A proteomic perspective on the resistance response of Klebsiella pneumoniae to antimicrobial peptide PaDBS1R1.

BACKGROUND: The synthetic antimicrobial peptide, PaDBS1R1, has been reported as a powerful anti-Klebsiella pneumoniae antimicrobial. However, there is only scarce knowledge about whether K. pneumoniae could develop resistance against PaDBS1R1 and which resistance mechanisms could be involved. OBJECTIVES: Identify via label-free shotgun proteomics the K. pneumoniae resistance mechanisms developed against PaDBS1R1. METHODS: An adaptive laboratory evolution experiment was performed to obtain a PaDBS1R1-resistant K. pneumoniae lineage. Antimicrobial susceptibility was determined through microdilution assay. Modifications in protein abundances between the resistant and sensitive lineages were measured via label-free quantitative shotgun proteomics. Enriched Gene Ontology terms and KEGG pathways were identified through over-representation analysis. Data are available via ProteomeXchange with identifier PXD033020. RESULTS: K. pneumoniae ATCC 13883 parental strain challenged with increased subinhibitory PaDBS1R1 concentrations allowed the PaDBS1R1-resistant K. pneumoniae lineage to emerge. Proteome comparisons between PaDBS1R1-resistant K. pneumoniae and PaDBS1R1-sensitive K. pneumoniae under PaDBS1R1-induced stress conditions enabled the identification and quantification of 1702 proteins, out of which 201 were differentially abundant proteins (DAPs). The profiled DAPs comprised 103 up-regulated proteins (adjusted P value < 0.05, fold change ≥ 2) and 98 down-regulated proteins (adjusted P value < 0.05, fold change ≤ 0.5). The enrichment analysis suggests that PhoPQ-guided LPS modifications and CpxRA-dependent folding machinery could be relevant resistance mechanisms against PaDBS1R1. CONCLUSIONS: Based on experimental evolution and a label-free quantitative shotgun proteomic approach, we showed that K. pneumoniae developed resistance against PaDBS1R1, whereas PhoPQ-guided LPS modifications and CpxRA-dependent folding machinery appear to be relevant resistance mechanisms against PaDBS1R1.

Preclinical data on morpholine (3,5-di-tertbutyl-4-hydroxyphenyl) methanone induced anxiolysis.

Trimetozine is used to be indicated for the treatment of mental illnesses, particularly anxiety. The present study provides data on the pharmacological profile of trimetozine derivative morpholine (3,5-di-tert-butyl-4-hydroxyphenyl) methanone (LQFM289) which was designed from molecular hybridization of trimetozine lead compound and 2,6-di-tert-butyl-hydroxytoluene to develop new anxiolytic drugs. Here, we conduct molecular dynamics simulations, docking studies, receptor binding assays, and in silico ADMET profiling of LQFM289 before its behavioral and biochemical assessment in mice within the dose range of 5-20 mg/kg. The docking of LQFM289 showed strong interactions with the benzodiazepine binding sites and matched well with receptor binding data. With the ADMET profile of this trimetozine derivative that predicts a high intestinal absorption and permeability to blood-brain barrier without being inhibited by the permeability glycoprotein, the oral administration of LQFM289 10 mg/kg consistently induced anxiolytic-like behavior of the mice exposed to the open field and light-dark box apparatus without eliciting motor incoordination in the wire, rotarod, and chimney tests. A decrease in the wire and rotarod´s fall latency coupled with an increase in the chimney test´s climbing time and a decrease in the number of crossings in the open field apparatus at the dose of 20 mg/kg of this trimetozine derivative suggest sedative or motor coordination impairment at this highest dose. The attenuation of the anxiolytic-like effects of LQFM289 (10 mg/kg) by flumazenil pretreatment implicates the participation of benzodiazepine binding sites. The lowering of corticosterone and tumor necrosis factor alpha (cytokine) in LQFM289-treated mice at a single oral (acute) dose of 10 mg/kg suggests that the anxiolytic-like effect of this compound also involves the recruitment of non-benzodiazepine binding sites/GABAergic molecular machinery.

