Simultaneous analysis of DL-Amino acids in foods and beverages using a highly sensitive chiral resolution labeling reagent.

Amino acids with various functions are abundant in living organisms and foods. Recent advances in analytical technology show that trace amounts of D-amino acids exist in living organisms and foods. In addition, studies show that these amino acids are involved in various physiological functions that differ from those of L-amino acids. Thus, a technique for analyzing DL-amino acids is required. However, the simultaneous separation and highly sensitive detection of DL-amino acids are complicated; therefore, highly sensitive analytical methods that can rapidly separate and identify compounds are required. We previously developed our original chiral resolution labeling reagents for the separation and highly sensitive detection of DL-amino acids. Here, we developed a simple method for the rapid separation and highly sensitive detection of DL-amino acids in various foods and beverages by liquid chromatography-mass spectrometry (LC-MS) using an octadecyl (C18) column after labeling with 1-fluoro-2,4-dinitrophenyl-5-D-leucine-N,N-dimethylethylenediamineamide (D-FDLDA; enantiomeric excess > 99.9 %). In addition, we synthesized a stable isotope (13C6)-labeled D-FDLDA (13C6-D-FDLDA) and established an analytical method that can accurately identify the peak of each DL-amino acid. MS sensitivity of DL-amino acids labeled with our labeling reagent was higher than that of conventional labeling reagents (Marfey's reagents). The labeling reagent was neither desorbed from each DL-amino acid nor degraded for at least 1 week at 4 °C. Furthermore, we determined the DL-amino acid contents in foods and beverages using the proposed method, and differences in the total amino acid content and D/L ratio in each food and beverage were observed.

Computational Insights into SARS-CoV-2 Main Protease Mutations and Nirmatrelvir Efficacy: The Effects of P132H and P132H-A173V.

Nirmatrelvir, a pivotal component of the oral antiviral Paxlovid for COVID-19, targets the SARS-CoV-2 main protease (Mpro) as a covalent inhibitor. Here, we employed combined computational methods to explore how the prevalent Omicron variant mutation P132H, alone and in combination with A173V (P132H-A173V), affects nirmatrelvir's efficacy. Our findings suggest that P132H enhances the noncovalent binding affinity of Mpro for nirmatrelvir, whereas P132H-A173V diminishes it. Although both mutants catalyze the rate-limiting step more efficiently than the wild-type (WT) Mpro, P132H slows the overall rate of covalent bond formation, whereas P132H-A173V accelerates it. Comprehensive analysis of noncovalent and covalent contributions to the overall binding free energy of the covalent complex suggests that P132H likely enhances Mpro sensitivity to nirmatrelvir, while P132H-A173V may confer resistance. Per-residue decompositions of the binding and activation free energies pinpoint key residues that significantly affect the binding affinity and reaction rates, revealing how the mutations modulate these effects. The mutation-induced conformational perturbations alter drug-protein local contact intensities and the electrostatic preorganization of the protein, affecting noncovalent binding affinity and the stability of key reaction states, respectively. Our findings inform the mechanisms of nirmatrelvir resistance and sensitivity, facilitating improved drug design and the detection of resistant strains.

Vesicle-like Nanocapsules Formed by Self-Assembly of Peptides with Oligoproline and -Leucine.

Since drug carriers are envisaged to be used in a wide variety of situations and environments, nanocarriers with diverse properties, such as biocompatibility, biodegradability, nonimmunogenicity, adequate particle size, robustness, and cell permeability, are required. Here, we report the construction of novel nanocapsules with the above-mentioned features by the self-assembly of peptides composed of oligoproline and oligoleucine (i.e., H-Pro10Leu4-NH2 and H-Pro10Leu6-NH2). The peptides self-organized via hydrogen bonds and hydrophobic interactions between oligoleucine moieties to form vesicle-like nanocapsules with cationic oligoproline exposed on the surface. The guest encapsulation experiments revealed that the nanocapsules were capable of uptake of both water-soluble and insoluble compounds. Furthermore, positively charged and/or oligoproline-based peptides are known to improve cell permeability and cellular uptake, suggesting that the peptide nanocapsules are good candidates for nanocarriers to complement liposomes and polymer micelles.

Size-Dependent Fullerenes for Enhanced Interaction of l-Leucine: A Combined DFT and MD Simulations Approach.

