Structure Elucidation and Functional Studies of a Novel β-hairpin Antimicrobial Peptide from the Marine Polychaeta Capitella teleta

Endogenous antimicrobial peptides (AMPs) are evolutionary ancient molecular factors of innate immunity that play a key role in host defense. Among the most active and stable under physiological conditions AMPs are the peptides of animal origin that adopt a β-hairpin conformation stabilized by disulfide bridges. In this study, a novel BRICHOS-domain related AMP from the marine polychaeta Capitella teleta, named capitellacin, was produced as the recombinant analogue and investigated. The mature capitellacin exhibits high homology with the known β-hairpin AMP family—tachyplesins and polyphemusins from the horseshoe crabs. The β-hairpin structure of the recombinant capitellacin was proved by CD and NMR spectroscopy. In aqueous solution the peptide exists as monomeric right-handed twisted β-hairpin and its structure does not reveal significant amphipathicity. Moreover, the peptide retains this conformation in membrane environment and incorporates into lipid bilayer. Capitellacin exhibits a strong antimicrobial activity in vitro against a wide panel of bacteria including extensively drug-resistant strains. In contrast to other known β-hairpin AMPs, this peptide acts apparently via non-lytic mechanism at concentrations inhibiting bacterial growth. The molecular mechanism of the peptide antimicrobial action does not seem to be related to the inhibition of bacterial translation therefore other molecular targets may be assumed. The reduced cytotoxicity against human cells and high antibacterial cell selectivity as compared to tachyplesin-1 make it an attractive candidate compound for an anti-infective drug design.     

Mechanism of Action and Therapeutic Potential of the β-Hairpin Antimicrobial Peptide Capitellacin from the Marine Polychaeta Capitella teleta

Among the most potent and proteolytically resistant antimicrobial peptides (AMPs) of animal origin are molecules forming a β-hairpin structure stabilized by disulfide bonds. In this study, we investigated the mechanism of action and therapeutic potential of the β-hairpin AMP from the marine polychaeta Capitella teleta, named capitellacin. The peptide exhibits a low cytotoxicity toward mammalian cells and a pronounced activity against a wide range of bacterial pathogens including multi-resistant bacteria, but the mechanism of its antibacterial action is still obscure. In view of this, we obtained analogs of capitellacin and tachyplesin-inspired chimeric variants to identify amino acid residues important for biological activities. A low hydrophobicity of the β-turn region in capitellacin determines its modest membranotropic activity and slow membrane permeabilization. Electrochemical measurements in planar lipid bilayers mimicking the E. coli membrane were consistent with the detergent-like mechanism of action rather than with binding to a specific molecular target in the cell. The peptide did not induce bacterial resistance after a 21-day selection experiment, which also pointed at a membranotropic mechanism of action. We also found that capitellacin can both prevent E. coli biofilm formation and destroy preformed mature biofilms. The marked antibacterial and antibiofilm activity of capitellacin along with its moderate adverse effects on mammalian cells make this peptide a promising scaffold for the development of drugs for the treatment of chronic E. coli infections, in particular those caused by the formation of biofilms.     

Ultrasound-guided injections of the equine head and neck: review and expert opinion

Ultrasound-guided injections can be used for a wide variety of conditions in the horse, including both diagnostic and therapeutic applications. Benefits of ultrasound guidance include more accurate deposition of injectate compared with blind approaches. Improved identification of vital structures, including nerves and blood vessels, allows their avoidance and thus reduces procedure-associated complications. Validation of such ultrasound-guided techniques has shown that they can be easily learnt by inexperienced veterinarians, assuming a proper knowledge of the sonographic anatomy. In many cases they can be employed in the field with a high level of accuracy, using widely available equipment, and with complete adherence to the sterility principles. Many ultrasound-guided injection techniques of the axial skeleton in the horse have been described in past years, enabling the equine veterinarian to perform more accurate treatments of specific anatomical areas. The goal of this review is to discuss diagnostic and therapeutic ultrasound-guided injection techniques of the skull and cervical spine in the horse, including those for the retrobulbar space, maxillary and inferior alveolar nerves, atlanto-occipital and atlanto-axial junctions, and cervical articular process joints, as well as the 1st cervical nerve, the C2 and C3 nerve plexus, and the 6th, 7th, and 8th cervical nerve roots.     

