin-containing monooxygenases (FMOs) are, after cytochromes P450, the most important monooxygenase system in humans and are involved in xenobiotics metabolism and variability in drug response. The x-ray structure of a soluble prokaryotic FMO from Methylophaga sp. strain SK1 has been solved at 2.6-A resolution and is now the protein of known structure with the highest sequence similarity to human FMOs. The structure possesses a two-domain architecture, with both FAD and NADP(+) well defined by the electron density maps. Biochemical analysis shows that the prokaryotic enzyme shares many functional properties with mammalian FMOs, including substrate specificity and the ability to stabilize the hydroperoxyflavin intermediate that is crucial in substrate oxygenation. On the basis of their location in the structure, the nicotinamide ring and the adjacent ribose of NADP(+) turn out to be an integral part of the catalytic site being actively engaged in the stabilization of the oxygenating intermediate. This feature suggests that NADP(H) has a moonlighting role, in that it adopts two binding modes that allow it to function in both flavin reduction and oxygen reactivity modulation, respectively. We hypothesize that a relative domain rotation is needed to bring NADP(H) to these distinct positions inside the active site. Localization of mutations in human FMO3 that are known to cause trimethylaminuria (fish-odor syndrome) in the elucidated FMO structure provides a structural explanation for their biological effects.

A methodology to investigate the intrinsic effect of the pulsed electric current during the spark plasma sintering of electrically conductive powders.

A novel methodology is proposed for investigating the effect of the pulsed electric current during the spark plasma sintering (SPS) of electrically conductive powders without potential misinterpretation of experimental results. First, ensemble configurations (geometry, size and material of the powder sample, die, plunger and spacers) are identified where the electric current is forced to flow only through either the sample or the die, so that the sample is heated either through the Joule effect or by thermal conduction, respectively. These ensemble configurations are selected using a recently proposed mathematical model of an SPS apparatus, which, once suitably modified, makes it possible to carry out detailed electrical and thermal analysis. Next, SPS experiments are conducted using the ensemble configurations theoretically identified. Using aluminum powders as a case study, we find that the temporal profiles of sample shrinkage, which indicate densification behavior, as well as the final density of the sample are clearly different when the electric current flows only through the sample or through the die containing it, whereas the temperature cycle and mechanical load are the same in both cases.

Electrospun PCL/PGS Composite Fibers Incorporating Bioactive Glass Particles for Soft Tissue Engineering Applications.

Poly(glycerol-sebacate) (PGS) and poly(epsilon caprolactone) (PCL) have been widely investigated for biomedical applications in combination with the electrospinning process. Among others, one advantage of this blend is its suitability to be processed with benign solvents for electrospinning. In this work, the suitability of PGS/PCL polymers for the fabrication of composite fibers incorporating bioactive glass (BG) particles was investigated. Composite electrospun fibers containing silicate or borosilicate glass particles (13-93 and 13-93BS, respectively) were obtained and characterized. Neat PCL and PCL composite electrospun fibers were used as control to investigate the possible effect of the presence of PGS and the influence of the bioactive glass particles. In fact, with the addition of PGS an increase in the average fiber diameter was observed, while in all the composite fibers, the presence of BG particles induced an increase in the fiber diameter distribution, without changing significantly the average fiber diameter. Results confirmed that the blended fibers are hydrophilic, while the addition of BG particles does not affect fiber wettability. Degradation test and acellular bioactivity test highlight the release of the BG particles from all composite fibers, relevant for all applications related to therapeutic ion release, i.e., wound healing. Because of weak interface between the incorporated BG particles and the polymeric fibers, mechanical properties were not improved in the composite fibers. Promising results were obtained from preliminary biological tests for potential use of the developed mats for soft tissue engineering applications.

First successful stabilization of consolidated amorphous calcium phosphate (ACP) by cold sintering: toward highly-resorbable reactive bioceramics.

