Amino Acid Residues Research Articles

Design, synthesis, molecular docking, dynamics simulations and antiviral activities of quinoline derivatives

The designed and synthesized quinoline derivatives were screened for their anti-RSV, anti-YFV and broad spectrum anti-viral activity. Initially, physicochemical and drug-like properties of all compounds were evaluated then molecular docking and molecular dynamics (MD) simulations were performed on the active site of RSV G (glycoproteins) and Mtase (methyltransferase) enzyme against RSV and YFV respectively. All compounds interacted well with amino acid residues Thr173 (2.85 Å), Ser123 (3.02 Å), Gln89 (3.00 Å), Tyr34 (3.62 Å), Val147 (3.08 Å), Leu46 (3.03 Å), Tyr34 (3.62 Å), Trp96 (2.70 Å) of RSV G protein and with amino acid residues Ile124 (2.94 Å), Phe126 (3.08 Å), Tyr89 (3.77 Å), Arg41 (2.91 Å), Arg37 (2.89 Å), Arg84 (2.97 Å) of the MTase protein, showing their nature as probable inhibitors of RSV and YFV. Antiviral screening revealed that compound 4 was an effective inhibitor against RSV, showing an SI value of 11.6 and an EC50 of 8.6 µg/mL (3.2 × 103 µM), while compound 6 was effective against YFV, with an SI value of 28.5 and an EC50 of 3.5 µg/mL (1.17 × 103 µM). Molecular dynamics parameters, such as RMSD, RMSF, and RGyr, were satisfactory for the 6BLH:4 complex, with a predicted binding energy of -5.64 kcal/mol, which was lower than that of the 6BLH: ribavirin complex (-4.13 kcal/mol). Similarly, the MD parameters for the 3EVA:6 complex were also highly satisfactory. Therefore, compounds 4 and 6 may serve as potential inhibitors against RSV and YFV, respectively.

Abstract Tu149: FDA-Approved Antidepressant Trazodone Potentially Increases the Risk of Dyslipidemia

Cardiovascular disease is the leading cause of death. Many cardiovascular health problems, such as atherosclerosis, are caused by dyslipidemia. One xenobiotic nuclear receptor, Pregnane X Receptor (PXR), plays a significant role in atherosclerosis and dyslipidemia, and is activated by various environmental chemicals, including endocrine-disrupting chemicals (EDCs). EDCs are found in common household items such as plastics, medications, and food. Trazodone is a clinically used medication to treat depression by aiding in restoring the balance of serotonin in the brain. But it is unclear if Trazodone has possible impacts on cardiovascular risk factors such as dyslipidemia. Our preliminary data suggested that Trazodone activated human PXR in both intestinal (LS180) and hepatic (HepG2) cells. We hypothesize that Trazodone could regulate the cholesterol uptake mediated by PXR signaling. In this study we use cell-based transfection assay to evaluate the underlying mechanisms by which Trazodone activates PXR. We found that Trazodone was a more potent agonist of human PXR than mouse PXR. Trazodone could activate PXR more intensely in human liver cells compared with human intestinal cells. Our data suggested that Trazodone was a selective PXR agonist and promoted the dissociation between PXR and its nuclear corepressors. Next, we are to identify the key amino acid residues within PXR ligand binding pocket that interact with Trazodone by using computational docking study along with site-mutagenesis assay. Furthermore, we plan to estimate if Trazodone altered cholesterol uptake by human intestinal cells using fluorescence-labeled cholesterol. In conclusion, we explore the potential molecular mechanisms of how FDA-approved antidepressant Trazodone activates human PXR and increases the possible risk of dyslipidemia, which provides potential evidence on future cardiovascular disease risk assessment for Trazodone as well as other antidepressant drugs.

Structural, Biochemical Characterization and Molecular Mechanism of Cerastokunin: A New Kunitz-Type Peptide with Potential Inhibition of Thrombin, Factor Xa and Platelets.

