Pap12-6: A host defense peptide with potent immunomodulatory activity in a chicken hepatic cell culture.

In the fight against antimicrobial resistance, host defense peptides (HDPs) are increasingly referred to as promising molecules for the design of new antimicrobial agents. In terms of their future clinical use, particularly small, synthetic HDPs offer several advantages, based on which their application as feed additives has aroused great interest in the poultry sector. However, given their complex mechanism of action and the limited data about the cellular effects in production animals, their investigation is of great importance in these species. The present study aimed to examine the immunomodulatory activity of the synthetic HDP Pap12-6 (PAP) solely and in inflammatory environments evoked by lipoteichoic acid (LTA) and polyinosinic-polycytidylic acid (Poly I:C), in a primary chicken hepatocyte-non-parenchymal cell co-culture. Based on the investigation of the extracellular lactate dehydrogenase (LDH) activity, PAP seemed to exert no cytotoxicity on hepatic cells, suggesting its safe application. Moreover, PAP was able to influence the immune response, reflected by the decreased production of interleukin (IL)-6, IL-8, and "regulated on activation, normal T cell expressed and secreted"(RANTES), as well as the reduced IL-6/IL-10 ratio in Poly I:C-induced inflammation. PAP also diminished the levels of extracellular H2O2 and nuclear factor erythroid 2-related factor 2 (Nrf2) when applied together with Poly I:C and in both inflammatory conditions, respectively. Consequently, PAP appeared to display potent immunomodulatory activity, preferring to act towards the cellular anti-inflammatory and antioxidant processes. These findings confirm that PAP might be a promising alternative for designing novel antimicrobial immunomodulatory agents for chickens, thereby contributing to the reduction of the use of conventional antibiotics.

Multimodal binding and inhibition of bacterial ribosomes by the antimicrobial peptides Api137 and Api88.

Proline-rich antimicrobial peptides (PrAMPs) inhibit bacterial protein biosynthesis by binding to the polypeptide exit tunnel (PET) near the peptidyl transferase center. Api137, an optimized derivative of honeybee PrAMP apidaecin, inhibits protein expression by trapping release factors (RFs), which interact with stop codons on ribosomes to terminate translation. This study uses cryo-EM, functional assays and molecular dynamic (MD) simulations to show that Api137 additionally occupies a second binding site near the exit of the PET and can repress translation independently of RF-trapping. Api88, a C-terminally amidated (-CONH2) analog of Api137 (-COOH), binds to the same sites, occupies a third binding pocket and interferes with the translation process presumably without RF-trapping. In conclusion, apidaecin-derived PrAMPs inhibit bacterial ribosomes by multimodal mechanisms caused by minor structural changes and thus represent a promising pool for drug development efforts.

Hepcidin, in contrast to heparin binding protein, does not portend acute kidney injury in patients with community acquired septic shock.

BACKGROUND: Acute kidney injury (AKI) is a common and severe complication in patients treated at an Intensive Care Unit (ICU). The pathogenesis of AKI has been reported to involve hypoperfusion, diminished oxygenation, systemic inflammation, and damage by increased intracellular iron concentration. Hepcidin, a regulator of iron metabolism, has been shown to be associated with sepsis and septic shock, conditions that can result in AKI. Heparin binding protein (HBP) has been reported to be associated with sepsis and AKI. The aim of the present study was to compare serum hepcidin and heparin binding protein (HBP) levels in relation to AKI in patients admitted to the ICU. METHODS: One hundred and forty patients with community acquired illness admitted to the ICU within 24 hours after first arrival to the hospital were included in the study. Eighty five of these patients were diagnosed with sepsis and 55 with other severe non-septic conditions. Logistic and linear regression models were created to evaluate possible correlations between circulating hepcidin and heparin-binding protein (HBP), stage 2-3 AKI, peak serum creatinine levels, and the need for renal replacement therapy (RRT). RESULTS: During the 7-day study period, 52% of the 85 sepsis and 33% of the 55 non-sepsis patients had been diagnosed with AKI stage 2-3 already at inclusion. The need for RRT was 20% and 15%, respectively, in the groups. Hepcidin levels at admission were significantly higher in the sepsis group compared to the non-sepsis group but these levels did not significantly correlate to the development of stage 2-3 AKI in the sepsis group (p = 0.189) nor in the non-sepsis group (p = 0.910). No significant correlation between hepcidin and peak creatinine levels, nor with the need for RRT was observed. Stage 2-3 AKI correlated, as expected, significantly with HBP levels at admission in both groups (Odds Ratio 1.008 (CI 1.003-1.014, p = 0.005), the need for RRT, as well as with peak creatinine in septic patients. CONCLUSION: Initial serum hepcidin, and HBP levels in patients admitted to the ICU are biomarkers for septic shock but in contrast to HBP, hepcidin does not portend progression of disease into AKI or a later need for RRT. Since hepcidin is a key regulator of iron metabolism our present data do not support a decisive role of initial iron levels in the progression of septic shock into AKI.

