Functional analysis of airway remodeling is related with fibrotic mediators in asthmatic children.

BACKGROUND: Asthmatic children present variable degrees of airway inflammation, remodeling, and resistance, which correlate with disease control and severity. The chronic inflammatory process of the airway triggers airway remodeling, which reflects the degree of airway resistance. Pro-inflammatory and pro-fibrotic mediators are centrally involved in this process. OBJECTIVE: To investigate whether the levels of pulmonary and systemic pro-inflammatory and pro-fibrotic mediators present a correlation with the resistance of the respiratory system and of the proximal and distal airways. METHODS: 39 Asthmatic children (persistent mild and moderate) and 39 non-asthmatic children (both between 6 and 13 years old) were evaluated for anthropometric characteristics, lung function and mechanics, and pulmonary and systemic immune responses. RESULTS: Asthmatic children showed an increased number of blood eosinophils (p < 0.04), basophils (p < 0.04), monocytes (p < 0.002) and lymphocytes (p < 0.03). In addition, asthmatic children showed impaired lung function, as demonstrated by FEV1 (p < 0.0005) and FEV1/FVC (p < 0.004), decreased total resistance of the respiratory system (R5Hz; p < 0.009), increased resistance of the proximal airways (R20Hz; p < 0.02), increased elastance (Z5Hz; p < 0.02) and increased reactance (X5Hz; p < 0.002) compared to non-asthmatic children. Moreover, the following inflammatory factors were significantly higher in asthmatic than non-asthmatic children: GM-CSF in the breath condensate (BC) (p < 0.0001) and in the serum (p < 0.0001); TGF-beta in the BC (p < 0.0001) and in the serum (p < 0.004); IL-5 in the BC (p < 0.02) and in the serum (p < 0.01); IL-4 in the serum (p < 0.0002). CONCLUSIONS: Impulse oscillometry is a sensitive method to detect airway resistance in persistent mild and moderate asthmatic children, an event followed by increased levels of pro-inflammatory and pro-fibrotic mediators.

Purification, Characterization and Evaluation of the Anticoagulant Effect of an Uncompetitive Trypsin Inhibitor obtained from Bauhinia pulchella (Benth) Seeds.

INTRODUCTION: Trypsin inhibitors (TIs) have the ability to competitively or non-competitively bind to trypsin and inhibit its action. These inhibitors are commonly found in plants and are used in protease inhibition studies involved in biochemical pathways of pharmacological interest. OBJECTIVES: This work aimed to purify a trypsin inhibitor from Bauhinia pulchella seeds (BpuTI), describing its kinetic mechanism and anticoagulant effect. METHODS: Affinity chromatography, protein assay, and SDS-PAGE were used to purify the inhibitor. Mass spectrometry, inhibition assays, and enzyme kinetics were used to characterize the inhibitor. In vitro assays were performed to verify its ability to prolong blood clotting time. RESULTS: Affinity chromatography on a Trypsin-Sepharose 4B column gave a yield of 43.1. BpuTI has an apparent molecular mass of 20 kDa with glycosylation (1.15%). Protein identification was determined by MS/MS, and BpuTI showed similarity to several Kunitz-type trypsin inhibitors. BpuTI inhibited bovine trypsin as an uncompetitive inhibitor with IC50 (3 x 10-6 M) and Ki (1.05 x 10-6 M). Additionally, BpuTI showed high stability to temperature and pH variations, maintaining its activity up to 100ºC and in extreme pH ranges. However, the inhibitor was susceptible to reducing agents, such as DTT, which completely abolished its activity. BpuTI showed an anticoagulant effect in vitro at a concentration of 33 µM, prolonging clotting time by 2.6 times. CONCLUSION: Our results suggest that BpuTI can be a biological tool to be used in blood clotting studies.

JcTI-PepI, a synthetic peptide bioinspired in the trypsin inhibitor from Jatropha curcas, presents potent inhibitory activity against C. krusei, a neglected pathogen.

