The intrinsic antimicrobial activity of citric acid-coated manganese ferrite nanoparticles is enhanced after conjugation with the antifungal peptide Cm-p5.

Diseases caused by bacterial and fungal pathogens are among the major health problems in the world. Newer antimicrobial therapies based on novel molecules urgently need to be developed, and this includes the antimicrobial peptides. In spite of the potential of antimicrobial peptides, very few of them were able to be successfully developed into therapeutics. The major problems they present are molecule stability, toxicity in host cells, and production costs. A novel strategy to overcome these obstacles is conjugation to nanomaterial preparations. The antimicrobial activity of different types of nanoparticles has been previously demonstrated. Specifically, magnetic nanoparticles have been widely studied in biomedicine due to their physicochemical properties. The citric acid-modified manganese ferrite nanoparticles used in this study were characterized by high-resolution transmission electron microscopy, which confirmed the formation of nanocrystals of approximately 5 nm diameter. These nanoparticles were able to inhibit Candida albicans growth in vitro. The minimal inhibitory concentration was 250 µg/mL. However, the nanoparticles were not capable of inhibiting Gram-negative bacteria (Escherichia coli) or Gram-positive bacteria (Staphylococcus aureus). Finally, an antifungal peptide (Cm-p5) from the sea animal Cenchritis muricatus (Gastropoda: Littorinidae) was conjugated to the modified manganese ferrite nanoparticles. The antifungal activity of the conjugated nanoparticles was higher than their bulk counterparts, showing a minimal inhibitory concentration of 100 µg/mL. This conjugate proved to be nontoxic to a macrophage cell line at concentrations that showed antimicrobial activity.

Cm-p5: an antifungal hydrophilic peptide derived from the coastal mollusk Cenchritis muricatus (Gastropoda: Littorinidae).

Antimicrobial peptides form part of the first line of defense against pathogens for many organisms. Current treatments for fungal infections are limited by drug toxicity and pathogen resistance. Cm-p5 (SRSELIVHQRLF), a peptide derived from the marine mollusk Cenchritis muricatus peptide Cm-p1, has a significantly increased fungistatic activity against pathogenic Candida albicans (minimal inhibitory concentration, 10 µg/ml; EC50, 1.146 µg/ml) while exhibiting low toxic effects against a cultured mammalian cell line. Cm-p5 as characterized by circular dichroism and nuclear magnetic resonance revealed an α-helical structure in membrane-mimetic conditions and a tendency to random coil folding in aqueous solutions. Additional studies modeling Cm-p5 binding to a phosphatidylserine bilayer in silico and isothermal titration calorimetry using lipid monophases demonstrated that Cm-p5 has a high affinity for the phospholipids of fungal membranes (phosphatidylserine and phosphatidylethanolamine), only moderate interactions with a mammalian membrane phospholipid, low interaction with ergosterol, and no interaction with chitin. Adhesion of Cm-p5 to living C. albicans cells was confirmed by fluorescence microscopy with FITC-labeled peptide. In a systemic candidiasis model in mice, intraperitoneal administration of Cm-p5 was unable to control the fungal kidney burden, although its low amphiphaticity could be modified to generate new derivatives with improved fungicidal activity and stability.

Antifungal nanofibers made by controlled release of sea animal derived peptide.

Candida albicans is a common human-pathogenic fungal species with the ability to cause several diseases including surface infections. Despite the clear difficulties of Candida control, antimicrobial peptides (AMPs) have emerged as an alternative strategy for fungal control. In this report, different concentrations of antifungal Cm-p1 (Cencritchis muricatus peptide 1) were electrospun into nanofibers for drug delivery. The nanofibers were characterized by mass spectrometry confirming the presence of the peptide on the scaffold. Atomic force microscopy and scanning electronic microscopy were used to measure the diameters, showing that Cm-p1 affects fiber morphology as well as the diameter and scaffold thickness. The Cm-p1 release behavior from the nanofibers demonstrated peptide release from 30 min to three days, leading to effective yeast control in the first 24 hours. Moreover, the biocompatibility of the fibers were evaluated through a MTS assay as well as ROS production by using a HUVEC model, showing that the fibers do not affect cell viability and only nanofibers containing 10% Cm-p1-PVA improved ROS generation. In addition, the secretion of pro-inflammatory cytokines IL-6 and TNF-α by the HUVECs was also slightly modified by the 10% Cm-p1-PVA nanofibers. In conclusion, the electrospinning technique applied here allowed for the manufacture of biodegradable biomimetic nanofibrous extracellular membranes with the ability to control fungal infection.