pDNA-tachyplesin treatment stimulates the immune system and increases the probability of apoptosis in MC4-L2 tumor cells.

Breast cancer is the most common cancer among women worldwide, and marine creatures are the most abundant reservoir of anticancer medicines. Tachyplesin peptides have shown antibacterial capabilities, but their potential to inhibit cancer growth and trigger cancer cell death has not been investigated. A synthetic tachyplesin nucleotide sequence was generated and inserted into the pcDNA3.1( +) Mammalian Expression Vector. PCR analysis and enzyme digesting procedures were used to evaluate the vectors' accuracy. The transfection efficiency of MCF-7 and MCF10-A cells was 57% and 65%, respectively. The proliferation of MCF-7 cancer cells was markedly suppressed. Administration of plasmid DNA (pDNA) combined with tachyplesin to mice with tumors did not cause any discernible morbidity or mortality throughout treatment. The final body weight curves revealed a significant reduction in weight among mice treated with pDNA/tachyplesin and tachyplesin at a dose of 100 µg/ml (18.4 ± 0.24 gr, P < 0.05; 11.4 ± 0.24 gr P < 0.01) compared to the control group treated with PBS (22 ± 0.31 gr). Animals treated with pDNA/tachyplesin and tachyplesin exhibited a higher percentage of CD4 + Foxp3 + Tregs, CD8 + Foxp3 + Tregs, and CD4 + and CD8 + T cell populations expressing CTLA-4 in their lymph nodes and spleen compared to the PBS group. The groups that received pDNA/tachyplesin exhibited a substantial upregulation in the expression levels of caspase-3, caspase-8, BAX, PI3K, STAT3, and JAK genes. The results offer new possibilities for treating cancer by targeting malignancies using pDNA/tachyplesin and activating the mTOR and NFκB signaling pathways.

Mycobacterium avium paratuberculosis Infection Suppresses Vitamin D Activation and Cathelicidin Production in Macrophages through Modulation of the TLR2-Dependent p38/MAPK-CYP27B1-VDR-CAMP Axis.

BACKGROUND: Vitamin D plays a vital role in modulating both innate and adaptive immune systems. Therefore, vitamin D deficiency has been associated with higher levels of autoimmune response and increased susceptibility to infections. CYP27B1 encodes a member of the cytochrome P450 superfamily of enzymes. It is instrumental in the conversion of circulating vitamin D (calcifediol) to active vitamin D (calcitriol). This is a crucial step for macrophages to express Cathelicidin Anti-microbial Peptide (CAMP), an anti-bacterial factor released during the immune response. Our recent study indicated that a Crohn's disease (CD)-associated pathogen known as Mycobacterium avium paratuberculosis (MAP) decreases vitamin D activation in macrophages, thereby impeding cathelicidin production and MAP infection clearance. The mechanism by which MAP infection exerts these effects on the vitamin D metabolic axis remains elusive. METHODS: We used two cell culture models of THP-1 macrophages and Caco-2 monolayers to establish the effects of MAP infection on the vitamin D metabolic axis. We also tested the effects of Calcifediol, Calcitriol, and SB203580 treatments on the relative expression of the vitamin D metabolic genes, oxidative stress biomarkers, and inflammatory cytokines profile. RESULTS: In this study, we found that MAP infection interferes with vitamin D activation inside THP-1 macrophages by reducing levels of CYP27B1 and vitamin D receptor (VDR) gene expression via interaction with the TLR2-dependent p38/MAPK pathway. MAP infection exerts its effects in a time-dependent manner, with the maximal inhibition observed at 24 h post-infection. We also demonstrated the necessity to have toll-like receptor 2 (TLR2) for MAP infection to influence CYP27B1 and CAMP expression, as TLR2 gene knockdown resulted in an average increase of 7.78 ± 0.88 and 13.90 ± 3.5 folds in their expression, respectively. MAP infection also clearly decreased the levels of p38 phosphorylation and showed dependency on the p38/MAPK pathway to influence the expression of CYP27B1, VDR, and CAMP which was evident by the average fold increase of 1.93 ± 0.28, 1.86 ± 0.27, and 6.34 ± 0.51 in their expression, respectively, following p38 antagonism. Finally, we showed that calcitriol treatment and p38/MAPK blockade reduce cellular oxidative stress and inflammatory markers in Caco-2 monolayers following macrophage-mediated MAP infection. CONCLUSIONS: This study characterized the primary mechanism by which MAP infection leads to diminished levels of active vitamin D and cathelicidin in CD patients, which may explain the exacerbated vitamin D deficiency state in these cases.