Antimicrobial resistance has been increasing globally, posing a global public health risk. It has prompted the scientific community to look for alternatives to traditional drugs. Antimicrobial Peptides (AMPs) have stood out in this context because they have the potential to control infectious diseases while causing no or little harm to mammalian cells. In the present study, three peptides, JcTI-PepI, JcTI-PepII, and JcTI-PepIII, were designed and tested for antimicrobial activity based on the primary sequence of JcTI-I, a 2S albumin with trypsin inhibitory activity from Jatropha curcas. JcTI-PepI strongly inhibited C. krusei growth, and it caused severe disruptions in cellular processes and cell morphology. C. krusei cells treated with JcTI-PepI showed indicative of membrane permeabilization and overproduction of Reactive Oxygen Species. Moreover, the yeast's ability to acidify the medium was severely compromised. JcTI-PepI was also effective against pre-formed biofilm and did not harm human erythrocytes and Vero cells. Overall, these characteristics indicate that JcTI-PepI is both safe and effective against C. krusei, an intrinsically resistant strain that causes serious health problems and is frequently overlooked. It implies that this peptide has a high potential for use as a new antimicrobial agent in the future.

New Insights into Anthelmintic Mechanisms of Action of a Synthetic Peptide: An Ultrastructural and Nanomechanical Approach.

Resistant nematodes are not affected by the most common drugs commercially available. In the search for new anthelmintics, peptides have been investigated. Here, a linear synthetic peptide named RcAlb-PepIII bioinspired from the antimicrobial protein Rc-2S-Alb was designed, synthesized, and tested against barber pole worm Haemonchus contortus. The physicochemical properties of the peptide, the 3D structure model, the egg hatch inhibition, and larval development inhibition of H. contortus were carried out. Additionally, the ultrastructure of the nematode after treatment with the peptide was evaluated by atomic force microscopy. The RcAlb-PepIII inhibited the larval development of H. contortus with an EC50 of 90 µM and did not affect egg hatch. Atomic force microscopy reveals the high affinity of RcAlb-PepIII with the cuticle of H. contortus in the L2 stage. It also shows the deposition of RcAlb-PepIII onto the surface of the cuticle, forming a structure similar to a film that reduces the roughness and mean square roughness (Rq) of it. In conclusion, the bioinspired RcAlb-PepIII has the potential to be used as a new anthelmintic compound to control gastrointestinal nematode parasites.

Antidermatophytic activity of synthetic peptides: Action mechanisms and clinical application as adjuvants to enhance the activity and decrease the toxicity of Griseofulvin.

BACKGROUND: Dermatophytes belonging to the Trichophyton genus are important human pathogens, but they have developed resistance to griseofulvin, the most common antifungal drug used to treat dermatophytosis. OBJECTIVE: This study was aimed to evaluate the antidermatophytic activity of synthetic peptides, as well as mechanisms of action and synergistic effect with griseofulvin. METHODS: Scanning electron microscopy (SEM), atomic force microscopy (AFM) and fluorescence microscopy (FM) were employed to understand the activity and the mechanism of action of peptides. RESULTS: Here we report that synthetic peptides at 50 µg/mL, a concentration 20-fold lower than griseofulvin, reduced the microconidia viability of T. mentagrophytes and T. rubrum by 100%, whereas griseofulvin decreased their viability by only 50% and 0%, respectively. The action mechanism of peptides involved cell wall damage, membrane pore formation and loss of cytoplasmic content. Peptides also induced overproduction of reactive oxygen species (ROS) and enhanced the activity of griseofulvin 10-fold against both fungi, suggesting synergistic effects, and eliminated the toxicity of this drug to human erythrocytes. Docking analysis revealed ionic and hydrophobic interactions between peptides and griseofulvin, which may explain the decline of griseofulvin toxicity when mixed with peptides. CONCLUSION: Therefore, our results strongly suggest six peptides with high potential to be employed alone as new drugs or as adjuvants to enhance the activity and decrease the toxicity of griseofulvin.

Machaerium acutifolium lectin alters membrane structure and induces ROS production in Candida parapsilosis.

Lectins are a group of widely distributed and structurally heterogeneous proteins of nonimmune origin. These proteins have the ability to interact with glycans present on cell surfaces and elicit diverse biological activities. Machaerium acutifolium lectin (MaL) is an N-acetyl-D-glucosamine-binding lectin that exhibits antinociceptive activity via transient receptor potential cation channel subfamily V member 1 (TRPV1). Lectins that have the ability to recognize and interact with N-acetyl-D-glucosamine residues are potential candidates for studies of fungicidal activity. In this work, we show that MaL has antifungal activity against Candida species, and we describe its mode of action towards Candida parapsilosis. MaL inhibited the growth of C. albicans and C. parapsilosis. However, MaL was more potent against C. parapsilosis. The candidacidal mode of action of MaL on C. parapsilosis involves enhanced cell permeabilization, alteration of the plasma membrane proton-pumping ATPase function (H+-ATPase), induction of oxidative stress, and DNA damage. MaL also exhibited antibiofilm activity and noncytotoxicity to Vero cells. These results indicate that MaL is a promising candidate for the future development of a new, natural, and safe drug for the treatment of infections caused by C. parapsilosis.