Fullerene-based biosensors have received great attention due to their unique electronic properties that allow them to transduce electrical signals by accepting electrons from amino acids. Babies with MSUD (maple syrup urine disease) are unable to break down amino acids such as l-leucine, and excess levels of the l-leucine are harmful. Therefore, sensing of l-leucine is foremost required. We aim to investigate the interaction tendencies of size-variable fullerenes (CX; X = 24, 36, 50, and 70) toward l-leucine (LEU) using density functional theory (DFT-D3) and classical molecular dynamics (MD) simulation. The C24 fullerene shows the highest affinity of the LEU biomolecule in the gas phase. Smaller fullerenes (C24 and C36) show stronger interactions with leucine due to their higher curvature in water environments. Moreover, recovery times in the ranges of 1010 and 104 s make it a viable candidate for the isolation application of LEU from the biological system. Further, the interaction between LEU and fullerenes is in line with the natural bond order (NBO) analysis, Mulliken charge analysis, quantum theory atom in molecule (QTAIM) analysis, and reduced density gradient (RDG) analysis. At 310 K, employing the explicit water model in classical MD simulations, fullerenes C24 and C36 demonstrate notably elevated binding free energies (-24.946 kJ/mol) in relation to LEU, showcasing their potential as sensors for l-leucine. Here, we demonstrate that the smaller fullerene exhibits a higher potential for l-leucine sensors than the larger fullerene.

Conformational Preferences of the Non-Canonical Amino Acids (2S,4S)-5-Fluoroleucine, (2S,4R)-5-Fluoroleucine, and 5,5'-Difluoroleucine in a Protein.

Proteins produced with leucine analogues, where CH2F groups substitute specific methyl groups, can readily be probed by 19F NMR spectroscopy. As CF and CH groups are similar in hydrophobicity and size, fluorinated leucines are expected to cause minimal structural perturbation, but the impact of fluorine on the rotational freedom of CH2F groups is unclear. We present high-resolution crystal structures of Escherichia coli peptidyl-prolyl cis-trans isomerase B (PpiB) prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5'-difluoroleucine. Apart from the fluorinated leucine residues, the structures show complete structural conservation of the protein backbone and the amino acid side chains except for a single isoleucine side chain located next to a fluorine atom in the hydrophobic core of the protein. The carbon skeletons of the fluorinated leucine side chains are also mostly conserved. The CH2F groups show a strong preference for staggered rotamers and often appear locked into single rotamers. Substitution of leucine CH3 groups for CH2F groups is thus readily tolerated in the three-dimensional (3D) structure of a protein, and the rotation of CH2F groups can be halted at cryogenic temperatures.

(2S,4S)-5-Fluoroleucine, (2S,4R)-5-Fluoroleucine, and 5,5'-Difluoroleucine in Escherichia coli PpiB: Protein Production, 19F NMR, and Ligand Sensing Enhanced by the γ-Gauche Effect.

Global substitution of leucine for analogues containing CH2F instead of methyl groups delivers proteins with multiple sites for monitoring by 19F nuclear magnetic resonance (NMR) spectroscopy. The 19 kDa Escherichia coli peptidyl-prolyl cis-trans isomerase B (PpiB) was prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5'-difluoroleucine. The stability of the samples toward thermal denaturation was little altered compared to the wild-type protein. 19F nuclear magnetic resonance (NMR) spectra showed large chemical shift dispersions between 6 and 17 ppm. The 19F chemical shifts correlate with the three-bond 1H-19F couplings (3JHF), providing the first experimental verification of the γ-gauche effect predicted by [Feeney, J. J. Am. Chem. Soc. 1996, 118, 8700-8706] and establishing the effect as the predominant determinant of the 19F chemical shifts of CH2F groups. Individual CH2F groups can be confined to single rotameric states by the protein environment, but most CH2F groups exchange between different rotamers at a rate that is fast on the NMR chemical shift scale. Interactions between fluorine atoms in 5,5'-difluoroleucine bias the CH2F rotamers in agreement with results obtained previously for 1,3-difluoropropane. The sensitivity of the 19F chemical shift to the rotameric state of the CH2F groups potentially renders them particularly sensitive for detecting allosteric effects.

Unravelling caramelization and Maillard reactions in glucose and glucose + leucine model cakes: Formation and degradation kinetics of volatile markers extracted during baking.