Myeloperoxidase concentration in bronchoalveolar lavage fluid from healthy horses and those with recurrent airway obstruction

The aim of this work was to measure the myeloperoxidase (MPO) concentration in bronchoalveolar lavage (BAL) fluid collected from horses with recurrent airway obstruction (RAO), both in crisis and in remission, as well as from healthy horses. Seven horses with RAO were exposed to moldy hay until the maximum change in pleural pressure was greater than 1.5 kPa. At that point, BAL was performed, and the total cell counts and percentages in the fluid were immediately determined. To measure the MPO concentration in BAL-fluid supernatant, we used a specific enzyme-linked immunosorbent assay with polyclonal antibodies against equine MPO. The tests were repeated on the horses with RAO after they had spent 2 mo on pasture. Six healthy horses serving as controls underwent the same tests. The absolute and relative neutrophil counts and the MPO concentration in the BAL fluid were significantly greater in the horses with an RAO crisis than in the control horses. After 2 mo on pasture, the horses that had been in RAO crisis were clinically normal, and their neutrophil counts and MPO levels in BAL fluid had significantly decreased; during remission their neutrophil counts were not significantly different from those in the healthy horses, but their MPO concentration remained significantly higher. This study showed that determining the MPO concentration in a horse's BAL fluid is technically possible and that during remission from RAO the concentration remains higher than normal. Thus, MPO may be a marker of neutrophil presence and activation in the lower airways.     

Direct determination of paclobutrazol residues in pear samples by liquid chromatography-electrospray tandem mass spectrometry

A rapid and sensitive liquid chromatography/electrospray ionization/tandem mass spectrometry (LC-ESI-MS-MS) method has been developed for the determination of the plant growth regulator paclobutrazol in pear samples. Extraction was performed with methanol by using a high-speed blender Ultra-Turrax, and 10 microL of pear extract was directly injected in the LC-ESI-MS-MS system without any previous sample treatment. The highest sensitivity of the method was achieved under MS-MS conditions obtaining a limit of detection of 0.7 microg/kg and a quantification limit of 5 microg/kg, with a run time of only 5.5 min. Recoveries for paclobutrazol from spiked pear samples at 0.005, 0.05, and 0.5 mg/kg were around 82-102% with relative standard deviations between 2 and 7%. The method was applied to real treated and untreated samples of pears, using quality control samples as a evaluation of the method reliability. Two MS-MS transitions were selected, one for quantification (294 > 70) and the other for confirmation of the analyte (296 > 70). All the experiments were performed in compliance with good laboratory practices.     

Low genetic diversity threatens imminent extinction for the Hungarian meadow viper (Vipera ursinii rakosiensis)

Meadow vipers (Vipera ursinii) are small venomous snakes whose range in Hungary has been greatly fragmented by anthropogenic habitat disturbance (especially, agriculture). We obtained DNA from a total of eight Hungarian snakes. Genetic variability at the major histocompatibility (Mhc) class I loci was much lower for these snakes than for specimens from two large Ukrainian populations. Within two Hungarian populations for which we had multiple individuals, band-sharing indices were 100 and 84.6% (versus 63.3 and 57% for the Ukraine populations). The Ukrainian snakes also displayed more RFLP fragments than the Hungarian vipers (mean 13.7 versus 9.0, respectively). In combination with reports of birth deformities, chromosomal abnormalities and low juvenile survival, these data strongly suggest that the Hungarian vipers are experiencing inbreeding depression. Genetic diversity is still present in the Hungarian vipers but among rather than within populations. Given the very low numbers of animals, the only feasible strategy to increase the genetic diversity and to save the Hungarian vipers from extinction is to implement a captive breeding program based on genetically screened animals.     

