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Host Defense Peptides (HDPs) have, in previous studies, been demonstrating antimicrobial, anti-inflammatory, and immunomodulatory capacity, important factors in the repair process. Knowing these characteristics, this article aims to evaluate the potential of HDPs IDR1018 and DJK-6 associated with MTA extract in the repair process of human pulp cells. Antibacterial activity of HDPs, MTA and HDPs combined with MTA in Streptococcus mutans planktonic bacteria and antibiofilm activity was evaluated. Cell toxicity was assayed with MTT and cell morphology was observed by scanning electron microscopy (SEM). Proliferation and migration of pulp cells were evaluated by trypan blue and wound healing assay. Inflammatory and mineralization related genes were evaluated by qPCR (IL-6, TNFRSF, DSPP, TGF-β). Alkaline phosphatase, phosphate quantification and alizarin red staining were also verified. The assays were performed in technical and biological triplicate (n = 9). Results were submitted for the calculation of the mean and standard deviation. Then, normality verification by Kolmogorov Smirnov test, analyzing one-way ANOVA. Analyses were considered at a 95% significance level, with a p-value < 0.05. Our study demonstrated that HDPs combined with MTA were able to reduce biofilms performed in 24 h and biofilm performed over 7 days S. mutans biofilm (p < 0.05). IDR1018 and MTA, as well as their combination, down-regulated IL-6 expression (p < 0.05). Tested materials were not cytotoxic to pulp cells. IDR1018 induced high cell proliferation and combined with MTA induced high cellular migration rates in 48 h (p < 0.05). Furthermore, the combination of IDR1018 and MTA also induced high expression levels of DSPP, ALP activity, and the production of calcification nodules. So, IDR-1018 and its combination with MTA could assist in pulp-dentine complex repair process in vitro., (© 2023. The Author(s).)

Klotho is an anti-aging protein with several therapeutic roles in the pathophysiology of different organs, such as the skeletal muscle and kidneys. Available evidence suggests that exercise increases Klotho levels, regardless of the condition or intervention, shedding some light on this anti-aging protein as an emergent and promising exerkine. Development of a systematic review and meta-analysis in order to verify the role of different exercise training protocols on the levels of circulating soluble Klotho (S-Klotho) protein. A systematic search of the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE through PubMed, EMBASE, CINAHL, CT.gov, and PEDro. Randomized and quasi-randomized controlled trials that investigated effects of exercise training on S-Klotho levels. We included 12 reports in the analysis, comprising 621 participants with age ranging from 30 to 65 years old. Klotho concentration increased significantly after chronic exercise training (minimum of 12 weeks) (Hedge' g [95%CI] 1.3 [0.69-1.90]; P < 0.0001). Moreover, exercise training increases S-Klotho values regardless of the health condition of the individual or the exercise intervention, with the exception of combined aerobic + resistance training. Furthermore, protocol duration and volume seem to influence S-Klotho concentration, since the effect of the meta-analysis changes when subgrouping these variables. Altogether, circulating S-Klotho protein is altered after chronic exercise training and it might be considered an exerkine. However, this effect may be influenced by different training configurations, including protocol duration, volume, and intensity., (© 2022. The Author(s).)

The main effectors in the innate immune system of Bombyx mori L. are antimicrobial peptides (AMPs). Here, we infected B. mori with varied inoculum sizes of Pseudomonas aeruginosa ATCC 25668 cells to investigate changes in morpho-anatomical responses, physiological processes and AMP production. Ultraviolet-visible spectra revealed a sharp change in λ max from 278 to 285 nm (bathochromic shift) in the hemolymph of infected B. mori incubated for 24 h. Further, Fourier Transform InfraRed studies on the hemolymph extracted from the infected B. mori showed a peak at 1550 cm -1 , indicating the presence of α-helical peptides. The peptide fraction was obtained through methanol, acetic acid and water mixture (90:1:9) extraction, followed by peptide purification using Reverse Phase High Performance Liquid Chromatography. The fraction exhibiting antibacterial properties was collected and characterized by Matrix-Assisted Laser Desorption/Ionization-Time of Flight. A linear α-helical peptide with flexible termini (LLKELWTKMKGAGKAVLGKIKGLL) was found, corresponding to a previously described peptide from ant venom and here denominated as Bm-ponericin-L1. The antibacterial activity of Bm-ponericin-L1 was determined against ESKAPE pathogens. Scanning electron microscopy confirmed the membrane disruption potential of Bm-ponericin-L1. Moreover, this peptide also showed promising antibiofilm activity. Finally, cell viability and hemolytic assays revealed that Bm-ponericin-L1 is non-toxic toward primary fibroblasts cell lines and red blood cells, respectively. This study opens up new perspectives toward an alternative approach to overcoming multiple-antibiotic-resistance by means of AMPs through invertebrates' infection with human pathogenic bacteria., (© 2022. The Author(s).)