Synthetic antimicrobial peptides: From choice of the best sequences to action mechanisms.

The emergence of antibiotic-resistant microbes has stimulated research worldwide seeking new biologically active molecules. In this respect, synthetic antimicrobial peptides (SAMPs) have been suggested to overcome this problem. Although there are some online servers that provide putative SAMPs from protein sequences, the choice of the best peptide sequences for further analysis is still difficult. Therefore, the goal of this paper is not to launch a new tool but to provide a friendly workflow to characterize and predict potential SAMPs by employing existing tools. Using this proposed workflow, two peptides (PepGAT and PepKAA) were obtained and extensively characterized. These peptides damaged microbial membranes and cell walls, and induced overproduction of reactive oxygen species (ROS). Both peptides were found to assume random coil secondary structure in aqueous solution, organic solvent, and upon binding to negatively charged lipid systems. Peptides were also able to degrade formed biofilms but not to prevent biofilm formation. PepGAT was not resistant to proteolysis, whereas PepKAA was resistant to pepsin but not to pancreatin. Furthermore, both presented no hemolytic activity against red blood cells, even at a 10-fold higher concentration than the antimicrobial concentration. The pipeline proposed here is an easy way to design new SAMPs for application as alternatives to develop new drugs against human pathogenic microorganisms.

Gene expression during development and overexpression after Cercospora kikuchii and salicylic acid challenging indicate defensive roles of the soybean toxin.

KEY MESSAGE: SBTX has defensive role against C. kikuchii, and therefore, its constituent genes SBTX17 and SBTX27 are promising candidates to engineer pathogen resistant plants. Soybean (Glycine max [L.] Merr.) is economically the most important legume crop in the world. Its productivity is strongly affected by fungal diseases, which reduce soybean production and seed quality and cause losses of billions of dollars worldwide. SBTX is a protein that apparently takes part in the defensive chemical arsenal of soybean against pathogens. This current study provides data that reinforce this hypothesis. Indeed, SBTX inhibited in vitro the mycelial growth of Cercospora kikuchii, it is constitutively located in the epidermal region of the soybean seed cotyledons, and it is exuded from mature imbibed seeds. Moreover, RT-qPCR analysis of the SBTX associated genes, SBTX17 and SBTX27, which encode for the 17 and 27 kDa polypeptide chains, showed that both genes are expressed in all studied plant tissues during the soybean development, with the highest levels found in the mature seeds and unifoliate leaves. In addition, to assess a local response of the soybean secondary leaves from 35-day-old plants, they were inoculated with C. kikuchii and treated with salicylic acid. It was verified using RT-qPCR that SBTX17 and SBTX27 genes overexpressed in leaves compared to controls. These findings strongly suggest that SBTX has defensive roles against C. kikuchii. Therefore, SBTX17 and SBTX27 genes are promising candidates to engineer pathogen resistant plants.

Anticandidal activity of synthetic peptides: Mechanism of action revealed by scanning electron and fluorescence microscopies and synergism effect with nystatin.

Candida albicans has emerged as a major public health problem in recent decades. The most important contributing factor is the rapid increase in resistance to conventional drugs worldwide. Synthetic antimicrobial peptides (SAMPs) have attracted substantial attention as alternatives and/or adjuvants in therapeutic treatments due to their strong activity at low concentrations without apparent toxicity. Here, two SAMPs, named Mo-CBP3 -PepI (CPAIQRCC) and Mo-CBP3 -PepII (NIQPPCRCC), are described, bioinspired by Mo-CBP3 , which is an antifungal chitin-binding protein from Moringa oleifera seeds. Furthermore, the mechanism of anticandidal activity was evaluated as well as their synergistic effects with nystatin. Both peptides induced the production of reactive oxygen species (ROS), cell wall degradation, and large pores in the C. albicans cell membrane. In addition, the peptides exhibited high potential as adjuvants because of their synergistic effects, by increasing almost 50-fold the anticandidal activity of the conventional antifungal drug nystatin. These peptides have excellent potential as new drugs and/or adjuvants to conventional drugs for treatment of clinical infections caused by C. albicans.