Antimicrobial Peptides towards Clinical Application-A Long History to Be Concluded.

Antimicrobial peptides (AMPs) are molecules with an amphipathic structure that enables them to interact with bacterial membranes. This interaction can lead to membrane crossing and disruption with pore formation, culminating in cell death. They are produced naturally in various organisms, including humans, animals, plants and microorganisms. In higher animals, they are part of the innate immune system, where they counteract infection by bacteria, fungi, viruses and parasites. AMPs can also be designed de novo by bioinformatic approaches or selected from combinatorial libraries, and then produced by chemical or recombinant procedures. Since their discovery, AMPs have aroused interest as potential antibiotics, although few have reached the market due to stability limits or toxicity. Here, we describe the development phase and a number of clinical trials of antimicrobial peptides. We also provide an update on AMPs in the pharmaceutical industry and an overall view of their therapeutic market. Modifications to peptide structures to improve stability in vivo and bioavailability are also described.

LL37 Microspheres Loaded on Activated Carbon-chitosan Hydrogel: Anti-bacterial and Anti-toxin Wound Dressing for Chronic Wound Infections.

Antimicrobial peptide LL37 is a promising antibacterial candidate due to its potent antimicrobial activity with no known bacterial resistance. However, intrinsically LL37 is susceptible to degradation in wound fluids limits its effectiveness. Bacterial toxins which are released after cell lysis are found to hinder wound healing. To address these challenges, encapsulating LL37 in microspheres (MS) and loading the MS onto activated carbon (AC)-chitosan (CS) hydrogel. This advanced wound dressing not only protects LL37 from degradation but also targets bacterial toxins, aiding in the healing of chronic wound infections. First, LL37 MS and LL37-AC-CS hydrogel were prepared and characterised in terms of physicochemical properties, drug release, and peptide-polymer compatibility. Antibacterial and antibiofilm activity, bacterial toxin elimination, cell migration, and cell cytotoxicity activities were investigated. LL37-AC-CS hydrogel was effective against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. LL37-AC-CS hydrogel bound more endotoxin than AC with CS hydrogel alone. The hydrogel also induced cell migration after 72 h and showed no cytotoxicity towards NHDF after 72 h of treatment. In conclusion, the LL37-AC-CS hydrogel was shown to be a stable, non-toxic advanced wound dressing method with enhanced antimicrobial and antitoxin activity, and it can potentially be applied to chronic wound infections to accelerate wound healing.

An Overview of Frog Skin-Derived Esc Peptides: Promising Multifunctional Weapons against Pseudomonas aeruginosa-Induced Pulmonary and Ocular Surface Infections.

Antimicrobial resistance is a silent pandemic harming human health, and Pseudomonas aeruginosa is the most common bacterium responsible for chronic pulmonary and eye infections. Antimicrobial peptides (AMPs) represent promising alternatives to conventional antibiotics. In this review, the in vitro/in vivo activities of the frog skin-derived AMP Esc(1-21) are shown. Esc(1-21) rapidly kills both the planktonic and sessile forms of P. aeruginosa and stimulates migration of epithelial cells, likely favoring repair of damaged tissue. However, to undertake preclinical studies, some drawbacks of AMPs (cytotoxicity, poor biostability, and limited delivery to the target site) must be overcome. For this purpose, the stereochemistry of two amino acids of Esc(1-21) was changed to obtain the diastereomer Esc(1-21)-1c, which is more stable, less cytotoxic, and more efficient in treating P. aeruginosa-induced lung and cornea infections in mouse models. Incorporation of these peptides (Esc peptides) into nanoparticles or immobilization to a medical device (contact lens) was revealed to be an effective strategy to ameliorate and/or to prolong the peptides' antimicrobial efficacy. Overall, these data make Esc peptides encouraging candidates for novel multifunctional drugs to treat lung pathology especially in patients with cystic fibrosis and eye dysfunctions, characterized by both tissue injury and bacterial infection.