The spread of multi-drug resistance and the slow pace at which antibiotics come onto the market are undermining our ability to treat human infections, leading to high mortality rates. Aiming to overcome this global crisis, antimicrobial peptides are considered promising alternatives to counter bacterial infections with multi-drug resistant bacteria. The cathelicidins comprise a well-studied class of AMPs whose members have been used as model molecules for sequence modifications, aiming at enhanced biological activities and stability, along with reduced toxic effects on mammalian cells. Here, we describe the antimicrobial activities, modes of action and structural characterization of two novel cathelicidin-like peptides, named BotrAMP14 and CrotAMP14, which were re-designed from snake batroxicidin and crotalicidin, respectively. BotrAMP14 and CrotAMP14 showed broad-spectrum antibacterial activity against susceptible microorganisms and clinical isolates with minimal inhibitory concentrations ranging from 2-35.1 μM. Moreover, both peptides had low cytotoxicity against Caco-2 cells in vitro. In addition, in vivo toxicity against Galleria mellonella moth larvae revealed that both peptides led to>76% larval survival after 144 h. Microscopy studies suggest that BotrAMP14 and CrotAMP14 destabilize E. coli membranes. Furthermore, circular dichroism and molecular dynamics simulations indicate that, in a membrane-like environment, both peptides adopt α-helical structures that interact with bilayer phospholipids through hydrogen bonds and electrostatic interaction. Thus, we concluded that BotrAMP14 and CrotAMP14 are helical membrane active peptides, with similar antibacterial properties but lower cytotoxicity than the larger parent peptides batroxicidin and crotalicidin, having advantages for drug development strategies.

Herein, we report the synthesis of silver nanoparticles (AgNPs) by a green route using the aqueous leaf extract of Morus indica L. V1. The synthesized AgNPs exhibited maximum UV-Vis absorbance at 460 nm due to surface plasmon resonance. The average diameter (~54 nm) of AgNPs was measured from HR-TEM analysis. EDX spectra also supported the formation of AgNPs, and negative zeta potential value (-14 mV) suggested its stability. Moreover, a shift in the carbonyl stretching (from 1639 cm -1 to 1630 cm -1 ) was noted in the FT-IR spectra of leaf extract after AgNPs synthesis which confirm the role of natural products present in leaves for the conversion of silver ions to AgNPs. The four bright circular rings (111), (200), (220) and (311) observed in the selected area electron diffraction pattern are the characteristic reflections of face centered cubic crystalline silver. LC-MS/MS study revealed the presence of phytochemicals in the leaf extract which is responsible for the reduction of silver ions. MTT assay was performed to investigate the cytotoxicity of AgNPs against two human cell lines, namely HepG2 and WRL-68. The antibacterial study revealed that MIC value of the synthesized AgNPs was 80 µg/ml against Escherichia coli K12 and Staphylococcus aureus (MTCC 96). Finally, the synthesized AgNPs at 10 µg/ml dosages showed beneficial effects on the survivability, body weights of the Bombyx mori L. larvae, pupae, cocoons and shells weights via enhancing the feed efficacy.

In order to study how acidic pro-peptides inhibit the antimicrobial activity of antimicrobial peptides, we introduce a simple model system, consisting of a 19 amino-acid long antimicrobial peptide, and an N-terminally attached, 10 amino-acid long acidic model pro-peptide. The antimicrobial peptide is a fragment of the crotalicidin peptide, a member of the cathelidin family, from rattlesnake venom. The model pro-peptide is a deca (glutamic acid). Attachment of the model pro-peptide only leads to a moderately large reduction in the binding to- and induced leakage of model liposomes, while the antimicrobial activity of the crotalicidin fragment is completely inhibited by attaching the model pro-peptide. Attaching the pro-peptide induces a conformational change to a more helical conformation, while there are no signs of intra- or intermolecular peptide complexation. We conclude that inhibition of antimicrobial activity by the model pro-peptide might be related to a conformational change induced by the pro-peptide domain, and that additional effects beyond induced changes in membrane activity must also be involved.

