Search Results (21)

The mevalonate pathway is crucial for synthesizing isopentenyl pyrophosphate (IPP), the universal precursor of terpenoids, with 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) serving as the rate-determining enzyme that catalyzes the reduction of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) to mevalonate, requiring NAD(P)H as an electron donor. Improving the cofactor promiscuity of HMGR can facilitate substrate utilization and terpenoid production by overcoming cofactor specificity limitations. In this study, we heterologously expressed rpHMGR from Ruegeria pomeroyi in Escherichia coli BL21(DE3) for the first time and established that it predominantly utilizes NADH. To broaden its cofactor usage, we employed Molecular Operating Environment (MOE)-assisted design to engineer the cofactor binding site, creating a dual-cofactor-utilizing mutant, D154K (the substitution of aspartic acid with lysine at residue 154). This mutant exhibited a significant 53.7-fold increase in activity toward NADPH, without compromising protein stability at physiological temperatures. The D154K mutant displayed an optimal pH of 6, maintaining over 80% of its catalytic activity across the pH range of 6–8, regardless of whether NADH or NADPH was the cofactor. These findings highlight the value of rational design, enhance our understanding of HMGR-cofactor recognition mechanisms, and provide a foundation for future efforts to optimize and engineer HMGR for broader cofactor flexibility. Full article

During the processing of marinated braised beef, excessive sodium intake is likely to occur, which can lead to various health issues. Exogenous L-lysine (L-Lys), as an essential amino acid for the human body, has the capability to enhance the quality of low-sodium meat products. This study aimed to investigate the effects of exogenous L-Lys on the quality of low-sodium plain boiled beef and marinated braised beef, as well as its underlying mechanisms of action. Among them, the substitution rate of KCl was 60%. This study was conducted with three batches of experiments, each batch serving as an independent parallel. For low-sodium plain boiled beef, the optimal addition level of L-Lys was screened out through the research on the effects on meat quality indicators, water distribution, microstructure, and sensory evaluation. For the quality of low-sodium plain boiled beef, in terms of microstructure, the addition of L-Lys reduced muscle fiber breakage and voids, thereby improving its microstructural characteristics. Combined with quantitative descriptive analysis (QDA), the optimal level of additional L-Lys was subsequently determined to be 0.6%. It was further processed into marinated braised beef in soy sauce, and a comparative analysis was conducted with low-sodium marinated braised beef in soy sauce without L-Lys addition for shear force, meat color, thiobarbituric acid reactive substances (TBARS), and total viable count (TVC) during the storage periods of 0, 3, 6, 9, and 12 d. The results show that the redness (a*) value significantly increased within 0–12 d (p < 0.05), leading to a more stable meat color. Moreover, the addition of L-Lys significantly reduced the shear force and thiobarbituric acid reactive species (TBARS) values in the marinated braised beef (p < 0.05), thereby optimizing the tenderness of the marinated braised beef and inhibiting lipid oxidation. Although the total viable count (TVC) of the L-Lys group was higher than that of conventional low-sodium marinated braised beef in soy sauce from 9 to 12 d, both groups of products had undergone spoilage by day 12; therefore, the addition of L-Lys had no effect on the shelf life of the products. Comprehensive analysis suggested that the addition of exogenous L-Lys could optimize beef quality by enhancing hydration, improving muscle structural properties, and exerting antioxidant synergistic effects. Full article