The current investigation focused on separating Cerastes cerastes venom to produce the first Kunitz-type peptide. Based on its anti-trypsin effect, Cerastokunin, a 7.75kDa peptide, was purified until homogenity by three steps of chromatography. Cerastokunin was found to include 67 amino acid residues that were obtained by de novo sequencing using LC-MALDI-MSMS. Upon alignment with Kunitz-type peptides, there was a high degree of similarity. Cerastokunin's 3D structure had 12% α-helices and 21% β-strands with pI 8.48. Cerastokunin showed a potent anticoagulant effect by inhibiting the protease activity of thrombin and trypsin as well as blocking the intrinsic and extrinsic coagulation pathways. In both PT and aPPT, Cerastokunin increased the blood clotting time in a dose-dependent way. Using Lys48 and Gln192 for direct binding, Cerastokunin inhibited thrombin, Factor Xa and trypsin as shown by molecular docking. Cerastokunin exhibited a dose-response blockade of PARs-dependent pathway platelet once stimulated by thrombin. An increased concentration of Cerastokunin resulted in a larger decrease of tail thrombus in the mice-carrageenan model in an in vivo investigation when compared to the effects of antithrombotic medications. At all Cerastokunin doses up to 6mg/kg, no in vivo toxicity was seen in challenged mice over the trial's duration.

Cationic homopolypeptides: A versatile tool to design multifunctional antimicrobial nanocoatings

Postoperative infections are the most common complications faced by surgeons after implant surgery. To address this issue, an emerging and promising approach is to develop antimicrobial coatings using antibiotic substitutes. We investigated the use of polycationic homopolypeptides in a layer-by-layer coating combined with hyaluronic acid (HA) to produce an effective antimicrobial shield. The three peptide-based polycations used to make the coatings, poly(l-arginine) (PAR), poly(l-lysine), and poly(l-ornithine), provided an efficient antibacterial barrier by a contact-killing mechanism against Gram-positive, Gram-negative, and antibiotic-resistant bacteria. Moreover, this activity was higher for homopolypeptides containing 30 amino-acid residues per polycation chain, emphasizing the impact of the polycation chain length and its mobility in the coatings to deploy its contact-killing antimicrobial properties. However, the PAR-containing coating emerged as the best candidate among the three selected polycations, as it promoted cell adhesion and epithelial monolayer formation. It also stimulated nitric oxide production in endothelial cells, thereby facilitating angiogenesis and subsequent tissue regeneration. More interestingly, bacteria did not develop a resistance to PAR and (PAR/HA) also inhibited the proliferation of eukaryotic pathogens, such as yeasts. Furthermore, in vivo investigations on a (PAR/HA)-coated hernia mesh implanted on a rabbit model confirmed that the coating had antibacterial properties without causing chronic inflammation. These impressive synergistic activities highlight the strong potential of PAR/HA coatings as a key tool in combating bacteria, including those resistant to conventional antibiotics and associated to medical devices.

Understanding protein adsorption on silica mesoporous materials through thermodynamic simulations

This work presents a molecular thermodynamic theory to study protein interactions within a charge-regulating silica-like nanopore structure. The theory accounts for electrostatic interactions, steric repulsions, finite-size entropic effects, and protonation equilibrium of silanol groups on silica and protein residue side-chains. Coarse-grained representations of proteins based on their 3D crystallographic structures are employed. We evaluate the influence of pH, salt concentration, and pore size on the adsorption of selected proteins (cytochrome c, lysozyme, and myoglobin) in both single- and binary-protein solutions. The surface charge density on the nanopore walls is less negative than on the adjacent planar surface, primarily influenced by pH and salt concentration. Adsorption within the nanopore from single protein solutions exhibits a non-monotonic behavior with pH, increasing with decreasing salt concentration and diminishing pore size. Analysis of the effective interactions between proteins and silica surfaces indicates that adsorption is enhanced when the protein size matches that of the nanopore. Evaluation of various adsorption pathways indicates minimized free energy when the protein enters the nanopore through its edge and contacts its cylindrical walls. In binary solutions, the presence of another protein significantly alters the affinity of a protein for the silica surfaces, potentially preventing its adsorption, and affects its organization on these surfaces upon adsorption.

Hydrogen-Bonding and Hydrophobic Interaction Networks as Structural Determinants of Microbial Rhodopsin Function.

