Search Results (3,547)

Polymyxin antibiotics are often the last line of defense against multidrug-resistant Gram-negative pathogens. A key resistance mechanism involves the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) to lipid A, mediated by the bifunctional enzyme ArnA. However, the evolutionary rationale and structural basis for ArnA’s domain fusion, hexameric assembly, and catalytic coordination remain mechanistically unresolved. Here, we integrate evolutionary genomics, high-resolution cryo-electron microscopy (cryo-EM), and computational protein design to provide a comprehensive mechanistic analysis of ArnA. Our evolutionary analysis reveals that the dehydrogenase (DH) and formyltransferase (TF) domains evolved independently and were selectively fused in Gammaproteobacteria, suggesting an adaptive advantage. A 2.89 Å cryo-EM structure of apo-ArnA resolves the flexible interdomain linker and reveals a DH-driven hexameric architecture essential for enzymatic activity. 3D variability analysis captures intrinsic conformational dynamics, indicating a molecular switch that may coordinate sequential catalysis and substrate channeling. Structure-based peptide inhibitors targeting the hexamerization and predicted ArnA–ArnB interaction interfaces were computationally designed, offering a novel strategy for disrupting L-Ara4N biosynthesis. These findings illuminate a previously uncharacterized structural mechanism of antimicrobial resistance and lay the groundwork for therapeutic intervention. Full article

Antimicrobial peptides (AMPs), evolutionarily conserved components of the immune system, have attracted considerable attention as promising therapeutic candidates. Derived from diverse organisms, AMPs represent a heterogeneous class of molecules, typically cationic, which facilitates their initial electrostatic interaction with anionic microbial membranes. Unlike conventional single-target antibiotics, AMPs utilize rapid, multi-target mechanisms, primarily physical membrane disruption, which results in a significantly lower incidence of resistance emergence. Their broad-spectrum antimicrobial activity, capacity to modulate host immunity, and unique mechanisms of action make them inherently less susceptible to resistance compared with traditional antibiotics. Despite these advantages, the clinical translation of natural AMPs remains limited by several challenges, including poor in vivo stability, and potential cytotoxicity. Bioengineering technology offers innovative solutions to these limitations of AMPs. Two techniques have demonstrated promise: (i) a chimeric recombinant of AMPs with stable scaffold, such as human serum albumin and antibody Fc domain and (ii) chemical modification approaches, such as lipidation. This review provides a comprehensive overview of AMPs, highlighting their origins, structures, and mechanisms of antimicrobial activity, followed by recent advances in bioengineering platforms designed to overcome their therapeutic limitations. By integrating natural AMPs with bioengineering and nanotechnologies, AMPs may be developed into next-generation antibiotics. Full article

Antibiotic resistance, especially among Gram-negative bacterial strains, places a massive burden on global healthcare systems as resistance development has outpaced antibiotic discovery. Protein–protein interactions, successful in other therapeutic contexts, are emerging as promising, yet underexplored, targets for the development of novel classes of antibacterials. Pathogen-specific protein–protein interactions are attractive targets because they are often structurally and functionally distinct from host proteins and are less likely to elicit rapid resistance. This review summarizes recent developments in targeting protein–protein interactions in Gram-negative bacteria, focusing on the modulation of five critical cellular processes: membrane regulation, replication, transcription, translation, and toxin-antitoxin systems. We highlight the design and discovery of both small-molecule and peptide-based inhibitors. While many identified modulators exhibit potent in vitro activity against their respective targets, achieving effective penetration of the complex Gram-negative cell envelope remains a major challenge. Nevertheless, the diverse and essential nature of these bacteria-specific protein–protein interactions represents an attractive strategy for developing next-generation antimicrobials to combat drug-resistant pathogens. Full article