In recent years the antimicrobial peptides (AMPs) have been prospected and designed as new alternatives to conventional antibiotics. Indeed, AMPs have presented great potential toward pathogenic bacterial strains by means of complex mechanisms of action. However, reports have increasingly emerged regarding the mechanisms by which bacteria resist AMP administration. In this context, we performed a comparative proteomic study by using the total bacterial lysate of magainin I-susceptible and -resistant E. coli strains. After nanoUPLC-MS E analyses we identified 742 proteins distributed among the experimental groups, and 25 proteins were differentially expressed in the resistant strains. Among them 10 proteins involved in bacterial resistance, homeostasis, nutrition and protein transport were upregulated, while 15 proteins related to bacterial surface modifications, genetic information and β-lactams binding-protein were downregulated. Moreover, 60 exclusive proteins were identified in the resistant strains, among which biofilm and cell wall formation and multidrug efflux pump proteins could be observed. Thus, differentially from previous studies that could only associate single proteins to AMP bacterial resistance, data here reported show that several metabolic pathways may be related to E. coli resistance to AMPs, revealing the crucial role of multiple "omics" studies in order to elucidate the global molecular mechanisms involved in this resistance.

The hemibiotrophic fungus Colletotrichum graminicola may cause severe damage to maize, affecting normal development of the plant and decreasing grain yield. In this context, understanding plant defense pathways at the inoculation site and systemically in uninoculated tissues can help in the development of genetic engineering of resistance against this pathogen. Previous work has discussed the molecular basis of maize - C. graminicola interaction. However, many genes involved in defense have not yet been exploited for lack of annotation in public databases. Here, changes in global gene expression were studied in root, male and female inflorescences of maize under local and systemic fungal infection treatments, respectively. RNA-Seq with qPCR was used to indicate genes involved in plant defense. We found that systemic acquired resistance induction in female inflorescences mainly involves accumulation of salicylic acid (SA)-inducible defense genes (ZmNAC, ZmHSF, ZmWRKY, ZmbZIP and PR1) and potential genes involved in chromatin modification. Furthermore, transcripts involved in jasmonic acid (JA) and ethylene (ET) signaling pathways were also accumulated and may participate in plant immunity. Moreover, several genes were functionally re-annotated based on domain signature, indicating novel candidates to be tested in strategies involving gene knockout and overexpression in plants.

ApoE3 is the major chylomicron apolipoprotein, binding in a specific liver peripheral cell receptor, allowing transport and normal catabolism of triglyceride-rich lipoprotein constituents. Point mutations in ApoE3 have been associated with Alzheimer's disease, type III hyperlipoproteinemia, atherosclerosis, telomere shortening and impaired cognitive function. Here, we evaluate the impact of missense SNPs in APOE retrieved from dbSNP through 16 computational prediction tools, and further evaluate the structural impact of convergent deleterious changes using 100 ns molecular dynamics simulations. We have found structural changes in four analyzed variants (Pro102Arg, Arg132Ser, Arg176Cys and Trp294Cys), two of them (Pro102Arg and Arg176Cys) being previously associated with human diseases. In all cases, except for Trp294Cys, there was a loss in the number of hydrogen bonds between CT and NT domains that could result in their detachment. In conclusion, data presented here could increase the knowledge of ApoE3 activity and be a starting point for the study of the impact of variations on APOE gene.

Antibiotic-resistant infections are predicted to kill 10 million people per year by 2050, costing the global economy $100 trillion. Therefore, there is an urgent need to develop alternative technologies. We have engineered a synthetic peptide called clavanin-MO, derived from a marine tunicate antimicrobial peptide, which exhibits potent antimicrobial and immunomodulatory properties both in vitro and in vivo. The peptide effectively killed a panel of representative bacterial strains, including multidrug-resistant hospital isolates. Antimicrobial activity of the peptide was demonstrated in animal models, reducing bacterial counts by six orders of magnitude, and contributing to infection clearance. In addition, clavanin-MO was capable of modulating innate immunity by stimulating leukocyte recruitment to the site of infection, and production of immune mediators GM-CSF, IFN-γ and MCP-1, while suppressing an excessive and potentially harmful inflammatory response by increasing synthesis of anti-inflammatory cytokines such as IL-10 and repressing the levels of pro-inflammatory cytokines IL-12 and TNF-α. Finally, treatment with the peptide protected mice against otherwise lethal infections caused by both Gram-negative and -positive drug-resistant strains. The peptide presented here directly kills bacteria and further helps resolve infections through its immune modulatory properties. Peptide anti-infective therapeutics with combined antimicrobial and immunomodulatory properties represent a new approach to treat antibiotic-resistant infections.