Using the framework of an investigation of the stimuli-responsive behavior of peptide assembly on a solid surface, this study on the behavior of a chemisorbed peptide on a gold surface was performed. The studied peptide is a dimeric form of the antimicrobial peptide Trichogin GAIV, which was also modified by substituting the glycine with lysine residues, while the N-terminus octanoyl group was replaced by a lipoic one that was able to bind to the gold surface. In this way, a chemically linked peptide assembly that is pH-responsive was obtained because of the protonation/deprotonation of the sidechains of the Lys residues. Information about the effect of protonation/deprotonation equilibria switching the pH from acid (pH = 3) to basic (pH = 11) conditions was obtained macroscopically by performing Quartz crystal microbalance with dissipation monitoring (QCM-D), Surface Plasmon Resonance (SPR), Nanoplasmonic Sensing (NPS), and FTIR techniques. Using molecular dynamics (MD) simulations, it is possible to explain, at the molecular level, our main experimental results: (1) pH changes induce a squeezing behavior in the system, consisting in thickness and mass variations in the peptide layer, which are mainly due to the pH-driven hydrophilic/hydrophobic character of the lysine residues, and (2) the observed hysteresis is due to small conformational rearrangements from helix to beta sheets occurring mainly on the first half of the peptide, closer to the surface, while the second half remains almost unaffected. The latter result, together with the evidence that the layer thickness is not simply double the assembly of the monomeric analog, indicates that the dimeric peptide does not behave as a double monomer, but assumes very peculiar features. Full article

Antibiotic resistance poses a serious threat to public health globally, reducing the effectiveness of conventional antibiotics in treating bacterial infections. ESKAPE pathogens are a group of highly transmissible bacteria that mainly contribute to the spread of antibiotic resistance and cause significant morbidity and mortality in humans. Phylloseptins, a class of antimicrobial peptides (AMPs) derived from Phyllomedusidae frogs, have been proven to have antimicrobial activity via membrane interaction. However, their relatively high cytotoxicity and low stability limit the clinical development of these AMPs. This project aims to study the antimicrobial activity and mechanisms of a phylloseptin-like peptide, phylloseptin-TO2 (PSTO2), following rational amino acid modification. Here, PSTO2 (FLSLIPHAISAVSALAKHL-NH₂), identified from the skin secretion of Phyllomedusa tomopterna, was used as the template for modification to enhance antimicrobial activity. Adding positive charges to PSTO2 through substitution with L-lysines enhanced the interaction of the peptides with cell membranes and improved their antimicrobial efficacy. The analogues SRD7 and SR2D10, which incorporated D-lysines, demonstrated significant antimicrobial effects against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) while also showing reduced haemolytic activity and cytotoxicity, resulting in a higher therapeutic index. Additionally, SRD7, modified with D-lysines, exhibited notable anti-proliferative properties against human lung cancer cell lines, including H838 and H460. This study thus provides a potential development model for new antibacterial and anti-cancer drugs combating antibiotic resistance. Full article

Antimicrobial peptides (AMPs) are a promising class of therapeutic alternatives with broad-spectrum activity against resistant pathogens. Small AMPs like temporin-SHa (1) and its first-generation analog [G10a]-SHa (2) possess notable efficacy against Gram-positive and Gram-negative bacteria. In an effort to further improve this antimicrobial activity, second-generation analogs of 1 were synthesised by replacing the natural glycine residue at position-10 of the parent molecule with atypical amino acids, such as D-Phenylalanine, D-Tyrosine and (2-Naphthyl)-D-alanine, to study the effect of hydrophobicity on antimicrobial efficacy. The resultant analogs (3–6) emerged as broad-spectrum antibacterial agents. Notably, the [G10K]-SHa analog (4), having a lysine substitution, demonstrated a 4-fold increase in activity against Gram-negative (Enterobacter cloacae DSM 30054) and Gram-positive (Enterococcus faecalis DSM 2570) bacteria relative to the parent peptide (1). Among all analogs, [G10f]-SHa peptide (3), featuring a D-Phe substitution, showed the most potent anticancer activity against lung cancer (A549), skin cancer (MNT-1), prostate cancer (PC-3), pancreatic cancer (MiaPaCa-2) and breast cancer (MCF-7) cells, achieving an IC₅₀ value in the range of 3.6–6.8 µM; however, it was also found to be cytotoxic against normal cell lines as compared to [G10K]-SHa (4). Peptide 4 also possessed good anticancer activity but was found to be less cytotoxic against normal cell lines as compared to 1 and 3. These findings underscore the potential of second-generation temporin-SHa analogs, especially analog 4, as promising leads to develop new broad-spectrum antibacterial and anticancer agents. Full article