Antimicrobial peptide thanatin fused endolysin PA90 (Tha-PA90) for the control of Acinetobacter baumannii infection in mouse model.

BACKGROUND: This study addresses the urgent need for infection control agents driven by the rise of drug-resistant pathogens such as Acinetobacter baumannii. Our primary aim was to develop and assess a novel endolysin, Tha-PA90, designed to combat these challenges. METHODS: Tha-PA90 incorporates an antimicrobial peptide (AMP) called thanatin at its N-terminus, enhancing bacterial outer membrane permeability and reducing host immune responses. PA90 was selected as the endolysin component. The antibacterial activity of the purified Tha-PA90 was evaluated using an in vitro colony-forming unit (CFU) reduction assay and a membrane permeability test. A549 cells were utilized to measure the penetration into the cytosol and the cytotoxicity of Tha-PA90. Finally, infection control was monitored in A. baumannii infected mice following the intraperitoneal administration of Tha-PA90. RESULTS: Tha-PA90 demonstrated remarkable in vitro efficacy, completely eradicating A. baumannii strains, even drug-resistant variants, at a low concentration of 0.5 µM. Notably, it outperformed thanatin, achieving only a < 3-log reduction at 4 µM. Tha-PA90 exhibited 2-3 times higher membrane permeability than a PA90 and thanatin mixture or PA90 alone. Tha-PA90 was found within A549 cells' cytosol with no discernible cytotoxic effects. Furthermore, Tha-PA90 administration extended the lifespan of A. baumannii-infected mice, reducing bacterial loads in major organs by up to 3 logs. Additionally, it decreased proinflammatory cytokine levels (TNF-α and IL-6), reducing the risk of sepsis from rapid bacterial lysis. Our findings indicate that Tha-PA90 is a promising solution for combating drug-resistant A. baumannii. Its enhanced efficacy, low cytotoxicity, and reduction of proinflammatory responses render it a potential candidate for infection control. CONCLUSIONS: This study underscores the significance of engineered endolysins in addressing the pressing challenge of drug-resistant pathogens and offers insights into improved infection management strategies.

Bactericidal Permeability-Increasing Protein (BPI) Inhibits Mycobacterium tuberculosis Growth.

Bactericidal permeability-increasing protein (BPI) is a multifunctional cationic protein produced by neutrophils, eosinophils, fibroblasts, and macrophages with antibacterial anti-inflammatory properties. In the context of Gram-negative infection, BPI kills bacteria, neutralizes the endotoxic activity of lipopolysaccharides (LPSs), and, thus, avoids immune hyperactivation. Interestingly, BPI increases in patients with Gram-positive meningitis, interacts with lipopeptides and lipoteichoic acids of Gram-positive bacteria, and significantly enhances the immune response in peripheral blood mononuclear cells. We evaluated the antimycobacterial and immunoregulatory properties of BPI in human macrophages infected with Mycobacterium tuberculosis. Our results showed that recombinant BPI entered macrophages, significantly reduced the intracellular growth of M. tuberculosis, and inhibited the production of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-α). Furthermore, BPI decreased bacterial growth directly in vitro. These data suggest that BPI has direct and indirect bactericidal effects inhibiting bacterial growth and potentiating the immune response in human macrophages and support that this new protein's broad-spectrum antibacterial activity has the potential for fighting tuberculosis.

Antibacterial and Hemolytic Activity of Antimicrobial Hydrogels Utilizing Immobilized Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are viewed as potential compounds for the treatment of bacterial infections. Nevertheless, the successful translation of AMPs into clinical applications has been impeded primarily due to their low stability in biological environments and potential toxicological concerns at higher concentrations. The covalent attachment of AMPs to a material's surface has been sought to improve their stability. However, it is still an open question what is required to best perform such an attachment and the role of the support. In this work, six different AMPs were covalently attached to a long-ranged ordered amphiphilic hydrogel, with their antibacterial efficacy evaluated and compared to their performance when free in solution. Among the tested AMPs were four different versions of synthetic end-tagged AMPs where the sequence was altered to change the cationic residue as well as to vary the degree of hydrophobicity. Two previously well-studied AMPs, Piscidin 1 and Omiganan, were also included as comparisons. The antibacterial efficacy against Staphylococcus aureus remained largely consistent between free AMPs and those attached to surfaces. However, the activity pattern against Pseudomonas aeruginosa on hydrogel surfaces displayed a marked contrast to that observed in the solution. Additionally, all the AMPs showed varying degrees of hemolytic activity when in solution. This activity was entirely diminished, and all the AMPs were non-hemolytic when attached to the hydrogels.