Scanning electron microscopy reveals deleterious effects of Moringa oleifera seed exuded proteins on root-knot nematode Meloidogyne incognita eggs.

Plant seeds can exudate active molecules with inhibitory effects against several soil pathogens, including nematodes. This study aimed to characterize and evaluate the nematicidal properties against Meloidogyne incognita of exuded proteins from Moringa oleifera seeds. M. oleifera seeds were soaked in distilled water, and exudates were harvested and analyzed for the presence of defense proteins and anthelmintic activity. Enzymatic assays revealed the existence of PR-proteins such as ß-1,3-glucanases (0.18 ± 0.003 nkatal mg-1 of protein), chitinases (0.22 ± 0.004 nkatal mg-1 of protein), proteases (261.30 ± 6.405 AU mg-1 of protein min-1), serine (190.30 ± 5.574 IA mg-1 of protein) and cysteine (231.70 ± 0.923 IA mg-1 of protein), protease inhibitors. The exuded proteins presented ovicidal activity and caused 100% mortality of second-stage juveniles (J2s). Scanning electron microscopy (SEM) revealed deleterious effects on M. incognita eggs, such as invaginations, cracks, scratched surface, and loss of internal content. These findings confirm the presence of anthelmintic proteins in M. oleifera seed exudate, possibly involved in plant defense during seed germination. Besides this, the exuded proteins exhibited strong biotechnological potential for use in the biocontrol of M. incognita infections, which are responsible for millions of dollars in staple crop losses every year.

RcAlb-PepII, a synthetic small peptide bioinspired in the 2S albumin from the seed cake of Ricinus communis, is a potent antimicrobial agent against Klebsiella pneumoniae and Candida parapsilosis.

Antimicrobial peptides (AMPs) are important constituents of the innate immunity system of all living organisms. They participate in the first line of defense against invading pathogens such as viruses, bacteria, and fungi. In view of the increasing difficulties to treat infectious diseases due to the emergence of antibiotic-resistant bacterial strains, AMPs have great potential to control infectious diseases in humans and animals. In this study, two small peptides, RcAlb-PepI and RcAlb-PepII, were designed based on the primary structure of Rc-2S-Alb, a 2S albumin from the seed cake of Ricinus communis, and their antimicrobial activity assessed. RcAlb-PepII strongly inhibited the growth of Klebsiella pneumoniae and Candida parapsilosis, and induced morphological alterations in their cell surface. C. parapsilosis exposed to RcAlb-PepII presented higher cell membrane permeabilization and elevated content of reactive oxygen species. RcAlb-PepII also degraded and reduced the biofilm formation in C. parapsilosis and in K. pneumonia cells. Experimentally, RcAlb-PepII was not hemolytic and had low toxicity to mammalian cells. These are advantageous characteristics, which suggest that RcAlb-PepII is safe and apparently effective for its intended use and has great potential for the future development of an antimicrobial agent with the ability to kill or inhibit K. pneumoniae and C. parapsilosis cells.

Identification, characterization, and antifungal activity of cysteine peptidases from Calotropis procera latex.

Cysteine peptidases (EC 3.4.22) are the most abundant enzymes in latex fluids. However, their physiological functions are still poorly understood, mainly related to defense against phytopathogens. The present study reports the cDNA cloning and sequencing of five undescribed cysteine peptidases from Calotropis procera (Aiton) Dryand (Apocynaceae) as well as some in silico analyses. Of these, three cysteine peptidases (CpCP1, CpCP2, and CpCP3) were purified. Their enzymatic kinetics were determined and they were assayed for their efficacy in inhibiting the hyphal growth of phytopathogenic fungi. The mechanism of action was investigated by fluorescence and atomic force microscopy as well as by induction of reactive oxygen species (ROS). The deduced amino acid sequences showed similar biochemical characteristics and high sequence homology with several other papain-like cysteine peptidases. Three-dimensional models showed two typical cysteine peptidase domains (L and R domains), forming a "V-shaped" active site containing the catalytic triad (Cys, His, and Asn). Proteolysis of CpCP1 was higher at pH 7.0, whereas for CpCP2 and CpCP3 it was higher at 7.5. All peptidases exhibited optimum activity at 35 °C and followed Michaelis-Menten kinetics. However, the major difference among them was that CpCP1 exhibited highest Vmax, Km, Kcat and catalytic efficiency. All peptidases were deleterious to the two fungi tested, with IC50 of around 50 µg/mL. The peptidases promoted membrane permeabilization, morphological changes with leakage of cellular content, and induction of ROS in F. oxysporum spores. These results corroborate the hypothesis that latex cysteine peptidases play a role in defense against fungi.