Microbial pump rhodopsins are highly versatile light-driven membrane proteins that couple protein conformational dynamics with ion translocation across the cell membranes. Understanding how microbial pump rhodopsins use specific amino acid residues at key functional sites to control ion selectivity and ion pumping direction is of general interest for membrane transporters, and could guide site-directed mutagenesis for optogenetics applications. To enable direct comparisons between proteins with different sequences we implement, for the first time, a unique numbering scheme for the microbial pump rhodopsin residues, NS-mrho. We use NS-mrho to show that distinct microbial pump rhodopsins typically have hydrogen-bond networks that are less conserved than anticipated from the amino acid residue conservation, whereas their hydrophobic interaction networks are largely conserved. To illustrate the role of the hydrogen-bond networks as structural elements that determine the functionality of microbial pump rhodopsins, we performed experiments, atomic-level simulations, and hydrogen bond network analyses on GR, the outward proton pump from Gloeobacter violaceus, and KR2, the outward sodium pump from Krokinobacter eikastus. The experiments indicate that multiple mutations that recover KR2 amino acid residues in GR not only fail to convert it into a sodium pump, but completely inactivate GR by abolishing photoisomerization of the retinal chromophore. This observation could be attributed to the drastically altered hydrogen-bond interaction network identified with simulations and network analyses. Taken together, our findings suggest that functional specificity could be encoded in the collective hydrogen-bond network of microbial pump rhodopsins.

Functional roles of the interaction of Moa1 with CENP-C and Rec8 in meiosis of Schizosaccharomyces pombe.

The localization of the meiotic specific regulatory molecule Moa1 to the centromere is regulated by the kinetochore protein CENP-C, and participates in the cohesion of sister chromatids in the centromere region mediated by the cohesin Rec8. To examine the interaction of these proteins, we analyzed the interactions between Moa1 and Rec8, CENP-C by yeast two-hybrid assays and identified several amino acid residues in Moa1 required for the interaction with CENP-C and Rec8. The results revealed that the interaction between Moa1 and CENP-C is crucial for the Moa1 to participate in the regulation of monopolar attachment of sister kinetochores. However, mutation at S143 and T150 of Moa1, which are required for interaction with Rec8 in the two-hybrid assay, did not show significant defects. Mutations in amino acid residues may not be sufficient to interfere with the interaction between Moa1 and Rec8 in vivo. Further research is needed to determine the interaction domain between Moa1 and Rec8. This study revealed specific amino acid sites at which Moa1 affects the meiotic homologous chromosome segregation, providing a deeper understanding of the mechanism of meiotic chromosome segregation.

Dissecting the role of the MS-ring protein FliF in Bacillus cereus flagella-related functions.

The flagellar MS-ring, uniquely constituted by FliF, is essential for flagellar biogenesis and functionality in several bacteria. The aim of this study was to dissect the role of FliF in the Gram-positive and peritrichously flagellated Bacillus cereus. We demonstrate that fliF forms an operon with the upstream gene fliE. In silico analysis of B. cereus ATCC 14579 FliF identifies functional domains and amino acid residues that are essential for protein functioning. The analysis of a ΔfliF mutant of B. cereus, constructed in this study using an in frame markerless gene replacement method, reveals that the mutant is unexpectedly able to assemble flagella, although in reduced amounts compared to the parental strain. Nevertheless, motility is completely abolished by fliF deletion. FliF deprivation causes the production of submerged biofilms and affects the ability of B. cereus to adhere to gastrointestinal mucins. We additionally show that the fliF deletion does not compromise the secretion of the three components of hemolysin BL, a toxin secreted through the flagellar type III secretion system. Overall, our findings highlight the important role of B. cereus FliF in flagella-related functions, being the protein required for complete flagellation, motility, mucin adhesion, and pellicle biofilms.