Synergistic resistance of honeybee (Apis mellifera) and their gut microorganisms to fluvalinate stress.

Fluvalinate is widely used in the control of Varroa destructor, but its residues in colonies threaten honeybees. The effect of fluvalinate-induced dysbiosis on honeybee-related gene expression and the gut microenvironment of honeybees has not yet been fully elucidated. In this study, two-day-old larvae to seven-day-old adult worker bees were continuously fed different amounts of fluvalinate-sucrose solutions (0, 0.5, 5, and 50 mg/kg), after which the expression levels of two immune-related genes (Hymenoptaecin and Defensin1) and three detoxication-related genes (GSTS3, CAT, and CYP450) in worker bees (1, 7, and 20 days old) were measured. The effect of fluvalinate on the gut microbes of worker bees at seven days old also was explored using 16S rRNA Illumina deep sequencing. The results showed that exposure of honeybees to the insecticide fluvalinate affected their gene expression and gut microbial composition. As the age of honeybees increased, the effect of fluvalinate on the expression of Hymenoptaecin, CYP450, and CAT decreased, and the abundance of honeybee gut bacteria was affected by increasing the fluvalinate concentration. These findings provide insights into the synergistic defense of honeybee hosts against exogenous stresses in conjunction with honeybee gut microbes.

[Progress of researches on the antiparasitic activity of antimicrobial peptide LL-37].

Parasitic diseases caused by protozoan and helminth infections are still widespread across the world, notably in tropical and subtropical areas, which threaten the children and adult health. Long-term use of anti-parasitic drugs may result in reduced drug susceptibility and even drug resistance. Antimicrobial peptides have been demonstrated to inhibit parasite growth and development, which has potential antiparasitic values. LL-37, the only human antimicrobial peptide in the cathelicidin family, has been widely investigated. This paper reviews the progress of researches on the antiparasitic activity of LL-37, and discusses the prospects of LL-37 in the research of parasites.

Vesicle protrusion induced by antimicrobial peptides suggests common carpet mechanism for short antimicrobial peptides.

Short-cationic alpha-helical antimicrobial peptides (SCHAMPs) are promising candidates to combat the growing global threat of antimicrobial resistance. They are short-sequenced, selective against bacteria, and have rapid action by destroying membranes. A full understanding of their mechanism of action will provide key information to design more potent and selective SCHAMPs. Molecular Dynamics (MD) simulations are invaluable tools that provide detailed insights into the peptide-membrane interaction at the atomic- and meso-scale level. We use atomistic and coarse-grained MD to look into the exact steps that four promising SCHAMPs-BP100, Decoralin, Neurokinin-1, and Temporin L-take when they interact with membranes. Following experimental set-ups, we explored the effects of SCHAMPs on anionic membranes and vesicles at multiple peptide concentrations. Our results showed all four peptides shared similar binding steps, initially binding to the membrane through electrostatic interactions and then flipping on their axes, dehydrating, and inserting their hydrophobic moieties into the membrane core. At higher concentrations, fully alpha-helical peptides induced membrane budding and protrusions. Our results suggest the carpet mode of action is fit for the description of SCHAMPs lysis activity and discuss the importance of large hydrophobic residues in SCHAMPs design and activity.

An esculentin-1 homolog from a dark-spotted frog (Pelophylax nigromaculatus) possesses antibacterial and immunoregulatory properties.

