Fluorescence decay of dyed protozoa: differences between stressed and non‐stressed cysts

Several series of tests have shown that fresh, intact samples of Giardia duodenalis and Cryptosporidium parvum (oo)cysts are not marked by fluorescent probes such as carboxyfluorcein‐succinimidyl‐diacetate‐ester (CFDA‐SE), C12‐resazurin and SYTOX® Green, probably because of their robust cell walls. These dyes fail to indicate the viability of such protozoa and allow negative responses to be recorded from living and infectious samples. Cryptosporidium parvum showed stronger isolation from chemicals, with living oocysts remaining unstained by the probe for up to 90 days after extraction. However, in further fluorescence decay (FD) experiments run with G. duodenalis samples stained using CFDA‐SE (comprising living, non‐stressed but aged cysts, heat‐killed samples and UV‐C‐stressed samples) each showed a different FD decay profile, here studied in seven series of tests of five replicates each. The FD profiles were fitted by double‐exponential decay kinetics, with the decay constant k₂ being five times higher than k₁. This FD procedure is fast and can be easily reproduced in 10 steps, taking ~ 1 h of laboratory work for already purified samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Peptaibol,Secondary‐Metabolite,and Hydrophobin Pattern of Commercial Biocontrol Agents Formulated with Species of the Trichoderma harzianum Complex

The production of bioactive polypeptides (peptaibiotics) in vivo is a sophisticated adaptation strategy of both mycoparasitic and saprotrophic Trichoderma species for colonizing and defending their natural habitats. This feature is of major practical importance, as the detection of peptaibiotics in plant‐protective Trichoderma species, which are successfully used against economically relevant bacterial and fungal plant pathogens, certainly contributes to a better understanding of these complex antagonistic interactions. We analyzed five commercial biocontrol agents (BCAs), namely Canna^®, Trichosan^®, Vitalin^®, Promot^® WP, and TrichoMax^®, formulated with recently described species of the Trichoderma harzianum complex, viz. T. afroharzianum, T. simmonsii, and T. guizhouense. By using the well‐established, HPLC/MS‐based peptaibiomics approach, it could unequivocally be demonstrated that all of these formulations contained new and recurrent peptaibols, i.e., peptaibiotics carrying an acetylated N‐terminus, the C‐terminus of which is reduced to a 1,2‐amino alcohol. Their chain lengths, including the amino alcohol, were 11, 14, and 18 residues, respectively. Peptaibols were also to be the dominating secondary metabolites in plate cultures of the four strains obtained from four of the Trichoderma‐ based BCAs, contributing 95% of the UHPLC‐UV/VIS peak areas and 99% of the total ion count MS peak area from solid media. Furthermore, species‐specific hydrophobins, as well as non‐peptaibiotic secondary metabolites, were detected, the latter being known for their antifungal, siderophore, or plant‐growth‐promoting activities. Notably, none of the isolates produced low‐molecular weight mycotoxins.     

Action of sisal (Agave sisalana, Perrine) extract in the in vitro development of sheep and goat gastrointestinal nematodes

Active compounds from Agave sisalana with antiparasitic action against gastrointestinal nematodes (GINs) could be an alternative to diversify the range of parasite management methods in the livestock sector. The objective of this study was to evaluate the in vitro action of A. sisalana extract on the development of sheep and goat GINs. The extract, obtained from shredded sisal leaves, was utilized at various concentrations in the egg hatch test (EHT), larval development test (LDT), larval feeding inhibition test (LFIT) and adult motility test (AMT). The LC(50) and LC(95) in the EHT were 6.90 and 24.79 mg/mL, in the LDT were 0.041 and 0.067 mg/mL and in the LFIT were 0.053 and 0.24 mg/mL, respectively, showing a dose-dependent relationship. The development and feeding inhibition on L(1) were both 100% at a dose of 0.12 mg/mL. In the AMT there was 100% inhibition at 75 mg/mL after 24h of exposure. The extract of A. sisalana therefore demonstrated significant action on L(1) at 0.12 mg/mL. So, if part of the A. sisalana extract passes through the animal's gastrointestinal system, this material can have a significant effect on the parasites in the feces. This is an interesting approach because it can drastically reduce the pasture contamination as well as the infection of herds.     