Bioactive peptides (BAPs) from dairy products have garnered increasing attention as natural agents with health-promoting properties, including antihypertensive, antioxidant, antimicrobial, immunomodulatory, opioid, and antidiabetic activities. This systematic review synthesizes research published between 2014 and 2024, retrieved from Scopus and PubMed, and selected according to PRISMA guidelines. A total of 192 studies met the inclusion criteria, collectively reporting over 3200 distinct peptides, with antihypertensive sequences, predominantly angiotensin-converting enzyme (ACE) inhibitors, constituting the largest category (n = 1237). β-casein was the principal precursor across bioactivities, followed by αs1-casein, β-lactoglobulin, and α-lactalbumin. Peptides were primarily produced via enzymatic hydrolysis, microbial fermentation, and gastrointestinal digestion, with peptide profiles influenced by the type of milk, microbial strains, and processing conditions. While cow’s milk remained the dominant source, investigations into goat, sheep, camel, buffalo, and donkey milk revealed species-specific biopeptides. Recent advances in proteomics have enhanced peptide identification and bioactivity prediction, enabling the discovery of novel sequences. These findings underscore the significant potential of dairy-derived BAPs as functional food components and nutraceutical ingredients, while highlighting the need for further in vivo validation, bioavailability studies, and broader exploration of underrepresented milk sources. Full article

Pacific pygmy octopus Paroctopus digueti is a promising model for cephalopod research and aquaculture; its feeding and nutritional biology remain poorly understood. The anterior salivary glands (ASG), posterior salivary glands (PSG), and digestive gland (DG) are central to these processes, but molecular comparisons are lacking. To address this gap, we performed a transcriptomic study to explore the enzymatic repertoire and functional specialization of these tissues. Total RNA was extracted from ASG, PSG, and DG of three pre-adult individuals collected in La Paz Bay, Mexico. RNA-Seq libraries were sequenced, and a non-redundant multi-tissue transcriptome was assembled. The ASG displayed high expression of neuropeptides, playing a role in neuroendocrine regulation. The PSG showed elevated protease expression, supporting its function in extracellular digestion, alongside toxins that reinforce its role as a venom gland. The DG was enriched in proteins linked to biomolecule catabolism and antimicrobial peptides, alluding to metabolic specialization and immune defense. These results were validated by qPCR, and target genes were also amplified in Octopus maya and O. hubbsorum, showing some similarities in expression patterns. Overall, our findings suggest strong glandular specialization in P. digueti, providing insights into cephalopod digestive physiology and supporting its value as a model species. Full article

Background/Objectives: Metabolic dysfunction-associated steatotic liver disease (MASLD) is becoming more common among adolescents, but non-invasive biomarkers for early detection are still limited. Liver-expressed antimicrobial peptide-2 (LEAP-2), a ghrelin receptor antagonist, has been connected to obesity and liver fat buildup in adults, but pediatric data are limited. This study investigates the hypothesis that higher levels of LEAP-2 are associated with hepatic steatosis and the role of LEAP-2 serum levels in the earlier and easier diagnosis of MASLD in children. Methods: In this cross-sectional study, 51 adolescents aged 12–18 were divided into three groups: one with MASLD and obesity (MASLD-Ob) (confirmed hepatosteatosis by imaging studies such as magnetic resonance or ultrasound, along with at least one cardiometabolic criterion and a body mass index (BMI) > 2 SD) (n = 19), another with obesity without any liver pathology or MASLD (BMI > 2 SD) (n = 14), and healthy controls (n = 18). The controlled attenuation parameter (CAP) was measured using FibroScan^® Mini + 430 (Echosens SA, Créteil, France), and serum ghrelin and LEAP-2 levels were determined via ELISA. Correlations between LEAP-2, ghrelin, CAP, BMI z-score, and metabolic parameters were analyzed. Results: LEAP-2 and ghrelin levels among the three groups were similar (p = 0.148, p = 0.515). A positive correlation was observed between LEAP-2 levels and CAP values in the obese group (both the MASLD-Ob and obesity groups) (r = 0.379, p = 0.030). When a cutoff of 240 dB/m was used, the median LEAP-2 level in cases above this value was 2.20 ng/mL, compared to 1.37 ng/mL in cases below it (p = 0.021), which was significantly different. When analyzing the obese group (both the MASLD-Ob and obese groups) a statistically significant correlation was found between serum LEAP-2 levels and CAP, AST, GGT, and total bilirubin values (r = 0.379, p = 0.030; r = 0.369, p = 0.035; r = 0.369, p = 0.035; r = 0.357, p = 0.049, respectively). Conclusions: Interventional imaging methods and biomarkers for diagnosing and monitoring hepatosteatosis have become well-established in the literature. However, since these tests are not available at all centers and can be costly, there is an increasing search for other easily accessible diagnostic and follow-up parameters. LEAP-2 could be a promising non-invasive biomarker for pediatric MASLD, especially when used alongside CAP measurements. The application of this biomarker in pediatric MASLD provides valuable data to help identify and monitor the condition in adolescents. We believe our study offers strong evidence to support further research and the development of drug treatments for MASLD that aim to reduce plasma LEAP-2. Full article