Antimicrobial peptides (AMPs), acknowledged as host defense peptides, constitute a category of predominant cationic peptides prevalent in diverse life forms. This study explored the antibacterial activity of α-conotoxin RgIA, and to enhance its stability and efficacy, D-amino acid substitution was employed, resulting in the synthesis of nine RgIA mutant analogs. Results revealed that several modified RgIA mutants displayed inhibitory efficacy against various pathogenic bacteria and fungi, including Candida tropicalis and Escherichia coli. Mechanistic investigations elucidated that these polypeptides achieved antibacterial effects through the disruption of bacterial cell membranes. The study further assessed the designed peptides’ hemolytic activity, cytotoxicity, and safety. Mutants with antibacterial activity exhibited lower hemolytic activity and cytotoxicity, with Pep 8 demonstrating favorable safety in mice. RgIA mutants incorporating D-amino acids exhibited notable stability and adaptability, sustaining antibacterial properties across diverse environmental conditions. This research underscores the potential of the peptide to advance innovative oral antibiotics, offering a novel approach to address bacterial infections. Full article

The current gold standard technique for the treatment of anterior cruciate ligament (ACL) injury is reconstruction with a tendon autograft. These treatments have a relatively high failure and re-rupture rate and are associated with early-onset osteoarthritis, developing within two decades of injury. Furthermore, both autografting and allografting come with several drawbacks. Tissue engineering and additive manufacturing present exciting new opportunities to explore 3D scaffolds as graft substitutes. We previously showed that 3D-printed scaffolds using low-cost equipment are suitable for tissue engineering approaches to regenerative medicine. Here, we hypothesize that Lay-Fomm 60, a commercially available nanoporous elastomer, may be a viable tissue engineering candidate for an ACL graft substitute. We first printed nanoporous thermoplastic elastomer scaffolds using low-cost desktop 3D printers and determined the mechanical and morphological properties. We then tested the impact of different surface coatings on primary human ACL fibroblast adhesion, growth, and ligamentous matrix deposition in vitro. Our data suggest that poly-L-lysine-coated Lay-Fomm 60 scaffolds increased ligament fibroblast activity and matrix formation when compared to uncoated scaffolds but did not have a significant effect on cell attachment and proliferation. Therefore, uncoated 3D printed Lay-Fomm 60 scaffolds may be viable standalone scaffolds and warrant further research as ligament tissue engineering and reconstruction grafts. Full article

Fasciola hepatica anthelmintic resistance may be associated with the catalytic activity of xenobiotic metabolizing enzymes. The gene expression of one of these enzymes, identified as carboxylesterase B (CestB), was previously described as inducible in adult parasites under anthelmintic treatment and exhibited a single nucleotide polymorphism at position 643 that translates into a radical amino acid substitution at position 215 from Glutamic acid to Lysine. Alphafold 3D models of both allelic sequences exhibited a significant affinity pocket rearrangement and different ligand-docking modeling results. Further bioinformatics analysis confirmed that the radical amino acid substitution is located at the ligand affinity site of the enzyme, affecting its affinity to serine hydrolase inhibitors and preferences for ester ligands. A field genotyping survey from parasite samples obtained from two developmental stages isolated from different host species from Argentina and Mexico exhibited a 37% allele distribution for 215E and a 29% allele distribution for 215K as well as a 34% E/K heterozygous distribution. No linkage to host species or geographic origin was found in any of the allele variants. Full article