ClTI, a Kunitz trypsin inhibitor purified from Cassia leiandra Benth. seeds, exerts a candidicidal effect on Candida albicans by inducing oxidative stress and necrosis.

Cassia leiandra is an Amazonian plant species that is used popularly for the treatment of mycoses. Recently, a protease inhibitor, named ClTI, with insecticidal activity against Aedes aegypti, was purified from the mature seeds of C. leiandra. In this work, we show that ClTI has antifungal activity against Candida species and describe its mode of action towards Candida albicans. This study is relevant because the nosocomial infections caused by Candida species are a global public health problem that, together with the growing resistance to current drugs, has increased the urgency of the search for new antifungal compounds. ClTI inhibited the growth of Candida albicans, Candida tropicalis, Candida parapsilosis, and Candida krusei. However, ClTI was more potent against C. albicans. The candidicidal mode of action of ClTI on C. albicans involves enhanced cell permeabilization, alteration of the plasma membrane proton-pumping ATPase function (H+ -ATPase), induction of oxidative stress, and DNA damage. ClTI also exhibited antibiofilm activity and non-cytotoxicity to mammalian cells. These results indicate that ClTI is a promising candidate for the future development of a new, natural, and safe agent for the treatment of infections caused by C. albicans.

ClCPI, a cysteine protease inhibitor purified from Cassia leiandra seeds has antifungal activity against Candida tropicalis by inducing disruption of the cell surface.

Infections caused by Candida tropicalis have increased significantly worldwide in parallel with resistance to antifungal drugs. To overcome resistance novel drugs have to be discovered. The objective of this work was to purify and characterize a cysteine protease inhibitor from the seeds of the Amazon rainforest tree Cassia leiandra and test its inhibitory effect against C. tropicalis growth. The inhibitor, named ClCPI, was purified after ion exchange and affinity chromatography followed by ultrafiltration. ClCPI is composed of a single polypeptide chain and is not a glycoprotein. The molecular mass determined by SDS-PAGE in the absence or presence of ß-mercaptoethanol and ESI-MS were 16.63 kDa and 18.362 kDa, respectively. ClCPI was stable in the pH range of 7.0-9.0 and thermostable up to 60 °C for 20 min. ClCPI inhibited cysteine proteases, but not trypsin, chymotrypsin neither alpha-amylase. Inhibition of papain was uncompetitive with a Ki of 4.1 × 10-7 M and IC50 of 8.5 × 10-7 M. ClCPI at 2.6 × 10-6 M reduced 50% C. tropicalis growth. ClCPI induced damages and morphological alterations in C. tropicalis cell surface, which led to death. These results suggest that ClCPI have great potential for the development of an antifungal drug against C. tropicalis.

Mo-CBP3-PepI, Mo-CBP3-PepII, and Mo-CBP3-PepIII are synthetic antimicrobial peptides active against human pathogens by stimulating ROS generation and increasing plasma membrane permeability.

The efficiency of current antimicrobial drugs is noticeably decreasing and thus the development of new treatments is necessary. Natural and synthetic antimicrobial peptides (AMPs) have attracted great attention as promising candidates. Inspired on Mo-CBP3, an antimicrobial protein from Moringa oleifera seeds, we designed and synthesized three AMPs named Mo-CBP3-PepI, Mo-CBP3-PepII, and Mo-CBP3-PepIII. All these three peptides inhibited the growth of Candida species and pathogenic bacteria, penetrate into microbial cells, but none is hemolytic or toxic to human cells. Mo-CBP3-PepIII, particularly, showed the strongest antimicrobial activity against Staphylococcus aureus and Candida species, important human pathogens. Additionally, Mo-CBP3-PepIII did not exhibit hemolytic or toxic activity to mammalian cells, but increased Staphylococcus aureus plasma membrane permeabilization. In Candida parapsilosis, Mo-CBP3-PepIII induced pore formation in the plasma membrane and overproduction of reactive oxygen species. Bioinformatics analysis suggested that Mo-CBP3-PepIII is resistant to pepsin digestion and other proteolytic enzymes present in the intestinal environment, which opens the possibility of oral delivery in future treatments. Together, these results suggest that Mo-CBP3-PepIII has great potential as an antimicrobial agent against the bacterium S. aureus and the fungi C. parapsilosis.