Background/Objectives: The global rise in antibiotic resistance necessitates the development of novel antimicrobial agents. Antimicrobial peptides (AMPs), key components of innate immunity, are promising candidates. This study aimed to develop novel therapeutic peptides with enhanced properties through the mutagenesis of natural AMPs and high-throughput screening. Methods: We constructed mutant libraries of three broad-spectrum AMPs—melittin, cecropin, and Hm-AMP2—using mutagenesis with partially degenerate oligonucleotides. Libraries were expressed in Escherichia coli, and antimicrobial activity was assessed through bacterial growth kinetics and droplet serial dilution assays. Candidate molecules were identified by DNA sequencing, and the most promising variants were chemically synthesized. Antimicrobial activity was determined by minimal inhibitory concentration (MIC) against E. coli and Bacillus subtilis, while cytotoxicity was evaluated in human Expi293F cells (IC₉₀) viability. The therapeutic index was calculated as the ratio of an AMP’s cytotoxic concentration to its effective antimicrobial concentration. Results: Mutant forms of melittin (MR1P7, MR1P8) showed significantly reduced cytotoxicity while retaining antimicrobial activity. Cecropin mutants exhibited reduced efficacy against E. coli, but variants CR2P2, CR2P7, and CR2P8 gained activity against Gram-positive bacteria. Mutagenesis of Hm-AMP2 generally decreased activity against E. coli, though two variants (A2R1P5 and A2R3P6) showed retained or enhanced efficacy against B. subtilis while maintaining low cytotoxicity. Conclusions: The proposed strategy successfully generated peptides with improved therapeutic profiles, including reduced toxicity or a broader spectrum of antimicrobial activity, despite not improving all parameters. This approach enables the discovery of novel bioactive peptides to combat antibiotic-resistant pathogens. Full article

Background/Objectives: There is a growing demand for the development of novel antimicrobial agents due to their efficacy being eroded by increasing antimicrobial resistance. Antimicrobial hydrogels have been reported as a method to treat bacterial infections. Methods: This study explores how different structural features are important for the hydrogelation properties of amphiphilic antimicrobial peptide-mimics through rheology and AFM, as well as properties important for antimicrobial activity measured through MIC. Results: Eleven novel peptide-mimicking anthranilamides containing various structural features were synthesised in 4–7 steps. Of these peptide-mimics, three novel compounds formed hydrogels, and it was identified that their mechanical strength, secondary structure, and fibre morphology could be tuned by altering the aromatic cap or the amino acid side chain. In conjunction, several structural features were identified that reduce hydrogelation strength and stiffness. Conclusions: This work provides an insight into how the structural features of low-molecular-weight self-assembling hydrogels can translate to differing physical and potent antimicrobial properties. This work provides a rational guide to optimising physical hydrogel properties, as well as highlighting features that may reduce hydrogelation. Full article

Among bioactive peptides, those with antimicrobial activity have attracted increasing attention due to their potential as alternatives to traditional antibiotics. Antimicrobial peptides (AMPs) are small molecules, typically composed of 6 to 60 amino acid residues, and some with low cytotoxicity and minimal side effects. They exhibit broad-spectrum activity against bacteria, viruses, fungi, and parasites through diverse mechanisms of action and interactions with the immune system. This review presents the main aspects of AMPs, including their biochemical characteristics, sources, mechanisms of action, and computational tools used for their identification and analysis. It also examines recent progress in clinical trials and the current limitations that restrict the development and commercialization of AMPs. The review discusses the application of AMPs beyond human medicine, including their use in food preservation to prevent microbial contamination and in veterinary medicine to control infections in livestock, thereby reducing dependence on conventional antibiotics. Overall, AMPs represent a versatile class of antimicrobial agents whose effective implementation across health, food, and veterinary sectors will require integrated multidisciplinary approaches. Full article