In the field of bone regeneration, some clinical conditions require highly-resorbable, reactive bone substitutes to rapidly initiate tissue neo-formation. In this view, Amorphous Calcium Phosphates (ACP) appear as well suited bioceramics taking into account their high metastability. However, the metastability also leads to difficulties of sintering without transformation into crystalline compounds. In this work, various calcium phosphate samples (co)doped with carbonate (CO32-) and magnesium ions were synthesized by the double decomposition method in alkaline media using ammonium and potassium hydroxide solutions. The obtained amorphous powders possess an exceptionally-high carbonate content up to 18.3 wt%. Spark Plasma Sintering (SPS) at very low temperature (150 °C) was then utilized to consolidate initial powders with the view to preserve their amorphous character. The influence of the introduction of different apatite growth inhibitors such as carbonate (CO32-) and magnesium ions was studied. XRD and FTIR analyses revealed that sintered ceramics generally consisted in highly carbonated low-crystallinity apatites, which are expected to have higher solubility than conventional apatite-based systems. However, most interestingly, modulation of the doping conditions allowed us to retain, for the first time, the amorphous character of ACP powders after SPS. Such consolidated ACP compounds may now be considered as a new family of bioceramics with high metastability allowing the fast release of bioactive ions upon resorption.

IM30 IDPs form a membrane-protective carpet upon super-complex disassembly.

Members of the phage shock protein A (PspA) family, including the inner membrane-associated protein of 30 kDa (IM30), are suggested to stabilize stressed cellular membranes. Furthermore, IM30 is essential in thylakoid membrane-containing chloroplasts and cyanobacteria, where it is involved in membrane biogenesis and/or remodeling. While it is well known that PspA and IM30 bind to membranes, the mechanism of membrane stabilization is still enigmatic. Here we report that ring-shaped IM30 super-complexes disassemble on membranes, resulting in formation of a membrane-protecting protein carpet. Upon ring dissociation, the C-terminal domain of IM30 unfolds, and the protomers self-assemble on membranes. IM30 assemblies at membranes have been observed before in vivo and were associated with stress response in cyanobacteria and chloroplasts. These assemblies likely correspond to the here identified carpet structures. Our study defines the thus far enigmatic structural basis for the physiological function of IM30 and related proteins, including PspA, and highlights a hitherto unrecognized concept of membrane stabilization by intrinsically disordered proteins.

3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1 Contains a Phosphatidylinositol Cofactor.

3-Hydroxybenzoate 6-hydroxylase (3HB6H, EC 1.13.14.26) is a FAD-dependent monooxygenase involved in the catabolism of aromatic compounds in soil microorganisms. 3HB6H is unique among flavoprotein hydroxylases in that it harbors a phospholipid ligand. The purified protein obtained from expressing the gene encoding 3HB6H from Rhodococcus jostii RHA1 in the host Escherichia coli contains a mixture of phosphatidylglycerol and phosphatidylethanolamine, which are the major constituents of E. coli's cytoplasmic membrane. Here, we purified 3HB6H (RjHB6H) produced in the host R. jostii RHA#2 by employing a newly developed actinomycete expression system. Biochemical and biophysical analysis revealed that Rj3HB6H possesses similar catalytic and structural features as 3HB6H, but now contains phosphatidylinositol, which is a specific constituent of actinomycete membranes. Native mass spectrometry suggests that the lipid cofactor stabilizes monomer-monomer contact. Lipid analysis of 3HB6H from Pseudomonas alcaligenes NCIMB 9867 (Pa3HB6H) produced in E. coli supports the conclusion that 3HB6H enzymes have an intrinsic ability to bind phospholipids with different specificity, reflecting the membrane composition of their bacterial host.

Processing, Mechanical and Optical Properties of Additive-Free ZrC Ceramics Prepared by Spark Plasma Sintering.