Evaluation of Gastroprotective Activity of Linoleic Acid on Gastric Ulcer in a Mice Model.

BACKGROUND: Gastric ulcer has been a major cause of morbidity and mortality worldwide, and it has been linked to factors such as nutritional deficiency, smoking, stress, and continuous intake of non-steroidal antiinflammatory drugs (NSAIDs). The search for new anti-ulcer therapeutic agents has been the subject of several studies. Recently, the gastroprotective effect of Celtis iguanaea has been reported, with linoleic acid (LA) responsible for many of the therapeutic effects of this medicinal plant. AIMS: This study aims to investigate the gastroprotective activity and the possible mechanisms in which LA may be involved through different experimental assays in mice. METHODS: The gastroprotective activity of LA was evaluated in the ulcer induced by indomethacin, HCl/EtOH, hypothermic-restraint stress and pyloric ligation. For the investigation of gastroprotective mechanisms, the quantification of the volume (mL), pH and total acidity of gastric secretion were considered. RESULTS: The oral administrations of 25 mg/kg, 50 mg/kg or 100 mg/kg of body weight of LA were capable of protecting the gastric mucosa against HCl/ethanol (10 mL/kg p.o.), and oral/intraduodenal treatment administrations of 50 mg/kg LA showed protection from ulcers induced by indomethacin, hypothermic-restraint stress and pyloric ligation. CONCLUSION: The results of this study show the gastroprotective role of LA in gastric mucosal damage induced by all assayed distresses. The observed gastroprotection possibly occurs due to the mediated increase of mucosal defensive factors.

The use of host defense peptides in root canal therapy in rats.

OBJECTIVES: In order to evaluate host defense peptides (HDPs) HHC-10 and synoeca-MP activity in in vitro osteoclastogenesis process and in vivo induced apical periodontitis, testing the effect of molecules in the inflammatory response and in apical periodontitis size/volume after root canal treatment. MATERIALS AND METHODS: In vitro osteoclastogenesis was assessed on bone marrow cell cultures extracted from mice, while in vivo endodontic treatment involved rats treated with Ca(OH)2 or HDPs. In vitro osteoclasts were subjected to TRAP staining, and in vivo samples were evaluated by radiographic and tomographic exams, as well as histologic analysis. RESULTS: None of the substances downregulated the in vitro osteoclastogenesis. Nevertheless, all treatments affected the average of apical periodontitis size in rats, although only teeth treated with HDPs demonstrated lower levels of the inflammatory process. These results demonstrated the in vivo potential of HDPs. Radiographic analysis suggested that HHC-10 and synoeca-MP-treated animals presented a similar lesion size than Ca(OH)2-treated animals after 7-day of endodontic treatment. However, tomography analysis demonstrated smaller lesion volume in synoeca-MP-treated animals than HHC-10 and Ca(OH)2-treated animals, after 7 days. CONCLUSIONS: These molecules demonstrated an auxiliary effect in endodontic treatment that might be related to its immunomodulatory ability, broad-spectrum antimicrobial activity, and possible induction of tissue repair at low concentrations. These results can encourage further investigations on the specific mechanisms of action in animal models to clarify the commercial applicability of these biomolecules for endodontic treatment. CLINICAL SIGNIFICANCE: HDPs have the potential to be adjuvant substances in endodontic therapy due to its potential to reduce inflammation in apical periodontitis.

Repurposing a peptide toxin from wasp venom into antiinfectives with dual antimicrobial and immunomodulatory properties.