A large number of volatile compounds are formed during the baking of foods by reactions such as caramelization and Maillard reactions. Elucidating the reaction mechanisms may be useful to predict and control food quality. Ten reaction volatile markers were extracted during baking of solid model cakes implemented with known amounts of precursors (glucose with or without leucine) and then quantified by Thermal desorption-Gas chromatography-Mass spectrometry. The kinetic data showed that the level of air convection in the oven had no significant influence on the reaction rates. In contrast, increasing baking temperatures had a nonlinear accelerating impact on the generation of newly formed volatile compounds with a bell-shaped kinetic curve found for most of the markers at 200 °C. The presence of leucine triggered the activation of the Maillard and Strecker routes with a specific and very rapid formation of 3-Methylbutanal and pyrazines. A dynamic model was developed, combining evaporation flow rate and kinetic formation and consumption of reaction markers. It can be used to describe, for two furanic compounds of different volatilities, the vapor concentrations in the oven from the concentrations measured in the model cakes.

Synthesis, taste characteristics and taste mechanism of N-lactoyl leucine from soy sauce using sensory analysis and UPLC-MS/MS.

Recently, amino acid derivatives gradually gained attention, but studies on N-lactoyl-leucine (Lac-Leu) and N-lactoyl-isoleucine (Lac-Ile) are limited. This study aims to explore the contributions of Lac-Leu and Lac-Ile to soy sauce. Lac-Leu and Lac-Ile were synthesized via enzymatic synthesis method catalyzed by Tgase. The mixed solutions containing Lac-Leu were found to have greater taste improvement than those containing Lac-Ile. Sensory evaluation indicated the sour, bitter, and astringent taste of Lac-Leu in water as well as its kokumi, astringent, and umami-enhancing taste in MSG solution. The taste threshold and umami-enhancing threshold of Lac-Leu measured by TDA and cTDA, respectively, were 0.08 mg/mL and 0.16 mg/mL. Molecular docking of Lac-Leu and Lac-Ile with the kokumi receptor CaSR and the umami receptors T1R1 and T1R3 indicated that Lac-Leu had higher affinities with receptors than Lac-Ile. These findings demonstrated the underlying contribution Lac-Leu made to soy sauce, indicating its potential to improve the flavor quality of soy sauce.

Stabilization of Prebiotic Vesicles by Peptides Depends on Sequence and Chirality: A Mechanism for Selection of Protocell-Associated Peptides.

Cells require oligonucleotides and polypeptides with specific, homochiral sequences to perform essential functions, but it is unclear how such oligomers were selected from random sequences at the origin of life. Cells were probably preceded by simple compartments such as fatty acid vesicles, and oligomers that increased the stability, growth, or division of vesicles could have thereby increased in frequency. We therefore tested whether prebiotic peptides alter the stability or growth of vesicles composed of a prebiotic fatty acid. We find that three of 15 dipeptides tested reduce salt-induced flocculation of vesicles. All three contain leucine, and increasing their length increases the efficacy. Also, leucine-leucine but not alanine-alanine increases the size of vesicles grown by multiple additions of micelles. In a molecular simulation, leucine-leucine docks to the membrane, with the side chains inserted into the hydrophobic core of the bilayer, while alanine-alanine fails to dock. Finally, the heterochiral forms of leucine-leucine, at a high concentration, rapidly shrink the vesicles and make them leakier and less stable to high pH than the homochiral forms do. Thus, prebiotic peptide-membrane interactions influence the flocculation, growth, size, leakiness, and pH stability of prebiotic vesicles, with differential effects due to sequence, length, and chirality. These differences could lead to a population of vesicles enriched for peptides with beneficial sequence and chirality, beginning selection for the functional oligomers that underpin life.

Genome Mining of a Fungal Polyketide Synthase-Nonribosomal Peptide Synthetase Hybrid Megasynthetase Pathway to Synthesize a Phytotoxic N-Acyl Amino Acid.

Guided by the retrobiosynthesis hypothesis, we characterized a fungal polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) hybrid megasynthetase pathway to generate 2-trans-4-trans-2-methylsorbyl-d-leucine (1), a polyketide amino acid conjugate that inhibits Arabidopsis root growth. The biosynthesis of 1 includes a PKS-NRPS enzyme to assemble an N-acyl amino alcohol intermediate, which is further oxidized to an N-acyl amino acid (NAAA), demonstrating a new biosynthetic logic for synthesizing NAAAs and expanding the chemical space of products encoded by fungal PKS-NRPS clusters.

Triple combination dry powder formulation of pretomanid, moxifloxacin, and pyrazinamide for treatment of multidrug-resistant tuberculosis.