Molecular detection of emerging porcine circovirus in Taiwan

Porcine Circovirus type (PCV) 2 is an important pathogen that has been circulating worldwide and has cuased serious economic loss in pig industry. However, both PCV3 and PCV4 are newly emerging viruses. In Taiwan, PCV2 has been one of the critical pathogens in pig frams and PCV3 has been detected since 2016; however, the epidemiolog of PCV3 in Taiwan remains unclear and PCV4 has yet to be identified. Therefore, in order to detect the positive rate of PCV2, to investigate the epidemiolog of PCV3 in the pig farms, and to examine whether pigs were infected with PCV4 in Taiwan, a total of 128 samples from 46 clinical cases of pigs were collected from September 2020 to December 2021. The case detection rates were 54.3 % for PCV2, 43.5 % for PCV3, and 2.2 % for PCV4. The results suggested that the positivity rates for both PCV2 and PCV3 were still high in Taiwan. In addition, PCV3 was detected among cases from all 7 sampled counties and in 11 of the 16 sampling months, suggesting that PCV3 may lead to endemic pig disease in Taiwan. Surprisingly, the PCV4 was also detected, suggesting the first PCV4 case in Taiwan. The complete genomes derived from the identified PCV3 and PCV4 strains were subsequently sequenced followed by phylogenetic analysis. The results suggested that the 17 identified PCV3 strains could be divided into Taiwanese-like and Japanese-like strains. In addition, the amino acid residues at positions 27, 80, and 212 in the identified PCV4 cap protein were asparagine, isoleucine, and methionine, respectively, and thus the identified PCV4 was catalorized into clade PCV4b. Consequently, it is concluded that (i) the prevalence of PCV2 and PCV3 is still high in Taiwanese pigs, (ii) PCV3 has may be an endemic infection in Taiwan and can be classified into Japanese-like and Taiwanese-like strains, (iii) PCV4 was detected for the first time in Taiwanese pigs and can be classified into PCV4b. It remains unclear how PCV2, PCV3, and PCV4 were introduced to Taiwan, and thus continuous investigation of emerging pathogens in pigs is needed.

Red/green cyanobacteriochromes acquire isomerization from phycocyanobilin to phycoviolobilin.

Cyanobacteriochromes (CBCRs) are unique cyanobacteria-specific photoreceptors that share a distant relation with phytochromes. Most CBCRs contain conserved cysteine residues known as canonical Cys, while some CBCRs have additional cysteine residues called second Cys within the DXCF motif, leading to their classification as DXCF CBCRs. They typically undergo a process where they incorporate phycocyanobilin (PCB) and subsequently isomerize it to phycoviolobilin (PVB). Conversely, CBCRs with conserved Trp residues and without the second Cys are called extended red/green (XRG) CBCRs. Typical XRG CBCRs bind PCB without undergoing PCB-to-PVB isomerization, displaying red/green reversible photoconversion, and there are also atypical CBCRs that exhibit diverse photoconversions. We discovered novel XRG CBCRs with Cys residue instead of the conserved Trp residue. These novel XRG CBCRs exhibited the ability to isomerize PCB to PVB, displaying green/teal reversible photoconversion. Through sequence- and structure-based comparisons coupled with mutagenesis experiments, we identified three amino acid residues, including the Cys residue, crucial for facilitating PCB-to-PVB isomerization. This research expands our understanding of the diversity of XRG CBCRs, highlighting the remarkable molecular plasticity of CBCRs.

An insight into structure-activity relationships in subclass IIb bacteriocins: Plantaricin EvF

This study reports the structure-activity relationships of a unique subclass IIb bacteriocin, plantaricin EvF, which consists of two peptide chains and possesses potent antimicrobial activity. Because the plantaricin Ev peptide chain lacks an α-helix structure, plantaricin EvF is unable to exert its antimicrobial activity through helix-helix interactions like typical subclass IIb bacteriocins. We have shown by various structural evaluation methods that plantaricin Ev can be stabilized by hydrogen bonding at amino acid residues R3, V12, and R13 to the N-terminal region of plantaricin F. This binding gives plantaricin EvF a special spade-shaped structure that exerts antimicrobial activity. In addition, the root-mean-square deviations (RMSDs) of the amino acid residues Y6, F8, and R13 of plantaricin Ev pre- and post-binding were 1.512, 1.723, and 1.369, respectively, indicating that they underwent large structural changes. The alanine scanning experiments demonstrated the important role of the above key amino acids in maintaining the structural integrity of plantaricin EvF. This study not only reveals the unique structural features of plantaricin EvF, but also provides an insight into the structure-activity relationships of subclass IIb bacteriocins.

Amino Acids at Positions 156 and 332 in the E Protein of the West Nile Virus Subtype Kunjin Virus Classical Strain OR393 Are Involved in Plaque Size, Growth, and Pathogenicity in Mice.