Novel antibiotics are urgently needed to combat multidrug-resistant pathogens. Venoms represent previously untapped sources of novel drugs. Here we repurposed mastoparan-L, the toxic active principle derived from the venom of the wasp Vespula lewisii, into synthetic antimicrobials. We engineered within its N terminus a motif conserved among natural peptides with potent immunomodulatory and antimicrobial activities. The resulting peptide, mast-MO, adopted an α-helical structure as determined by NMR, exhibited increased antibacterial properties comparable to standard-of-care antibiotics both in vitro and in vivo, and potentiated the activity of different classes of antibiotics. Mechanism-of-action studies revealed that mast-MO targets bacteria by rapidly permeabilizing their outer membrane. In animal models, the peptide displayed direct antimicrobial activity, led to enhanced ability to attract leukocytes to the infection site, and was able to control inflammation. Permutation studies depleted the remaining toxicity of mast-MO toward human cells, yielding derivatives with antiinfective activity in animals. We demonstrate a rational design strategy for repurposing venoms into promising antimicrobials.

Involvement of the gabaergic, serotonergic and glucocorticoid mechanism in the anxiolytic-like effect of mastoparan-L.

Mastoparan-L (mast-L) is a cell-penetrating tetradecapeptide and stimulator of monoamine exocytosis. In the present study, we evaluated the anxiolytic-like effect of mast-L. Preliminary pharmacological tests were conducted to determine the most appropriate route of administration, extrapolate dose and detect potential toxic effects of this peptide. Oral and intracerebroventricular administration of mast-L (0.1, 0.3 or 0.9 mg.kg-1), diazepam (1 or 5 mg.kg-1), buspirone (10 mg.kg-1) or vehicle 10 mL.kg-1 was carried out prior to the exposure of mice to the anxiety models: open field, light-dark box and elevated plus-maze. To characterize the mechanism underlying the antianxiety-like effect of mast-L, pharmacological antagonism, blood plasma analysis, molecular docking, and receptor binding assays were performed. The absence of a neurotoxic sign, animal's death as well as lack of significant changes in the relative organ weight, hematological and biochemical parameters suggest that mast-L is relatively safe. The anxiolytic-like effect of mast-L was attenuated by flumazenil (antagonist of benzodiazepine binding site) and WAY100635 (selective antagonist of 5-HT1A receptors) pretreatments. Mast-L reduced plasma corticosterone and lowered the scoring function at GABAA -18.48 kcal/mol (Ki = 155 nM), 5-HT1A -22.39 kcal/mol (Ki = 130 nM), corticotropin-releasing factor receptor subtype 1 (CRF1) -11.95 kcal/mol (Ki = 299 nM) and glucocorticoid receptors (GR) -14.69 kcal/mol (Ki = 3552 nM). These data fit the binding affinity (Ki) and demonstrate the involvement of gabaergic, serotonergic and glucocorticoid mechanisms in the anxiolytic-like property of mast-L.

Selective antibacterial activity of the cationic peptide PaDBS1R6 against Gram-negative bacteria.

Infections caused by Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa foremost among them, constitute a major worldwide health problem. Bioinformatics methodologies are being used to rationally design new antimicrobial peptides, a potential alternative for treating these infections. One of the algorithms used to develop antimicrobial peptides is the Joker, which was used to design the peptide PaDBS1R6. This study evaluates the antibacterial activities of PaDBS1R6 in vitro and in vivo, characterizes the peptide interaction to target membranes, and investigates the PaDBS1R6 structure in contact with mimetic vesicles. Moreover, we demonstrate that PaDBS1R6 exhibits selective antimicrobial activity against Gram-negative bacteria. In the presence of negatively charged and zwitterionic lipids the structural arrangement of PaDBS1R6 transits from random coil to α-helix, as characterized by circular dichroism. The tertiary structure of PaDBS1R6 was determined by NMR in zwitterionic dodecylphosphocholine (DPC) micelles. In conclusion, PaDBS1R6 is a candidate for the treatment of nosocomial infections caused by Gram-negative bacteria, as template for producing other antimicrobial agents.

Perspectives on the Therapeutic Benefits of Arginine Supplementation in Cancer Treatment.