Both latent and multidrug-resistant tuberculosis (TB) have been causing significant concern worldwide. A novel drug, pretomanid (PA-824), has shown a potent bactericidal effect against both active and latent forms of Mycobacterium tuberculosis (MTb) and a synergistic effect when combined with pyrazinamide and moxifloxacin. This study aimed to develop triple combination spray dried inhalable formulations composed of antitubercular drugs, pretomanid, moxifloxacin, and pyrazinamide (1:2:8 w/w/w), alone (PaMP) and in combination with an aerosolization enhancer, L-leucine (20 % w/w, PaMPL). The formulation PaMPL consisted of hollow, spherical, dimpled particles (<5 µm) and showed good aerosolization behaviour with a fine particle fraction of 70 %. Solid-state characterization of formulations with and without L-leucine confirmed the amorphous nature of moxifloxacin and pretomanid and the crystalline nature of pyrazinamide with polymorphic transformation after the spray drying process. Further, the X-ray photoelectron spectroscopic analysis revealed the predominant surface composition of L-leucine on PaMPL dry powder particles. The dose-response cytotoxicity results showed pyrazinamide and moxifloxacin were non-toxic in both A549 and Calu-3 cell lines up to 150 µg/mL. However, the cell viability gradually decreased to 50 % when the pretomanid concentration increased to 150 µg/mL. The in vitro efficacy studies demonstrated that the triple combination formulation had more prominent antibacterial activity with a minimum inhibitory concentration (MIC) of 1 µg/mL against the MTb H37Rv strain as compared to individual drugs. In conclusion, the triple combination of pretomanid, moxifloxacin, and pyrazinamide as an inhalable dry powder formulation will potentially improve treatment efficacy with fewer systemic side effects in patients suffering from latent and multidrug-resistant TB.

13 Cß-Valine and 13 Cγ-Leucine Methine Labeling To Probe Protein Ligand Interaction.

Precise information regarding the interaction between proteins and ligands at molecular resolution is crucial for effectively guiding the optimization process from initial hits to lead compounds in early stages of drug development. In this study, we introduce a novel aliphatic side chain isotope-labeling scheme to directly probe interactions between ligands and aliphatic sidechains using NMR techniques. To demonstrate the applicability of this method, we selected a set of Brd4-BD1 binders and analyzed 1 H chemical shift perturbation resulting from CH-π interaction of Hß -Val and Hγ -Leu as CH donors with corresponding ligand aromatic moieties as π acceptors.

Leucine Motifs Stabilize Residual Helical Structure in Disordered Proteins.

Many examples are known of regions of intrinsically disordered proteins that fold into α-helices upon binding to their targets. These helical binding motifs (HBMs) can be partially helical also in the unbound state, and this so-called residual structure can affect binding affinity and kinetics. To investigate the underlying mechanisms governing the formation of residual helical structure, we assembled a dataset of experimental helix contents of 65 peptides containing HBM that fold-upon-binding. The average residual helicity is 17% and increases to 60% upon target binding. The helix contents of residual and target-bound structures do not correlate, however the relative location of helix elements in both states shows a strong overlap. Compared to the general disordered regions, HBMs are enriched in amino acids with high helix preference and these residues are typically involved in target binding, explaining the overlap in helix positions. In particular, we find that leucine residues and leucine motifs in HBMs are the major contributors to helix stabilization and target-binding. For the two model peptides, we show that substitution of leucine motifs to other hydrophobic residues (valine or isoleucine) leads to reduction of residual helicity, supporting the role of leucine as helix stabilizer. From the three hydrophobic residues only leucine can efficiently stabilize residual helical structure. We suggest that the high occurrence of leucine motifs and a general preference for leucine at binding interfaces in HBMs can be explained by its unique ability to stabilize helical elements.

Evaluation of protein undernourishment on the condylar process of the Wistar rat mandible correlation with insulin receptor expression

The mandible condylar process cartilage (CP) of Wistar rats is a secondary cartilage and acts as a mandibular growth site. This phenomenon depends on adequate proteins intake and hormone actions, including insulin. Objectives The present study evaluated the morphological aspects and the expression of the insulin receptor (IR) in the cartilage of the condylar process (CP) of rats subjected to protein undernourishment. Material and Methods The nourished group received a 20% casein diet, while the undernourished group (U) received a 5% casein diet. The re-nourished groups, R and RR, were used to assess the effects of re-nutrition during puberty and adulthood, respectively. CPs were processed and stained with picro-sirius red, safranin-O and azocarmine. Scanning electron microscopy and immunohistochemistry were also performed. Results The area of the CP cartilage and the number of cells in the chondroblastic layer decreased in the U group, as did the thickness of the CP layer in the joint and hypertrophic layer. Renourishment during the pubertal stage, but not during the adult phase, restored these parameters. The cell number was restored when re-nutrition occurred in the pubertal stage, but not in the adult phase. The extracellular matrix also decreased in the U group, but was restored by re-nutrition during the pubertal stage and further increased in the adult phase. IR expression was observed in all CPs, being higher in the chondroblastic and hypertrophic cartilage layers. The lowest expression was found in the U and RR groups. Conclusions Protein malnutrition altered the cellularity, the area, and the fibrous cartilage complex, as well as the expression of the IRs. .