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.

Toward in vitro fertilization in Brachiaria spp.

Brachiaria are forage grasses widely cultivated in tropical areas. In vitro pollination was applied to accessions of Brachiaria spp. by placing pollen of non-dehiscent anthers on a solid medium near isolated ovaries. Viability and in vitro germination were tested in order to establish good conditions for pollen development. Comparing sexual to apomictic plants, apomictic pollen has more abortion after meiosis during the microspore stage and a lower viability and, of both types, only some plants have sufficient germination in a high sugar concentration. Using in vitro pollination with the sexual plant, the pollen tube penetrates into the nucellus and micropyle, but the embryo sac degenerates and collapses. In the apomictic B. decumbens, in vitro pollination leads to the transfer of the sperm nuclei into the egg cell and the central cell. The results are discussed according to normal fertilization and barriers in sexual and apomictic plants.

Morphology, distribution and abundance of antennal sensilla in three stink bug species (Hemiptera: Pentatomidae).

The neotropical stink bugs, Euschistus heros, Piezodorus guildinii and Edessa meditabunda, are important pests of soybean and other crops throughout Central America and in South America from Northern Argentina to Brazil. Mate finding and host plant location in these species depend largely on their chemical communication, and semiochemicals are important mediators of these behaviors. In this study scanning electron microscopy was used to examine the external morphology, distribution and abundance of antennal sensilla on males, females and 5th instar nymphs of these species. Nine morphologically different sensilla types were found: trichod sensilla, type 1 and 2 (ST1 and ST2), long and short basiconic sensilla (SB1, SB2, and SB3), slit-tipped and knob-shaped basiconic sensilla, long chaetic sensilla (Sch) and coeloconic sensilla (Sco). Differences were detected in the abundance and arrangement of the sensilla over the antennal segments in individuals of the same species and among the species studied. The Sch, Sco and the slit-tipped and knob-shaped basiconic sensilla accounted for the major difference in sensilla types among the species. The ST1 was the most abundant type and was restricted to the flagellum. The pedicel of E. heros differs from the pedicels of P. guildinii and E. meditabunda mainly by the absence of Sch. There was a sexual dimorphism in ST1, SB1 and SB2, and this may be an indicative of their roles in detection of male-produced sex pheromone and odors derived from the host plants. The SB2 was lacking in the antennal tip of both sexes and 5th instar nymphs, but was abundant on the second flagellar segment of females of the three species. The same types of sensilla were found on 5th instar nymphs, but always in significantly lower numbers. The morphology and putative functions of each sensilla were compared and discussed.

Molecular cloning of alpha-amylases from cotton boll weevil, Anthonomus grandis and structural relations to plant inhibitors: an approach to insect resistance.

Anthonomus grandis, the cotton boll weevil, causes severe cotton crop losses in North and South America. Here we demonstrate the presence of starch in the cotton pollen grains and young ovules that are the main A. grandis food source. We further demonstrate the presence of alpha-amylase activity, an essential enzyme of carbohydrate metabolism for many crop pests, in A. grandis midgut. Two alpha-amylase cDNAs from A. grandis larvae were isolated using RT-PCR followed by 5' and 3' RACE techniques. These encode proteins with predicted molecular masses of 50.8 and 52.7kDa, respectively, which share 58% amino acid identity. Expression of both genes is induced upon feeding and concentrated in the midgut of adult insects. Several alpha-amylase inhibitors from plants were assayed against A. grandis alpha-amylases but, unexpectedly, only the BIII inhibitor from rye kernels proved highly effective, with inhibitors generally active against other insect amylases lacking effect. Structural modeling of Amylag1 and Amylag2 showed that different factors seem to be responsible for the lack of effect of 0.19 and alpha-AI1 inhibitors on A. grandis alpha-amylase activity. This work suggests that genetic engineering of cotton to express alpha-amylase inhibitors may offer a novel route to A. grandis resistance.