Plant extracts and probiotics play a vital role in maintaining animal intestinal health. However, their joint compatibility program still needs to be further explored. In our study, thirty two piglets (Duroc × Landrace × Yorkshire) were selected to divided into four treatments, which included basal diet, basal diet + 1000 mg/kg probiotics with added glucose oxidase (PGO), basal diet + 500 mg/kg Illicium verum extracts (IVE), and basal diet + 500 mg/kg IVE + 1000 mg/kg PGO. All the piglets were housed individually for the 42-d trial period after 7-d adaptation. Results showed that dietary supplementation of PGO and IVE increased the digestibility of ether extract (EE), crude protein (CP), and lysine (p < 0.05). Likewise, the net protein utilization (NPU) was also improved (p < 0.05). What is more, adding PGO and IVE reduced crypt depth, increased villus length, and chrionic gland ratio of piglets (p < 0.05). Additionally, IVE or PGO that was applied alone can increase the expression of Occludin, Zona occludens 1 (ZO-1), and Sodium-dependent glucose transporters 1 (SGLT1) in jejunum (p < 0.05). Our results strongly suggest that the combination of IVE and PGO can improve the nutrient digestibility of weaned piglets by increasing the expression of nutrient transport vectors (SGLT1 and CAT1) and tight junction proteins (Occludin and ZO-1) in the jejunum. In conclusion, the combination of plant extracts and probiotics is a vital strategy to improve animal health before the advent of antibiotic substitutes with absolute advantages. Full article

There is a significant and urgent need for the development of novel antibacterial agents to tackle the increasing incidence of antibiotic resistance. Cholic acid-based small molecular antimicrobial peptide mimics are reported as potential new leads to treat bacterial infection. Here, we describe the design, synthesis and biological evaluation of cholic acid-based small molecular antimicrobial peptide mimics. The synthesis of cholic acid analogues involves the attachment of a hydrophobic moiety at the carboxyl terminal of the cholic acid scaffold, followed by the installation of one to three amino acid residues on the hydroxyl groups present on the cholic acid scaffold. Structure–activity relationship studies suggest that the tryptophan moiety is important for high antibacterial activity. Moreover, a minimum of +2 charge is also important for antimicrobial activity. In particular, analogues containing lysine-like residues showed the highest antibacterial potency against Gram-positive S. aureus. All di-substituted analogues possess high antimicrobial activity against both Gram-positive S. aureus as well as Gram-negative E. coli and P. aeruginosa. Analogues 17c and 17d with a combination of these features were found to be the most potent in this study. These compounds were able to depolarise the bacterial membrane, suggesting that they are potential antimicrobial pore forming agents. Full article

Proline-rich antimicrobial peptides (PrAMPs) are promising candidates to treat bacterial infections. The designer peptide ARV-1502 exhibits strong antimicrobial effects against Enterobacteriaceae both in vitro and in vivo. Since the inhibitory effects of ARV-1502 reported for the 70 kDa heat-shock protein DnaK do not fully explain the antimicrobial activity of its 176 substituted analogs, we further studied their effect on the bacterial 70S ribosome of Escherichia coli, a known target of PrAMPs. ARV-1502 analogues, substituted in positions 3, 4, and 8 to 12 (underlined) of the binding motif D³KPRPYLPRP¹² with aspartic acid, lysine, serine, phenylalanine or leucine, were tested in a competitive fluorescence polarization (FP) binding screening assay using 5(6)-carboxyfluorescein-labeled (Cf-) ARV-1502 and the 70S ribosome isolated from E. coli BW25113. While their effect on ribosomal protein expression was studied for green fluorescent protein (GFP) in a cell-free expression system (in vitro translation), the importance of known PrAMP transporters SbmA and MdtM was investigated using E. coli BW25113 and the corresponding knockout mutants. The dissociation constant (K_d) of 201 ± 16 nmol/L obtained for Cf-ARV-1502 suggests strong binding to the E. coli 70S ribosome. An inhibitory binding assay indicated that the binding site overlaps with those of other PrAMPs including Onc112 and pyrrhocoricin as well as the non-peptidic antibiotics erythromycin and chloramphenicol. All these drugs and drug candidates bind to the exit-tunnel of the 70S ribosome. Substitutions of the C-terminal fragment of the binding motif YLPRP reduced binding. At the same time, inhibition of GFP expression increased with net peptide charge. Interestingly, the MIC values of wild-type and ΔsbmA and ΔmdtM knockout mutants indicated that substitutions in the ribosomal binding motif altered also the bacterial uptake, which was generally improved by incorporation of hydrophobic residues. In conclusion, most substituted ARV-1502 analogs bound weaker to the 70S ribosome than ARV-1502 underlining the importance of the YLPRP binding motif. The weaker ribosomal binding correlated well with decreased antimicrobial activity in vitro. Substituted ARV-1502 analogs with a higher level of hydrophobicity or positive net charge improved the ribosome binding, inhibition of translation, and bacterial uptake. Full article