In the present study, nearly fully dense monolithic ZrC samples are produced and broadly characterized from microstructural, mechanical and optical points of view. Specifically, 98% dense products are obtained by Spark Plasma Sintering (SPS) after 20 min dwell time at 1850 °C starting from powders preliminarily prepared by Self-propagating High-temperature Synthesis (SHS) followed by 20 min ball milling. A prolonged mechanical treatment up to 2 h of SHS powders does not lead to appreciable benefits. Vickers hardness of the resulting samples (17.5 ± 0.4 GPa) is reasonably good for monolithic ceramics, but the mechanical strength (about 250 MPa up to 1000 °C) could be further improved by suitable optimization of the starting powder characteristics. The very smoothly polished ZrC specimen subjected to optical measurements displays high absorption in the visible-near infrared region and low thermal emittance at longer wavelengths. Moreover, the sample exhibits goodspectral selectivity (2.1-2.4) in the 1000-1400 K temperature range. These preliminary results suggest that ZrC ceramics produced through the two-step SHS/SPS processing route can be considered as attractive reference materials for the development of innovative solar energy absorbers.

Tuning Cysteine Reactivity and Sulfenic Acid Stability by Protein Microenvironment in Glyceraldehyde-3-Phosphate Dehydrogenases of Arabidopsis thaliana.

AIMS: Cysteines and H2O2 are fundamental players in redox signaling. Cysteine thiol deprotonation favors the reaction with H2O2 that generates sulfenic acids with dual electrophilic/nucleophilic nature. The protein microenvironment surrounding the target cysteine is believed to control whether sulfenic acid can be reversibly regulated by disulfide formation or irreversibly oxidized to sulfinates/sulfonates. In this study, we present experimental oxidation kinetics and a quantum mechanical/molecular mechanical (QM/MM) investigation to elucidate the reaction of H2O2 with glycolytic and photosynthetic glyceraldehyde-3-phosphate dehydrogenase from Arabidopsis thaliana (cytoplasmic AtGAPC1 and chloroplastic AtGAPA, respectively). RESULTS: Although AtGAPC1 and AtGAPA have almost identical 3D structure and similar acidity of their catalytic Cys149, AtGAPC1 is more sensitive to H2O2 and prone to irreversible oxidation than AtGAPA. As a result, sulfenic acid is more stable in AtGAPA. INNOVATION: Based on crystallographic structures of AtGAPC1 and AtGAPA, the reaction potential energy surface for Cys149 oxidation by H2O2 was calculated by QM. In both enzymes, sulfenic acid formation was characterized by a lower energy barrier than sulfinate formation, and sulfonate formation was prevented by very high energy barriers. Activation energies for both oxidation steps were lower in AtGAPC1 than AtGAPA, supporting the higher propensity of AtGAPC1 toward irreversible oxidation. CONCLUSIONS: QM/MM calculations coupled to fingerprinting analyses revealed that two Arg of AtGAPA (substituted by Gly and Val in AtGAPC1), located at 8-15 Å distance from Cys149, are the major factors responsible for sulfenic acid stability, underpinning the importance of long-distance polar interactions in tuning sulfenic acid stability in native protein microenvironments.

Development of a molecular method for the rapid screening and identification of the three functionally relevant polymorphisms in the human TAS2R38 receptor gene in studies of sensitivity to the bitter taste of PROP.

The objective of this work was to develop a rapid screening method to identify the three single nucleotide polymorphisms (SNPs) in the TAS2R38 gene, with the aim of providing a significant contribution to studies designed to assess sensitivity to the bitter taste of 6-n-propylthiouracil (PROP). Specifically, the objective of this study was to characterize the TAS2R38 gene haplotypes in a group of 60 subjects with variable sensitivity to PROP and preliminarily genotyped for the rs2274333 allele (A/G) of carbonic anhydrase isoform VI gene (CA6). The molecular characterization of the TAS2R38 gene was conducted using the PCR-restriction fragment length polymorphism technique after creating artificial restriction sites upstream or downstream of the SNPs, as none of the three polymorphisms contributes to the formation of a restriction site for a specific endonuclease. The results indicate that the method described in this paper could be a valid and simple experimental strategy to identify genetic differences related to taste sensitivity to bitter taste, and could be applied as a nutrigenetics test in studies aimed at understanding people's eating behaviors.