BACKGROUND: Esculentin-1, initially discovered in the skin secretions of pool frogs (Pelophylax lessonae), has demonstrated broad-spectrum antimicrobial activity; however, its immunomodulatory properties have received little attention. RESULTS: In the present study, esculentin-1 cDNA was identified by analysing the skin transcriptome of the dark-spotted frog (Pelophylax nigromaculatus). Esculentin-1 from this species (esculentin-1PN) encompasses a signal peptide, an acidic spacer peptide, and a mature peptide. Sequence alignments with other amphibian esculentins-1 demonstrated conservation of the peptide, and phylogenetic tree analysis revealed its closest genetic affinity to esculentin-1P, derived from the Fukien gold-striped pond frog (Pelophylax fukienensis). Esculentin-1PN transcripts were observed in various tissues, with the skin exhibiting the highest mRNA levels. Synthetic esculentin-1PN demonstrated antibacterial activity against various pathogens, and esculentin-1PN exhibited bactericidal activity by disrupting cell membrane integrity and hydrolyzing genomic DNA. Esculentin-1PN did not stimulate chemotaxis in RAW264.7, a murine leukemic monocyte/macrophage cell line. However, it amplified the respiratory burst and augmented the pro-inflammatory cytokine gene (TNF-α and IL-1ß) expression in RAW264.7 cells. CONCLUSIONS: This novel finding highlights the immunomodulatory activity of esculentin-1PN on immune cells.

Screening and Application of DNA Aptamers for Heparin-Binding Protein.

Rapid detection of heparin-binding protein (HBP) is essential for timely intervention in sepsis cases. Current detection techniques are usually antibody-based immunological methods, which have certain problems, such as complexity and slow detection, and fall short in meeting the urgency of clinical needs. The application of an aptamer can address these concerns well. In this study, HBP-specific DNA aptamers were screened first. Among which, Apt-01, Apt-02, and Apt-13 had a high affinity for HBP, exhibiting impressive KD values of 3.42, 1.44, and 1.04 nmol/L, respectively. Then, the aptamer of HBP and its partially complementary primer probe were combined to form double-stranded DNA (dsDNA) and synthesize a circular DNA template. The template is complementary to the primer probe, but due to the presence of dsDNA, ExoIII cleaves C2-13 as an RCA primer probe, rendering the template unable to recognize the primer probe and preventing the RCA reaction from proceeding. When the target is present, it competes with the adapter for recognition and releases C2-13, exposing its 3' end. After initiating the RCA at room temperature and reacting with SYBR GreenII at 37 °C for 20 min, fluorescence changes can be observed and quantitatively analyzed at a 530 nm wavelength, achieving quantitative biological analysis. Apt-01 was used to develop a fluorescent biosensor for HBP detection, which exhibited a good linear range (0.01 nmol/L to 10 nmol/L) and detection limit (0.0056 nmol/L). This advancement holds the potential to lay a solid groundwork for pioneering sensitive and specific methods for HBP detection and to significantly enhance the diagnostic processes for sepsis.

Susceptibility of Legionella gormanii Membrane-Derived Phospholipids to the Peptide Action of Antimicrobial LL-37-Langmuir Monolayer Studies.

LL-37 is the only member of the cathelicidin-type host defense peptide family in humans. It exhibits broad-spectrum bactericidal activity, which represents a distinctive advantage for future therapeutic targets. The presence of choline in the growth medium for bacteria changes the composition and physicochemical properties of their membranes, which affects LL-37's activity as an antimicrobial agent. In this study, the effect of the LL-37 peptide on the phospholipid monolayers at the liquid-air interface imitating the membranes of Legionella gormanii bacteria was determined. The Langmuir monolayer technique was employed to prepare model membranes composed of individual classes of phospholipids-phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CL)-isolated from L. gormanii bacteria supplemented or non-supplemented with exogenous choline. Compression isotherms were obtained for the monolayers with or without the addition of the peptide to the subphase. Then, penetration tests were carried out for the phospholipid monolayers compressed to a surface pressure of 30 mN/m, followed by the insertion of the peptide into the subphase. Changes in the mean molecular area were observed over time. Our findings demonstrate the diversified effect of LL-37 on the phospholipid monolayers, depending on the bacteria growth conditions. The substantial changes in membrane properties due to its interactions with LL-37 enable us to propose a feasible mechanism of peptide action at a molecular level. This can be associated with the stable incorporation of the peptide inside the monolayer or with the disruption of the membrane leading to the removal (desorption) of molecules into the subphase. Understanding the role of antimicrobial peptides is crucial for the design and development of new strategies and routes for combating resistance to conventional antibiotics.

Antibacterial activity of a short de novo designed peptide against fish bacterial pathogens.