BACKGROUND: Arginine is considered a semi-essential amino acid in healthy adults and the elderly. This amino acid seems to improve the immune system, stimulate cell growth and differentiation, and increase endothelial permeability, among other effects. For those reasons, it has been theorized that arginine supplementation may be used as an adjuvant to conventional cancer therapy treatments. OBJECTIVE: This review aims to evaluate the existing knowledge of the scientific community on arginine supplementation in order to improve the efficacy of current cancer treatment. RESULTS: Despite the continued efforts of science to improve treatment strategies, cancer remains one of the greatest causes of death on the planet in adults and elderly people. Chemo and radiotherapy are still the most effective treatments but at the cost of significant side effects. CONCLUSION: Thus, new therapeutic perspectives have been studied in recent years, to be used in addition to traditional treatments or not, seeking to treat or even cure the various types of cancer with fewer side effects.

Recombinant Inga Laurina Trypsin Inhibitor (ILTI) Production in Komagataella Phaffii Confirms Its Potential Anti-Biofilm Effect and Reveals an Anti-Tumoral Activity.

Protease inhibitors have a broad biotechnological application ranging from medical drugs to anti-microbial agents. The Inga laurina trypsin inhibitor (ILTI) previously showed a great in vitro inhibitory effect under the adherence of Staphylococcus species, being a strong candidate for use as an anti-biofilm agent. Nevertheless, this is found in small quantities in its sources, which impairs its utilization at an industrial scale. Within this context, heterologous production using recombinant microorganisms is one of the best options to scale up the recombinant protein production. Thus, this work aimed at utilizing Komagataella phaffii to produce recombinant ILTI. For this, the vector pPIC9K+ILTI was constructed and inserted into the genome of the yeast K. phaffii, strain GS115. The protein expression was highest after 48 h using methanol 1%. A matrix-assisted laser desorption ionization⁻time-of-flight (MALDI⁻TOF) analysis was performed to confirm the production of the recombinant ILTI and its activity was investigated trough inhibitory assays using the synthetic substrate Nα-Benzoyl-D,L-arginine p-nitroanilide hydrochloride (BAPNA). Finally, recombinant ILTI (rILTI) was used in assays, showing that there was no significant difference between native and recombinant ILTI in its inhibitory activity in biofilm formation. Anti-tumor assay against Ehrlich ascites tumor (EAT) cells showed that rILTI has a potential anti-tumoral effect, showing the same effect as Melittin when incubated for 48 h in concentrations above 25 µg/mL. All together the results suggests broad applications for rILTI.

Host-defense peptides and their potential use as biomarkers in human diseases.

Since the early 19th century, host-defense peptides (HDPs) have been known to play a crucial role in innate host defense. Subsequent work has demonstrated their role in adaptive immunity as well as their involvement in cancer and also a number of inflammatory and/or autoimmune diseases. In addition to these multiple functional activities, several studies have shown that HDP accumulation might be correlated with various human diseases and, therefore, could be used as a biomarkers for such. Thus, research has aimed to validate the clinical use of HDPs for diagnosis, prognosis, and further treatment. In this review, we outline the most recent findings related to the use of HDPs as biomarkers, their clinical and epidemiological value, and the techniques used to determine the levels of HDPs.

Evaluation of the antimicrobial activity of the mastoparan Polybia-MPII isolated from venom of the social wasp Pseudopolybia vespiceps testacea (Vespidae, Hymenoptera).