Protected areas and frugivorous fish in tropical rivers: Small-scale fisheries,conservation and ecosystem services

1. Frugivorous fish provide often conflicting ecosystem services of seed dispersal and food provisioning in tropical rivers. Fishing may reduce the size and abundance of frugivorous fish, thus affecting their potential as seed dispersers, which could affect the conservation of these fish and of floodplain forests.
2. The goal of this study was to assess the influence of co-managed protected areas in the form of extractive reserves (RESEX) and small-scale fisheries on frugivorous fish in the Tapajós and Negro rivers, in the Brazilian Amazon. The study examined whether: (i) frugivorous fish are important for fisheries and selectively caught; and (ii) frugivorous fish abundance, size and fisheries catch per unit of effort are higher inside the RESEX than outside.
3. The analyses included fisheries-dependent data (3,753 fish landings) and independent data (12,730 sampled fish) collected in 16 fishing communities (eight for each river). In both rivers, frugivorous fish are among the 10 species caught the most and frugivorous biomass was proportionately higher in landings than in samplings, indicating fisheries selectivity towards these fishes.
4. In both rivers, catches of frugivorous fish were higher outside the RESEX than inside. Catch per unit effort and the proportion of frugivores in the catch were higher outside the RESEX in the Tapajós River but did not vary between sites inside and outside the RESEX in the Negro River. Frugivorous fish were larger inside the RESEX in the Negro, but smaller inside the RESEX in the Tapajós.
5. The results indicated that the ecosystem services of seed dispersal and food source provided by frugivorous fish are not in conflict in the tropical rivers studied. Therefore, these clearwater and blackwater rivers in the Brazilian Amazon show a balance between fisheries and conservation of frugivorous fish, which play an essential role in the functioning of tropical floodplain forest ecosystems.

     

Self-inhibition of spore germination via reactive oxygen in the fungus Cladosporium cucumerinum, causal agent of cucurbit scab

Cladosporium cucumerinum spore germination in vitro depended on spore suspension density. Different fungal isolates displayed germination maxima at different spore concentrations. For one isolate, the maximum was observed at the same spore density at both 18 and 25°C, although germination percentage increased slightly at the higher temperature. Diffusates originating in other spore suspensions of the same isolate reduced germination percentage of spores taken at optimal concentration. The least effect occurred in diffusate taken from spores kept at their optimal concentration. Self-suppression of spore germination at unfavourable concentrations was diminished more or less by antioxidants (superoxide dismutase, catalase, mannitol or formate). The same compounds, added to spore diffusates, reduced their fungitoxicities. All diffusates generated superoxide radical (assayed by adrenalin oxidation sensitive to superoxide dismutase). This activity correlated positively with diffusate toxicity. Leaf inoculation of the susceptible cucumber cultivar at 18°C with spore suspensions at extreme densities, at which they germinated poorly in vitro, led to less disease severity then that at optimal density. In contrast, no disease symptoms appeared at 25°C. It is suggested that spores germinating at their extreme concentrations produced reactive oxygen species, suppressing the pathogen; this effect could reduce disease development at low temperatures. At high temperatures, however, this mechanism seems not to work, suggesting that plant infection may be reduced by other disease inhibiting factors.     

In Vitro Elution and Antibacterial Activity of Clindamycin,Amikacin, and Vancomycin from R‐gel Polymer

Objective: To characterize the in vitro elution and bioactivity of 2 formulations of antibiotics in a novel, dissolvable, cross‐linked dextran polymer matrix: Formulation 1—amikacin and clindamycin (AC); Formulation 2—amikacin, clindamycin, and vancomycin (ACV). Study Design: Prospective, in vitro, experimental study. Methods: Aliquots of the antibiotic impregnated polymer were incubated in PBS buffer for 10 days. PBS was changed every 24 hours and concentrations of the antibiotics eluted into saline were quantified. Antimicrobial activity of the eluent from each sampling period was tested for growth inhibition of Staphylococcus aureus. Results: Both formulations of R‐gel^? had a rapid initial release of antibiotics within the first 24 hours and then the concentrations decreased gradually over 10 days. The concentration of amikacin, clindamycin, and vancomycin remained above the breakpoint minimum inhibitory concentration of each drug for a minimum of 9 days. No significant difference (P=.9938, P=.9843) was present in the elution pattern or total amount of antibiotic eluted from clindamycin or amikacin, respectively. Eluent from both groups demonstrated bioactivity against S. aureus for the entire 10‐day study period. Conclusions: Amikacin and clindamycin together, or in combination with vancomycin, elute from R‐gel^? effectively and at gradually decreasing concentrations for at least 10 days. The antibiotics maintained their bioactivity following polymerization and elution from the R‐gel^?.