The West Nile virus (WNV) subtype Kunjin virus (WNVKUN) is endemic to Australia. Here, we characterized the classical WNVKUN strain, OR393. The original OR393 strain contained two types of viruses: small plaque-forming virus (SP) and large plaque-forming virus (LP). The amino acid residues at positions 156 and 332 in the E protein (E156 and E332) of SP were Ser and Lys (E156S/332K), respectively, whereas those in LP were Phe and Thr (E156F/332T). SP grew slightly faster than LP in vitro. The E protein of SP was N-glycosylated, whereas that of LP was not. Analysis using two recombinant single-mutant LP viruses, rKUNV-LP-EF156S and rKUNV-LP-ET332K, indicated that E156S enlarged plaques formed by LP, but E332K potently reduced them, regardless of the amino acid at E156. rKUNV-LP-EF156S showed significantly higher neuroinvasive ability than LP, SP, and rKUNV-LP-ET332K. Our results indicate that the low-pathogenic classical WNVKUN can easily change its pathogenicity through only a few amino acid substitutions in the E protein. It was also found that Phe at E156 of the rKUNV-LP-ET332K was easily changed to Ser during replication in vitro and in vivo, suggesting that E156S is advantageous for the propagation of WNVKUN in mammalian cells.

The importance of stratum corneum ω-linoleoyloxyacylceramides in human skin barrier health: their biochemistry, processing enzymes and metabolites involved in corneocyte lipid envelope maturation.

Over the past 50 years there have been great strides made in the discovery of the composition and relevance of the total stratum corneum (SC) ceramide matrix. However, the focus of this review is on the free intercellular class of ω-linoleoyloxyacylceramides, corneocyte-bound ceramides and associated lipids known as the corneocyte lipid envelope (CLE) together with their processing enzymes involved in aiding ceramide attachment the corneocyte protein envelope (CPE). Two structural models and partially shared biosynthetic pathways have been proposed for the attachment of CPE-bound O-ceramides (ω-hydroxyceramides attached to glutamate residues of proteins in the (CPE) using the 12R-lipoxygenase (12R-LOX)/epidermal lipoxygenase-3 (eLOX3)/epoxide hydrolase-3 (EPHX3)/unknown esterase/ transglutaminase-1 (TG1) attachment pathway) and CPE-bound EO-ceramides (epoxy-enone ceramides attached to cysteine residues of proteins in the CPE using the 12R-LOX/eLOX3/short chain dehydrogenase/reductase family 9C member 7 (SDR9C7)/non-enzymatic attachment pathway), i.e. there is a bifurcation step beyond epidermal eLOX3. Their formation and structures will be discussed as well as their relevance in compromised skin barrier conditions together with our own work on SC maturation examined by proteomics, lipidomics, enzyme immunolocalization studies, mechanical fragility assays and Nile red staining of corneocyte envelopes (CE). Reduced levels of 12R-LOX, eLOX3, SDR9C7 and TG1 were observed in photodamaged skin of the cheeks that were associated with reduced SC maturation as evidenced by Nile red staining and increased CE fragility. In the severely photodamaged cheeks of Albino African SC we also observed increased levels of acylceramides. Concomitantly by reducing the activity of 12R-LOX by antibody inhibition and TG1 inhibition with a known chemical inhibitor, we demonstrated in a humidity-based exvivo SC maturation model that these enzymes contributed to increased CE hydrophobicity and mechanical integrity. We hypothesize that at least the CPE-bound O-ceramide pathway is operational in the SC. Nevertheless, our understanding of the full complexity of ω-linoleoyloxyacylceramides and the composition of the CLE is limited particularly on cosmetically relevant body sites such as the face.

An Integrated Metabolomics Study on Antidiabetic Activity of Christia vespertilionis Leaves Extract Using Chemometric and Molecular Docking Analysis.