Mastoparans, a class of peptides found in wasp venom, have significant effects following a sting as well as useful applications in clinical practice. Among these is their potential use in the control of micro-organisms that cause infectious diseases with a significant impact on society. Thus, the present study describes the isolation and identification of a mastoparan peptide from the venom of the social wasp Pseudopolybia vespiceps and evaluated its antimicrobial profile against bacteria (Staphylococcus aureus and Mycobacterium abscessus subsp. massiliense), fungi (Candida albicans and Cryptococcus neoformans) and in vivo S. aureus infection. The membrane pore-forming ability was also assessed. The mastoparan reduced in vitro and ex vivo mycobacterial growth by 80% at 12.5 µM in infected peritoneal macrophages but did not affect the shape of bacterial cells at the dose tested (6.25 µM). The peptide also showed potent action against S. aureus in vitro (EC50 and EC90 values of 1.83 µM and 2.90 µM, respectively) and reduced the in vivo bacterial load after 6 days of topical treatment (5 mg/kg). Antifungal activity was significant, with EC50 and EC90 values of 12.9 µM and 15.3 µM, respectively, for C. albicans, and 11 µM and 22.70 µM, respectively, for C. neoformans. Peptides are currently attracting interest for their potential in the design of antimicrobial drugs, particularly due to the difficulty of micro-organisms in developing resistance to them. In this respect, Polybia-MPII proved to be highly effective, with a lower haemolysis rate compared with peptides of the same family.

Characterization of a Bioactive Acyclotide from Palicourea rigida.

The extraction and purification of parigidin-br3, a cyclotide analogue belonging to the "bracelet" subfamily, from Palicourea rigida leaves is discussed. Unlike conventional cyclotides, parigidin-br3 has free N- and C-termini, as identified by MALDI-TOF/TOF analysis and confirmed by gene structure elucidation, and is one of a small number of acyclotides discovered during recent years. Parigidin-br3 showed cytotoxic activity against MCF-7 (breast cancer) and CACO2 (colorectal adenocarcinoma) cells, with IC50 values of ∼2.5 µM and less than 10% hemolytic activity. Overall, parigidin-br3 is a promising new molecule with cytotoxic properties against tumor cell lines and, unlike many synthetic acyclic analogues, demonstrates that cytotoxic activity is not limited to conventional (i.e., cyclic) cyclotides.

Structural Studies of a Lipid-Binding Peptide from Tunicate Hemocytes with Anti-Biofilm Activity.

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.

Structural and functional evaluation of the palindromic alanine-rich antimicrobial peptide Pa-MAP2.

Recently, several peptides have been studied regarding the defence process against pathogenic microorganisms, which are able to act against different targets, with the purpose of developing novel bioactive compounds. The present work focuses on the structural and functional evaluation of the palindromic antimicrobial peptide Pa-MAP2, designed based on the peptide Pa-MAP from Pleuronectes americanus. For a better structural understanding, molecular modelling analyses were carried out, together with molecular dynamics and circular dichroism, in different media. Antibacterial activity against Gram-negative and positive bacteria was evaluated, as well as cytotoxicity against human erythrocytes, RAW 264.7, Vero and L6 cells. In silico docking experiments, lipid vesicle studies, and atomic force microscopy (AFM) imaging were carried out to explore the activity of the peptide. In vivo studies on infected mice were also done. The palindromic primary sequence favoured an α-helix structure that was pH dependent, only present on alkaline environment, with dynamic N- and C-terminals that are stabilized in anionic media. Pa-MAP2 only showed activity against Gram-negative bacteria, with a MIC of 3.2 µM, and without any cytotoxic effect. In silico, lipid vesicles and AFM studies confirm the preference for anionic lipids (POPG, POPS, DPPE, DPPG and LPS), with the positively charged lysine residues being essential for the initial electrostatic interaction. In vivo studies showed that Pa-MAP2 increases to 100% the survival rate of mice infected with Escherichia coli. Data here reported indicated that palindromic Pa-MAP2 could be an alternative candidate for use in therapeutics against Gram-negative bacterial infections.

Effects of cyclotides against cutaneous infections caused by Staphylococcus aureus.

The main bacterium associated with skin infection is Staphylococcus aureus, occurring especially in infections acquired via surgical wounds, commonly leading to lethal hospital-acquired infections, emphasizing the importance of identifying new antimicrobial compounds. Among them, cyclotides have gained interest due to their high stability and multifunctional properties. Here, cycloviolacin 2 (CyO2) and kalata B2 (KB2) were evaluated to determinate their anti-staphylococcal activities using a subcutaneous infection model. Anti-staphylococcal activities of 50mM for KB2 and 25mM for CyO2 were detected with no cytotoxic activities against RAW 264.7 monocytes. In the in vivo assays, both cyclotides reduced bacterial load and CyO2 demonstrated an increase in the phagocytosis index, suggesting that the CyO2 in vivo anti-staphylococcal activity may be associated with phagocytic activity, additionally to direct anti-pathogenic activity.