Clavanins is a class of peptides (23aa) histidine-rich, free of post-translational modifications. Clavanins have been studied largely for their ability to disrupt bacterial membranes. In the present study, the interaction of clavanin A with membranes was assessed by dynamic light scattering, zeta potential and permeabilization assays. We observed through those assays that clavanin A lysis bacterial cells at concentrations corresponding to its MIC. Further, the structure and function of clavanin A was investigated. To better understand how clavanin interacted with bacteria, its NMR structure was elucidated. The solution state NMR structure of clavanin A in the presence of TFE-d3 indicated an α-helical conformation. Secondary structures, based on circular dichroism measurements in anionic sodium dodecyl sulfate (SDS) and TFE (2,2,2-trifluorethanol), in silico lipid-peptide docking and molecular simulations with lipids DPPC and DOPC revealed that clavanin A can adopt a variety of folds, possibly influencing its different functions. Microcalorimetry assays revealed that clavanin A was capable of discriminating between different lipids. Finally, clavanin A was found to eradicate bacterial biofilms representing a previously unrecognized function.

Stingrays commonly cause human envenoming related accidents in populations of the sea, near rivers and lakes. Transcriptomic profiles have been used to elucidate components of animal venom, since they are capable of providing molecular information on the biology of the animal and could have biomedical applications. In this study, we elucidated the transcriptomic profile of the venom glands from two different freshwater stingray species that are endemic to the Paraná-Paraguay basin in Brazil, Potamotrygon amandae and Potamotrygon falkneri. Using RNA-Seq, we identified species-specific transcripts and overlapping proteins in the venom gland of both species. Among the transcripts related with envenoming, high abundance of hyaluronidases was observed in both species. In addition, we built three-dimensional homology models based on several venom transcripts identified. Our study represents a significant improvement in the information about the venoms employed by these two species and their molecular characteristics. Moreover, the information generated by our group helps in a better understanding of the biology of freshwater cartilaginous fishes and offers clues for the development of clinical treatments for stingray envenoming in Brazil and around the world. Finally, our results might have biomedical implications in developing treatments for complex diseases.

Due to the growing concern about antibiotic-resistant microbial infections, increasing support has been given to new drug discovery programs. A promising alternative to counter bacterial infections includes the antimicrobial peptides (AMPs), which have emerged as model molecules for rational design strategies. Here we focused on the study of Pa-MAP 1.9, a rationally designed AMP derived from the polar fish Pleuronectes americanus. Pa-MAP 1.9 was active against Gram-negative planktonic bacteria and biofilms, without being cytotoxic to mammalian cells. By using AFM, leakage assays, CD spectroscopy and in silico tools, we found that Pa-MAP 1.9 may be acting both on intracellular targets and on the bacterial surface, also being more efficient at interacting with anionic LUVs mimicking Gram-negative bacterial surface, where this peptide adopts α-helical conformations, than cholesterol-enriched LUVs mimicking mammalian cells. Thus, as bacteria present varied physiological features that favor antibiotic-resistance, Pa-MAP 1.9 could be a promising candidate in the development of tools against infections caused by pathogenic bacteria.

Peptide rational design was used here to guide the creation of two novel short β-lactamase inhibitors, here named dBLIP-1 and -2, with length of five amino acid residues. Molecular modeling associated with peptide synthesis improved bactericidal efficacy in addition to amoxicillin, ampicillin and cefotaxime. Docked structures were consistent with calorimetric analyses against bacterial β-lactamases. These two compounds were further tested in mice. Whereas commercial antibiotics alone failed to cure mice infected with Staphylococcus aureus and Escherichia coli expressing β-lactamases, infection was cleared when treated with antibiotics in combination with dBLIPs, clearly suggesting that peptides were able to neutralize bacterial resistance. Moreover, immunological assays were also performed showing that dBLIPs were unable to modify mammalian immune response in both models, reducing the risks of collateral effects. In summary, the unusual peptides here described provide leads to overcome β-lactamase-based resistance, a remarkable clinical challenge.