In the face of increasing antimicrobial resistance in aquaculture, researchers are exploring novel substitutes to customary antibiotics. One potential solution is the use of antimicrobial peptides (AMPs). We aimed to design and evaluate a novel, short, and compositionally simple AMP with potent activity against various bacterial pathogens in aquaculture. The resulting peptide, KK12YW, has an amphipathic nature and net charge of + 7. Molecular docking experiments disclosed that KK12YW has a strong affinity for aerolysin, a virulence protein produced by the bacterial pathogen Aeromonas sobria. KK12YW was synthesized using Fmoc chemistry and tested against a range of bacterial pathogens, including A. sobria, A. salmonicida, A. hydrophila, Edwardsiella tarda, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, and methicillin-resistant S. aureus. The AMP showed promising antibacterial activity, with MIC and MBC values ranging from 0.89 to 917.1 µgmL-1 and 3.67 to 1100.52 µgmL-1, respectively. In addition, KK12YW exhibited resistance to high temperatures and remained effective even in the presence of serum and salt, indicating its stability. The peptide also demonstrated minimal hemolysis toward fish RBCs, even at higher concentrations. Taken together, these findings indicate that KK12YW could be a highly promising and viable substitute for conventional antibiotics to combat microbial infections in aquaculture.

Decoding the influence of central LEAP2 on food intake and its effect on accumbal dopamine release.

The gut-brain peptide ghrelin and its receptor are established as a regulator of hunger and reward-processing. However, the recently recognized ghrelin receptor inverse agonist, liver-expressed antimicrobial peptide 2 (LEAP2), is less characterized. The present study aimed to elucidate LEAP2s central effect on reward-related behaviors through feeding and its mechanism. LEAP2 was administrated centrally in mice and effectively reduced feeding and intake of palatable foods. Strikingly, LEAP2s effect on feeding was correlated to the preference of the palatable food. Further, LEAP2 reduced the rewarding memory of high preference foods, and attenuated the accumbal dopamine release associated with palatable food exposure and eating. Interestingly, LEAP2 was widely expressed in the brain, and particularly in reward-related brain areas such as the laterodorsal tegmental area (LDTg). This expression was markedly altered when allowed free access to palatable foods. Accordingly, infusion of LEAP2 into LDTg was sufficient to transiently reduce acute palatable food intake. Taken together, the present results show that central LEAP2 has a profound effect on dopaminergic reward signaling associated with food and affects several aspects of feeding. The present study highlights LEAP2s effect on reward, which may have applications for obesity and other reward-related psychiatric and neurological disorders.

Broad-spectrum activity of membranolytic cationic macrocyclic peptides against multi-drug resistant bacteria and fungi.

The emergence of multidrug-resistant (MDR) strains causes severe problems in the treatment of microbial infections owing to limited treatment options. Antimicrobial peptides (AMPs) are drawing considerable attention as promising antibiotic alternative candidates to combat MDR bacterial and fungal infections. Herein, we present a series of small amphiphilic membrane-active cyclic peptides composed, in part, of various nongenetically encoded hydrophilic and hydrophobic amino acids. Notably, lead cyclic peptides 3b and 4b showed broad-spectrum activity against drug-resistant Gram-positive (MIC = 1.5-6.2 µg/mL) and Gram-negative (MIC = 12.5-25 µg/mL) bacteria, and fungi (MIC = 3.1-12.5 µg/mL). Furthermore, lead peptides displayed substantial antibiofilm action comparable to standard antibiotics. Hemolysis (HC50 = 230 µg/mL) and cytotoxicity (>70 % cell viability against four different mammalian cells at 100 µg/mL) assay results demonstrated the selective lethal action of 3b against microbes over mammalian cells. A calcein dye leakage experiment substantiated the membranolytic effect of 3b and 4b, which was further confirmed by scanning electron microscopy. The behavior of 3b and 4b in aqueous solution and interaction with phospholipid bilayers were assessed by employing nuclear magnetic resonance (NMR) spectroscopy in conjunction with molecular dynamics (MD) simulations, providing a solid structural basis for understanding their membranolytic action. Moreover, 3b exhibited stability in human blood plasma (t1/2 = 13 h) and demonstrated no signs of resistance development against antibiotic-resistant S. aureus and E. coli. These findings underscore the potential of these newly designed amphiphilic cyclic peptides as promising anti-infective agents, especially against Gram-positive bacteria.