Clavanin A improves outcome of complications from different bacterial infections.

The rapid increase in the incidence of multidrug-resistant infections today has led to enormous interest in antimicrobial peptides (AMPs) as suitable compounds for developing unusual antibiotics. In this study, clavanin A, an antimicrobial peptide previously isolated from the marine tunicate Styela clava, was selected as a purposeful molecule that could be used in controlling infection and further synthesized. Clavanin A was in vitro evaluated against Staphylococcus aureus and Escherichia coli as well as toward L929 mouse fibroblasts and skin primary cells (SPCs). Moreover, this peptide was challenged here in an in vivo wound and sepsis model, and the immune response was also analyzed. Despite displaying clear in vitro antimicrobial activity toward Gram-positive and -negative bacteria, clavanin A showed no cytotoxic activities against mammalian cells, and in acute toxicity tests, no adverse reaction was observed at any of the concentrations. Moreover, clavanin A significantly reduced the S. aureus CFU in an experimental wound model. This peptide also reduced the mortality of mice infected with E. coli and S. aureus by 80% compared with that of control animals (treated with phosphate-buffered saline [PBS]): these data suggest that clavanin A prevents the start of sepsis and thereby reduces mortality. These data suggest that clavanin A is an AMP that could improve the development of novel peptide-based strategies for the treatment of wound and sepsis infections.

Clavanin bacterial sepsis control using a novel methacrylate nanocarrier.

Controlling human pathogenic bacteria is a worldwide problem due to increasing bacterial resistance. This has prompted a number of studies investigating peptides isolated from marine animals as a possible alternative for control of human pathogen infections. Clavanins are antimicrobial peptides isolated from the marine tunicate Styela clava, showing 23 amino acid residues in length, cationic properties, and also high bactericidal activity. In spite of clear benefits from the use of peptides, currently 95% of peptide properties have limited pharmaceutical applicability, such as low solubility and short half-life in the circulatory system. Here, nanobiotechnology was used to encapsulate clavanin A in order to develop nanoantibiotics against bacterial sepsis. Clavanin was nanostructured using EUDRAGIT(®) L 100-55 and RS 30 D solution (3:1 w:w). Atomic force, scanning electron microscopy and dynamic light scattering showed nanoparticles ranging from 120 to 372 nm in diameter, with a zeta potential of -7.16 mV and a polydispersity index of 0.123. Encapsulation rate of 98% was assessed by reversed-phase chromatography. In vitro bioassays showed that the nanostructured clavanin was partially able to control development of Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Furthermore, nanostructures did not show hemolytic activity. In vivo sepsis bioassays were performed using C57BL6 mice strain inoculated with a polymicrobial suspension. Assays led to 100% survival rate under sub-lethal sepsis assays and 40% under lethal sepsis assays in the presence of nanoformulated clavanin A until the seventh day of the experiment. Data here reported indicated that nanostructured clavanin A form shows improved antimicrobial activity and has the potential to be used to treat polymicrobial infections.

Investigating specific bacterial resistance to AMPs by using a magainin I-resistant Escherichia coli model.