Pulses and pseudocereals are sustainable protein sources of bioactive peptides (BAPs) with potential antioxidant, antihypertensive, antidiabetic, antimicrobial, and immunomodulatory activities. BAPs are typically liberated during gastrointestinal digestion or through bio-based processes, among which enzymatic hydrolysis and microbial fermentation represent the most widely applied strategies. Enzymatic hydrolysis provides controlled and reproducible release of short peptide motifs; recent advances such as ultrasound- or high-pressure–assisted hydrolysis enhance yield and bioactivity. Fermentation exploits microbial proteolytic activity to generate complex peptide mixtures, while improving sensory quality, reducing antinutritional compounds, and responding to consumer demand for natural and “clean-label” products. In silico tools increasingly complement these approaches by accelerating peptide discovery, predicting interactions with molecular targets, and guiding process design. This review provides an updated overview of bio-based methods to produce BAPs from pulses and pseudocereals, emphasizing the comparative advantages of enzymatic and fermentation technologies and their integration with computational tools. Moreover, it examines regulatory frameworks in the European Union, the United States, Japan, and China, while discussing current challenges for industrial scale-up and application in functional foods and nutraceuticals. These combined strategies offer a promising pathway to unlock the health and sustainability potential of plant proteins. Full article

Background/Objectives: The rapid emergence of antibiotic-resistant oral pathogens has rendered many conventional therapies increasingly ineffective. Antimicrobial peptides (AMPs) have emerged as a promising therapeutic alternative due to their unique mechanisms of action and low propensity for inducing resistance. The delivery of novel therapeutic AMPs against oral cavity bacterial infections requires effective pharmaceutical dosage formulations. This study investigated the potential of two liposomal formulations for the association and delivery of the antimicrobial peptide (AMP) LL17-32 against the dental bacterial pathogen Porphyromonas gingivalis. Methods: Liposomes composed of either negatively charged soya lecithin (SL) or neutrally charged dioleoyl-phosphatidylcholine (DOPC) phospholipids were formulated and characterized based on their hydrodynamic size distribution, ζ-potential, morphology, membrane fluidity, peptide association efficiency, stability and release of peptide in vitro under physiological conditions. The characterization of their biological activity included efficiency of bacterial killing, bacterial adherence, and mammalian cell cytotoxicity using human gingival keratinocyte (TIGK) cells. Results: Both liposomal formulations exhibited spherical morphology with hydrodynamic diameters smaller than ~170 nm and demonstrated good colloidal stability. LL17-32 showed high association efficiency with both liposomal membranes, with no detectable LL17-32 in vitro release. In biological assays, peptide-loaded DOPC liposomes exhibited dose-dependent bactericidal activity against P. gingivalis, whereas SL liposomes significantly attenuated the bactericidal effect of LL17-32. Both formulations displayed reduced cytotoxicity toward human gingival keratinocyte (TIGK) cells versus free peptide. Conclusions: These findings suggest that DOPC liposomes represent a promising delivery system for LL17-32 by adhering to P. gingivalis and exhibiting minimal cytotoxicity to mammalian cells. This study emphasises the critical role of lipid charge in designing AMP delivery systems for antibacterial applications, while it additionally demonstrates the utility of flow cytometry as a quantitative tool to assess liposome–bacteria association. Full article

The increasing prevalence of antimicrobial resistance in livestock pathogens necessitates the development of effective alternatives to conventional antibiotics. This review aims to assess the potential of antimicrobial peptides (AMPs) as alternatives to traditional antibiotics in farm animal production systems, examining their mechanisms of action, efficacy, and applications. A thorough examination of recent literature was conducted to evaluate the properties, classification, and mechanisms of action of AMPs, their natural occurrence, and their applications in poultry, swine, and ruminant production. The review also compared AMPs with conventional antibiotics, antifungals, and antiparasitic drugs. Specific AMPs have shown effectiveness against livestock pathogens, including Escherichia coli, Salmonella, and Clostridium perfringens, along with drug-resistant fungi. One of the primary benefits of AMPs is their strong antimicrobial activity against a wide range of pathogens relevant to farm animal health. Innovative delivery systems, such as self-assembly techniques and nanoparticle encapsulation, can tackle the stability and bioavailability issues associated with AMP administration in farm settings. AMPs represent promising alternatives to conventional antimicrobials in livestock production, offering significant benefits, including a reduced risk of resistance development, immunomodulatory effects, and broad-spectrum activity. However, addressing disadvantages related to production costs, stability, and delivery systems is crucial for their successful commercial application. Integrating AMPs into sustainable farming, after overcoming their shortcomings, could significantly contribute to global efforts to combat antimicrobial resistance. Full article