Estratégias para o enfrentamento do absenteísmo em consultas odontológicas nas Unidades de Saúde da Família de um município de grande porte: uma pesquisa-ação / Strategies for tackling absenteeism in dental appointments in the Family Health Units of a large municipality: action research

O objetivo deste trabalho foi analisar os motivos das faltas às consultas odontológicas em Unidades de Saúde da Família (USF) e implementar estratégias para sua redução por meio da pesquisa-ação. O estudo foi realizado em 12 USF de Piracicaba/SP, de 01 de janeiro a 31 de dezembro de 2010. A amostra se consistiu de 385 usuários, entrevistados por telefone, sobre os motivos das faltas, além de 12 cirurgiões-dentistas e 12 enfermeiras. Realizaram-se duas oficinas com os profissionais: uma para problematização dos dados coletados nas entrevistas e elaboração de estratégias; e outra após 4 meses, para avaliação. O maior motivo de faltas foi a coincidência do horário de funcionamento das unidades com o de trabalho dos usuários. Dentre as estratégias ressaltou-se a realização de palestras sobre saúde bucal, educação permanente nas reuniões de equipe, capacitação dos Agentes Comunitários de Saúde, participação em grupos terapêuticos e parcerias entre Equipe de Saúde Bucal e equipamentos sociais da comunidade. A adoção de prontuário único foi a estratégia desafiadora encontrada pelos profissionais. Concluiu-se que as estratégias implementadas levaram à diminuição das faltas em 66,6% e o caráter motivador das oficinas possibilitou a reflexão crítica para o redirecionamento da prática em saúde.

The aim of this study was to analyze the reasons for missed appointments in dental Family Health Units (FHU) and implement strategies to reduce same through action research. This is a study conducted in 12 FHUs in Piracicaba in the State of São Paulo from January, 1 to December, 31 2010. The sample was composed of 385 users of these health units who were interviewed over the phone and asked about the reasons for missing dental appointments, as well as 12 dentists and 12 nurses. Two workshops were staged with professionals: the first to assess the data collected in interviews and develop strategy, and the second for evaluation after 4 months. The primary cause for missed appointments was the opening hours of the units coinciding with the work schedule of the users. Among the strategies suggested were lectures on oral health, ongoing education in team meetings, training of Community Health Agents, participation in therapeutic groups and partnerships between Oral Health Teams and the social infrastructure of the community. The adoption of the single medical record was the strategy proposed by professionals. The strategies implemented led to a 66.6% reduction in missed appointments by the units and the motivating nature of the workshops elicited critical reflection to redirect health practices.

Comer en tiempos de "crisis": nuevos contextos alimentarios y de salud en España / Eating in a time of "crisis": New health and dietary contexts in Spain

En las sociedades industrializadas se está reflexionando cada vez más sobre el impacto de la inseguridad alimentaria, entendida como la dificultad para asegurar la accesibilidad de una parte de la población a los recursos alimentarios suficientes para garantizar su subsistencia y bienestar. Con base en datos recogidos a partir de una investigación en curso en España, este artículo discute, por un lado, si la actual crisis económica está revirtiendo algunas de las tendencias positivas que el sistema agroalimentario industrial había favorecido, como la disminución de las diferencias sociales en el consumo y el derecho a la alimentación. Por otro lado, reflexiona acerca de la creciente precarización en las estrategias alimentarias y en el estado de salud de la población, así como sobre la necesidad de considerar la desigualdad social como variable explicativa de las diversas maneras de alimentarse.

This article analyzes the reasons why food insecurity in Spain must increasingly be understood as lack of access to sufficient food resources to guarantee the survival and wellbeing of part of the population. Using data collected in an ongoing research project, two possible causes for this are explored. First, it is argued that certain positive developments that seemed firmly established, such as recognition of the right to an adequate diet and the leveling out of social differences in food consumption, are now being reversed by the current economic crisis. Second, the analysis focuses on strategies people in precarious circumstances use to obtain food, their relationship to health, and the need to take social inequality into consideration as an explanatory variable in accounting for different ways of procuring daily sustenance.