In disease treatment, the utilisation of medicinal plants has witnessed a discernible rise, driven by concerns over the adverse effects associated with synthetic drugs available in the market. Analyses of the plant Christia vespertilionis (L.f.) Bakh. F., indigenous to Malaysia, has suggested its antidiabetic property linked to α-glucosidase inhibition, but metabolites responsible for antidiabetic are unexplored. The metabolomics approaches and molecular docking simulations were integrated to identify the putative α-glucosidase inhibitors and their enzyme interaction. In this study, the crude leaves extracted from this plant were extracted using solvents of varying polarity, followed by gas and liquid chromatography coupled with mass spectrometry metabolomics. The highest inhibition activity in a mixture of n-hexane and ethyl acetate (1:1, v/v)) was observed. Six putative metabolites corresponding to antidiabetic activity were identified: palmitic acid (2), linolenic acid (4), 7-tetradecenal (5), aloeemodin-8-monoglucoside (14), bruceine I (15), and sanjidin B (16). The mechanism of action of all the identified compounds is competitive, mainly involving hydrophobic and hydrogen bonding interactions with the protein residues. Compounds 14, 15, and 16 exhibited strong binding capabilities with both enzyme crystal structures compared to the positive control, quercetin. The metabolites extracted from C. vespertilionis leaves have demonstrated promising antidiabetic effects. These antidiabetic compounds can potentially commercialise new drug candidates in managing diabetes conditions.

An Infrared Nanospectroscopy Technique for the Study of Electric-Field-Induced Molecular Dynamics.

Static electric fields play a considerable role in a variety of molecular nanosystems as diverse as single-molecule junctions, molecules supporting electrostatic catalysis, and biological cell membranes incorporating proteins. External electric fields can be applied to nanoscale samples with a conductive atomic force microscopy (AFM) probe in contact mode, but typically, no structural information is retrieved. Here we combine photothermal expansion infrared (IR) nanospectroscopy with electrostatic AFM probes to measure nanometric volumes where the IR field enhancement and the static electric field overlap spatially. We leverage the vibrational Stark effect in the polymer poly(methyl methacrylate) for calibrating the local electric field strength. In the relevant case of membrane protein bacteriorhodopsin, we observe electric-field-induced changes of the protein backbone conformation and residue protonation state. The proposed technique also has the potential to measure DC currents and IR spectra simultaneously, insofar enabling the monitoring of the possible interplay between charge transport and other effects.

The conformational properties of alamethicin in ethanol studied by NMR.

Alamethicin, a peptide consisted of 20 amino acid residues, has been known to function as an antibiotic. The peptides self-associate in biological membranes, form an ion channel, and then induce cell death by leaking intracellular contents through a transmembrane pore of an ion channel. We investigated conformation and its thermal stability of alamethicin-A6 and -U6 in ethanol using proton nuclear magnetic resonance (NMR) spectroscopy; alamethicin-A6 and -U6 have the amino acid sequences of UPUAUAQUVUGLUPVUUQQO and UPUAUUQUVUGLUPVUUQQO, respectively, where U and O represent α-aminoisobutyric acid and phenylalaninol, respectively. As indicated by the under bars in the sequences, only the residue 6 differs between the alamethicins. We show that the alamethicins in ethanol form helix conformation in the region of the residues 2-11 and a non-regular conformation in the regions of the N- and C-termini, and that the helices are maintained up to 66°C at least. Conformations in the region of the residues 12-18 of the alamethicins, however, are not well identified due to the lack of NMR data. In addition, we demonstrate that the amide proton chemical shift temperature coefficients' method, which is known as an indicator for intramolecular hydrogen bonds in peptides and proteins in aqueous solutions, can be also applied to the alamethicins in ethanol. Further, we show that the conformation around the C-terminus of alamethicin-A6 is restrained by intramolecular hydrogen bonds, whereas that of alamethicin-U6 is either restrained or unrestrained by intramolecular hydrogen bonds; the alamethicin-U6 molecules having the restrained and unrestrained conformations coexist in ethanol. We discuss the two types of conformations using a model chain consisting of particles linked by rigid bonds called as the free jointed chain.

Low Salivary IgA Levels Against PAc (361-386) as a Risk Factor for Root Caries in Older Adults.