In this study, we successfully cloned the fluorescent proteins eGFP and DsRed in-frame with the antimicrobial peptide epinecidin-1 (FIFHIIKGLFHAGKMIHGLV) at the N-terminal. The cloning strategy involved inserting the fluorescent reporters into the expression vector, followed by screening for positive clones through visual fluorescence detection and molecular validation. The visually identified fluorescent colonies were confirmed as positive by PCR and plasmid migration assays, indicating successful cloning. This fusion of fluorescent reporters with a short antimicrobial peptide enables real-time visualization and monitoring of the peptide’s mechanism of action on membranes and within cells, both in vivo and in vitro. The fusion of eGFP and DsRed to epinecidin-1 did not impair the expression or fluorescence of the reporter protein. Full article

Treating bacterial infections has become increasingly difficult due to the rise in antibiotic-resistant bacterial strains. Strategies involving the targeted delivery of antibiotics have been proposed to minimize the administered antibiotic doses. This study aims to develop the first double-modified nanovehicle capable of increasing bacterial membranes’ permeability while specifically targeting Staphylococcus aureus, one of the foremost pathogens responsible for global mortality rates. Thus, polymeric NPs composed of poly(lactic-co-glycolic acid) (PLGA) were produced, and their surface was modified with TAT peptide to increase the membranes’ permeability and folic acid (FA) to direct the NPs to S. aureus. The nanosystem showed spherical morphology with sizes of 174 ± 4 nm, a monodisperse population (polydispersity index of 0.08 ± 0.02), and a zeta potential of −2.5 ± 0.1 mV. The NPs remained stable for up to four months during storage. Fluorescence-based flow cytometry analysis proved that the double modification of PLGA NPs increased the interaction of the NPs with S. aureus, with fluorescence increasing from 71 ± 3% to 87 ± 1%. The nanosystem slightly affected the growth curve of S. aureus by extending both the lag time (from 2.5 ± 0.2 to 2.88 ± 0.4 h) and the exponential phase, as evidenced by an increase in the half-maximum growth time (from 3.9 ± 0.2 to 4.4 ± 0.1 h). Furthermore, the nanocarrier showed no toxicity for human dermal fibroblast cells, maintaining a 100% cell viability at the highest concentration tested (100 µM). Therefore, the proposed FA/TAT-functionalized nanocarrier presented promising features to be successfully used as a delivery vehicle of antimicrobials to fight S. aureus. Full article

Canine otitis externa (OE) presents a significant challenge in veterinary medicine due to its complex, multifactorial nature and the growing issue of antimicrobial resistance (AMR) associated with conventional antibiotic use. The objective of this study was to compare the efficacy of a novel, antibiotic-free topical ear solution (Therapy A) containing antimicrobial peptides and encapsulated plant extracts (chamomile, calendula, rosemary, and hops) against a standard conventional treatment (Therapy B) composed of gentamicin, betamethasone valerate, and clotrimazole. A longitudinal, randomized study was conducted over four weeks with 40 domestic dogs diagnosed with OE. The dogs were divided into two groups, each receiving one of the therapies. Evaluations were performed weekly, assessing clinical signs using the Otitis Index Scoring System (OTIS-3) and a pruritus visual analog scale (pVAS), as well as ear canal pH and cytology. The results showed that Therapy A provided similar clinical efficacy in OTIS-3 and pVAS scores that were comparable to Therapy B. Cytological analysis also revealed a significant reduction in microbial presence for both groups. Notably, Therapy A was clinically effective in two of the three dogs presenting multi-drug resistant (MDR) bacterial infections. The novel formulation also demonstrated a favorable safety profile, with no adverse drug reactions reported, in contrast to one dog in the conventional treatment group that experienced an adverse reaction. These findings suggest that the plant-based formulation is a safe and effective alternative for managing canine OE, offering a promising solution to reduce the reliance on antibiotics and corticosteroids. Full article