Antimicrobial peptides (AMPs) are multifunctional compounds that may show antimicrobial and immunomodulatory activities. With the rapid increase in the incidence of multidrug-resistant bacteria, there is an enormous interest in AMPs as templates for the production of new antibiotics. However, there are concerns that the therapeutic administration of AMPs can select resistant strains. In order to distinguish between resistant and non-resistant strains and verify resistance specificity to AMPs, in this study a magainin I-resistant Escherichia coli model was used. First, the identity of all strains was confirmed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)-MS, VITEK 2 and MicroScan, and the susceptible and magainin-resistant strains were successfully differentiated by MALDI-TOF-MS analysis. Furthermore, cross-resistances to a broad spectrum of antibiotics were evaluated, showing that all E. coli strains are susceptible to the drugs tested, suggesting that the resistance seems to be specific to AMPs. Finally, the specific resistance to magainin I compared with other AMPs was checked by microdilution. This experiment showed that the magainin MICs were 62 and 104 µM for susceptible and resistant strains, respectively. The other AMPs MICs were 3.4 µM to proline-arginine-rich 39-amino-acid peptide, 43 µM to porcine myeloid antimicrobial 23-amino-acid peptide-23 and 1.2 µM to cecropin P1 for all strains, demonstrating any additional resistance to peptides here evaluated, confirming that the resistance seems to be essentially specific to magainin I. In summary, the data reported here reinforce the proposal that magainin I seems not to be merely a membrane disruptor, probably showing additional molecular targets in pathogenic bacteria.

Dentistry proteomics: from laboratory development to clinical practice.

Despite all the dental information acquired over centuries and the importance of proteome research, the cross-link between these two areas only emerged around mid-nineties. Proteomic tools can help dentistry in the identification of risk factors, early diagnosis, prevention, and systematic control that will promote the evolution of treatment in all dentistry specialties. This review mainly focuses on the evolution of dentistry in different specialties based on proteomic research and how these tools can improve knowledge in dentistry. The subjects covered are an overview of proteomics in dentistry, specific information on different fields in dentistry (dental structure, restorative dentistry, endodontics, periodontics, oral pathology, oral surgery, and orthodontics) and future directions. There are many new proteomic technologies that have never been used in dentistry studies and some dentistry areas that have never been explored by proteomic tools. It is expected that a greater integration of these areas will help to understand what is still unknown in oral health and disease.

Characterization and pharmacological properties of a novel multifunctional Kunitz inhibitor from Erythrina velutina seeds.

Inhibitors of peptidases isolated from leguminous seeds have been studied for their pharmacological properties. The present study focused on purification, biochemical characterization and anti-inflammatory and anticoagulant evaluation of a novel Kunitz trypsin inhibitor from Erythrina velutina seeds (EvTI). Trypsin inhibitors were purified by ammonium sulfate (30-60%), fractionation followed by Trypsin-Sepharose affinity chromatography and reversed-phase high performance liquid chromatography. The purified inhibitor showed molecular mass of 19,210.48 Da. Furthermore, a second isoform with 19,228.16 Da was also observed. The inhibitor that showed highest trypsin specificity and enhanced recovery yield was named EvTI (P2) and was selected for further analysis. The EvTI peptide fragments, generated by trypsin and pepsin digestion, were further analyzed by MALDI-ToF-ToF mass spectrometry, allowing a partial primary structure elucidation. EvTI exhibited inhibitory activity against trypsin with IC50 of 2.2×10(-8) mol.L(-1) and constant inhibition (Ki) of 1.0×10(-8) mol.L(-1), by a non-competitive mechanism. In addition to inhibit the activity of trypsin, EvTI also inhibited factor Xa and neutrophil elastase, but do not inhibit thrombin, chymotrypsin or peptidase 3. EvTI was investigated for its anti-inflammatory and anti-coagulant properties. Firstly, EvTI showed no cytotoxic effect on human peripheral blood cells. Nevertheless, the inhibitor was able to prolong the clotting time in a dose-dependent manner by using in vitro and in vivo models. Due to anti-inflammatory and anticoagulant EvTI properties, two sepsis models were here challenged. EvTI inhibited leukocyte migration and specifically acted by inhibiting TNF-α release and stimulating IFN-α and IL-12 synthesis. The data presented clearly contribute to a better understanding of the use of Kunitz inhibitors in sepsis as a bioactive agent capable of interfering in blood coagulation and inflammation.