Eight dual-flow continuous culture fermenters were used in three periods to study the effects of diets containing heat-treated soyabean meal (HSBM) or corn gluten meal (CGM) on ruminal microbial fermentation and the degradation of individual amino acids (AA). Treatments were a mix of non-protein nitrogen (N; urea and tryptone) that were progressively substituted (0, 33, 67 and 100%) for HSBM or CGM. Ruminal escape of AA was calculated with the slope ratio technique. Total volatile fatty acids (95.0 mM) and molar proportions (mol/100 mol) of acetate (59.3), propionate (21.8) and butyrate (10.5) were not affected by the treatments. As the level of HSBM or CGM increased, the concentration of ammonia-N and the degradation of protein decreased (p < 0.01), and the flows of nonammonia and dietary N increased (p < 0.01) quadratically. Compared with HSBM, CGM provided the highest flow (g/d) of total (20.6 vs. 18.3, p < 0.01), essential (9.04 vs. 8.25, p < 0.04) and nonessential (11.5 vs. 10.0, p < 0.01) AA, and increased linearly (p < 0.01) as the level of supplemental protein increased. Ruminal degradation of essential AA was higher (p < 0.04) than nonessential AA in CGM, but not in HSBM. Degradation of lysine was higher (p < 0.01) in both proteins, and degradation of methionine was higher in CGM. Ruminal degradation of individual AAs differ within and between protein sources and needs to be considered in precision feeding models. Full article

This study was conducted to evaluate the effects of lysine cell mass (LCM) as an alternative lysine source in diets for weaning pigs on growth performance, diarrhea incidence, and blood profiles. In experiment 1, a total of 200 weaning pigs, with an average body weight (BW) of 6.89 ± 1.04 kg, were allotted into one of five treatments with four replicates of 10 pigs per pen in a randomized complete block design (RCBD). The dietary treatments were composed of LCM supplementation (0, 0.25, 0.5, 0.75, or 1.0%) with partial replacement of L-lysine·HCl (0 to 0.8% for phase 1 diets and 0 to 0.07% for phase 2 diets). The BW and feed intake were recorded at the end of each phase (d 0 to 14 for phase 1, d 14 to 35 for phase 2), and diarrhea incidence was checked daily throughout the experimental period. Blood samples were taken from the jugular vein of pigs at 2 weeks and 5 weeks to determine the blood profiles of weaning pigs. In experiment 2, a total of 144 weaning pigs with an average BW of 6.44 ± 1.19 kg were allotted into one of six treatments with six replicates of four pigs per pen in RCBD. The dietary treatments were composed of LCM supplementation (0 to 3.5% for phase 1 diets and 0 to 2.2% for phase 2 diets) with replacement of L-lysine·HCl from 0 to 100%. In experiment 1, partial replacement of L-lysine·HCl with 0 to 1% LCM did not affect growth performance and diarrhea incidence of pigs. An increase in the LCM supplementation from 0 to 1% with partial replacement of L-lysine·HCl had no influence on the blood urea nitrogen concentrations, whereas it resulted in a linear decrease (p < 0.05) in the serum IgG concentrations for 5 weeks. In experiment 2, increasing the dietary level of LCM with replacement of L-lysine·HCl quadratically decreased (p < 0.05) ADG and G–F ratio for phase 2 and G–F ratio for the overall period such that 100% replacement of L-lysine·HCl with LCM decreased ADG and G–F ratio of weaning pigs. An increase in the LCM supplementation with replacement of L-lysine·HCl tended to decrease linearly (p < 0.10) the diarrhea incidence of weaning pigs for the overall period and linearly decrease (p < 0.05) the serum IgG concentrations for 2 weeks. In conclusion, partial replacement of L-lysine·HCl with LCM from 0 to 1% had no negative impacts on the growth performance, but 100% replacement of L-lysine·HCl with LCM decreased the growth performance of weaning pigs. Therefore, LCM could be included in the diets for weaning pigs up to 2.8% and 1.76% for phase 1 and phase 2, respectively, as a substitute for L-lysine·HCl without detrimental effects on the performance of weaning pigs. Full article