This study aimed to assess the intricate relationship between salivary IgA antibody levels to PAc (361-386) (PPA), mutans streptococci colonization, and root caries development in older adults. This study included 307 participants aged 76 years residing in Niigata city, Japan. Clinical oral examinations were performed at baseline in 2004 and 1 year later, during which the total number of untreated and treated root caries was assessed using the root decayed, filled tooth (DFT) index. The stimulated saliva samples were collected using the spitting method during the baseline survey. Salivary IgA antibody levels to amino acid residues 361-386 of Streptococcus mutans PAc were quantified using an enzyme-linked immunosorbent assay. Statistical analyses, including the χ2 test, Mann-Whitney U test, and logistic regressions, were performed to examine the association of increased root DFT with the independent variables. Among the 307 participants (53.1% men), the mean root DFT at baseline was 3.77 ± 3.66, and 36.5% of the study sample exhibited increased root DFT after 1 year with a mean increment of 0.36 ± 0.48. Participants with increase in root DFT after 1 year had significantly higher rates of low PPA levels (≤ 25th percentile) than those without increased root DFT (p = 0.020). Low PPA levels (≤ 25th percentile) were significantly more likely to have an increased risk of root caries development compared with PPA levels > 25th percentile (adjusted OR: 1.88, 95% CI: 1.09-3.25). Low PPA levels and root caries incidence correlated significantly, suggesting that low levels of salivary IgA antibody to PAc (361-386) may serve as a risk factor for increased root caries in older adults.

CYP74B34 Enzyme from Carrot (Daucus carota) with a Double Hydroperoxide Lyase/Epoxyalcohol Synthase Activity: Identification and Biochemical Properties.

The lipoxygenase cascade in plants is a source of oxylipins (oxidized fatty acid derivatives), which play an important role in regulatory processes and formation of plant response to stress factors. Some of the most common enzymes of the lipoxygenase cascade are 13-specific hydroperoxide lyases (HPLs, also called hemiacetal synthases) of the CYP74B subfamily. In this work, we identified and cloned the CYP74B34 gene from carrot (Daucus carotaL.) and described the biochemical properties of the corresponding recombinant enzyme. TheCYP74B34 enzyme was active towards 9- and 13-hydroperoxides of linoleic (9-HPOD and 13-HPOD, respectively) and α-linolenic (9-HPOT and 13-HPOT, respectively) acids. CYP74B34 specifically converted 9-HPOT and 13-HPOT into aldo acids (HPL products). The transformation of 13-HPOD led to the formation of aldo acids and epoxyalcohols [products of epoxyalcohol synthase (EAS) activity] as major and minor products, respectively. Atthe same time, conversion of 9-HPOD resulted in the formation of epoxyalcohols as the main products and aldo acids as the minor ones. Therefore, CYP74B34 is the first enzyme with a double HPL/EAS activity described in carrot. The presence of these catalytic activities was confirmed by analysis of the oxylipin profiles for the roots from young seedlings and mature plants. In addition, we substituted amino acid residues in one of the catalytically essential sites of the CYP74B34 and CYP74B33 proteins and investigated the properties of the obtained mutant enzymes.

A Comprehensive Study of Factors Affecting the Prediction of the pKa Shift of Asp26 in Thioredoxin Protein.

The stable protonation state of ionizable amino acids in a protein can be predicted by computing the pKa shift of that residue within the protein environment. Thermodynamic Integration (TI) is an ideal molecular dynamics-based approach for predicting the pKa shift of ionizable protein residues. Here, we probe TI-based simulation protocols for their ability to accurately predict the pKa shift of Asp26 in thioredoxin. While implicit solvent models can predict the pKa shift accurately, explicit solvent models result in substantial errors. To understand the underlying reason for this surprising discrepancy, we investigate the role of various factors such as solvent models, conformational sampling, background charges, and polarization.

Discovery of Novel Lysine Methyltransferase (SMYD3) Inhibitors by Utilizing 3D-QSAR, Molecular Docking and Molecular Dynamics Simulation

Aim: To investigate novel isoxazole amide SMYD3 inhibitors as adjuvant anticancer agents for multiple cancers. Background: SET and MYND Domain-Containing Protein 3 is a hopeful therapeutic target for breast, liver, colon, and prostate cancer. Objective: Novel SMYD3 inhibitors were predicted by the 3D-QSAR models. Methods: In this present work, 3D-QSAR, molecular docking and molecular dynamics (MD) simulations were performed on a series of isoxazole amides-based SMYD3 inhibitors. Results: Molecular docking revealed residues important to protein-compound interactions, indicating that SMYD3 inhibitors have a strong affinity with and bind to key protein residues such as TYR239, MET190, LYS297 and VAL368. The molecular docking results were further validated by molecular dynamics simulations. Conclusion: The above information provided significant guidance for the design of novel SMYD3 inhibitors.