The Maillard reaction is a simple but ubiquitous reaction that occurs both in vivo and ex vivo during the cooking or processing of foods under high-temperature conditions, such as baking, frying, or grilling. Glycation of proteins is a post-translational modification that forms temporary adducts, which, on further crosslinking and rearrangement, form permanent residues known as advanced glycation end products (AGEs). Cooking at high temperature results in various food products having high levels of AGEs. This review underlines the basis of AGE formation and their corresponding deleterious effects on the body. Glycated Maillard products have a direct association with the pathophysiology of some metabolic diseases, such as diabetes mellitus type 2 (DM2), acute renal failure (ARF), Alzheimer’s disease, dental health, allergies, and polycystic ovary syndrome (PCOS). The most glycated and structurally abundant protein is collagen, which acts as a marker for diabetes and aging, where decreased levels indicate reduced skin elasticity. In diabetes, high levels of AGEs are associated with carotid thickening, ischemic heart disease, uremic cardiomyopathy, and kidney failure. AGEs also mimic hormones or regulate/modify their receptor mechanisms at the DNA level. In women, a high AGE diet directly correlates with high levels of androgens, anti-Müllerian hormone, insulin, and androstenedione, promoting ovarian dysfunction and/or infertility. Vitamin D3 is well-associated with the pathogenesis of PCOS and modulates steroidogenesis. It also exhibits a protective mechanism against the harmful effects of AGEs. This review elucidates and summarizes the processing of infant formula milk and the associated health hazards. Formulated according to the nutritional requirements of the newborn as a substitute for mother’s milk, formula milk is a rich source of primary adducts, such as carboxy-methyl lysine, which render an infant prone to inflammation, dementia, food allergies, and other diseases. We therefore recommend that understanding this post-translational modification is the key to unlocking the mechanisms and physiology of various metabolic syndromes. Full article

The increasing emergence of multi-drug resistant bacteria is a serious threat to public health worldwide. Antimicrobial peptides have attracted attention as potential antibiotics since they are present in all multicellular organisms and act as a first line of defence against invading pathogens. We have previously identified a small all-d antimicrobial octapeptide amide kk(1-nal)fk(1-nal)k(nle)-NH₂ (D2D) with promising antimicrobial activity. In this work, we have performed a structure-activity relationship study of D2D based on 36 analogues aimed at discovering which elements are important for antimicrobial activity and toxicity. These modifications include an alanine scan, probing variation of hydrophobicity at lys⁵ and lys⁷, manipulation of amphipathicity, N-and C-termini deletions and lys-arg substitutions. We found that the hydrophobic residues in position 3 (1-nal), 4 (phe), 6 (1-nal) and 8 (nle) are important for antimicrobial activity and to a lesser extent cationic lysine residues in position 1, 2, 5 and 7. Our best analogue 5, showed MICs of 4 µg/mL against A. baumannii, E. coli, P. aeruginosa and S. aureus with a hemolytic activity of 47% against red blood cells. Furthermore, compound 5 kills bacteria in a concentration-dependent manner as shown by time-kill kinetics. Circular dichroism (CD) spectra of D2D and compounds 1–8 showed that they likely fold into α-helical secondary structure. Small angle x-ray scattering (SAXS) experiments showed that a random unstructured polymer-like chains model could explain D2D and compounds 1, 3, 4, 6 and 8. Solution structure of compound 5 can be described with a nanotube structure model, compound 7 can be described with a filament-like structure model, while compound 2 can be described with both models. Lipid interaction probed by small angle X-ray scattering (SAXS) showed that a higher amount of compound 5 (~50–60%) inserts into the bilayer compared to D2D (~30–50%). D2D still remains the lead compound, however compound 5 is an interesting antimicrobial peptide for further investigations due to its nanotube structure and minor improvement to antimicrobial activity compared to D2D. Full article