CHIP protects from the neurotoxicity of expanded and wild-type ataxin-1 and promotes their ubiquitination and degradation

CHIP (C terminus of Hsc-70 interacting protein) is an E3 ligase that links the protein folding machinery with the ubiquitin-proteasome system and has been implicated in disorders characterized by protein misfolding and aggregation. Here we investigate the role of CHIP in protecting from ataxin-1-induced neurodegeneration. Ataxin-1 is a polyglutamine protein whose expansion causes spinocerebellar ataxia type-1 (SCA1) and triggers the formation of nuclear inclusions (NIs). We find that CHIP and ataxin-1 proteins directly interact and co-localize in NIs both in cell culture and SCA1 postmortem neurons. CHIP promotes ubiquitination of expanded ataxin-1 both in vitro and in cell culture. The Hsp70 chaperone increases CHIP-mediated ubiquitination of ataxin-1 in vitro, and the tetratricopeptide repeat domain, which mediates CHIP interactions with chaperones, is required for ataxin-1 ubitiquination in cell culture. Interestingly, CHIP also interacts with and ubiquitinates unexpanded ataxin-1. Overexpression of CHIP in a Drosophila model of SCA1 decreases the protein steady-state levels of both expanded and unexpanded ataxin-1 and suppresses their toxicity. Finally we investigate the ability of CHIP to protect against toxicity caused by expanded polyglutamine tracts in different protein contexts. We find that CHIP is not effective in suppressing the toxicity caused by a bare 127Q tract with only a short hemagglutinin tag, but it is very efficient in suppressing toxicity caused by a 128Q tract in the context of an N-terminal huntingtin backbone. These data underscore the importance of the protein framework for modulating the effects of polyglutamine-induced neurodegeneration.     

A common mode of attraction of larvae and adults of insect predators to the sex pheromone of their prey (Hemiptera: Matsucoccidae)

The attraction of several adult predators, genera Elatophilus, Hemerobius and Sympherobius, to the sex pheromones of pine bast scales, Matsucoccus Cockerell, has already been demonstrated. Here, the hypothesis that the larvae of these predators are similarly attracted to the host prey sex pheromone is tested. The response of predators was tested in field trials using pine tree arenas baited with the sex pheromones of M. josephi Bodenheimer & Harpaz, M. feytaudi Ducasse and M. matsumurae Kuwana. Experiments were conducted in Israel in stands of Pinus halepensis infested by M. josephi and in Portugal in stands of P. pinaster infested by M. feytaudi, respectively. The selectivity of larvae for the three sex pheromones was tested in Petri dish arenas in the laboratory. In the field, the larval stages exhibited similar modes of attraction to those of the conspecific adults: Elatophilus hebraicus Pericart in Aleppo pine forest, E. crassicornis Reuter and Hemerobius stigma Stephens in the maritime pine forests. Laboratory choice tests confirmed the kairomonal selectivity of larvae. Both forest and laboratory tests demonstrated the response of a coccinellid of the genus Rhyzobius to the sex pheromones of M. feytaudi and M. matsumurae. A unique chemical communication system among several taxa of predators of Matsucoccus spp. was highlighted that may be attributed to their coevolution on a geological time scale.     

Shape and size of red blood cells from the Pygoscelid penguins of Antarctica using atomic force microscopy

Antarctic biodiversity is evolutionarily complex, reflecting the extreme ambient conditions. Therefore, Antarctic organisms exhibit sophisticated adaptations in all organization levels, including organs, tissues, and cells. Since red blood cells (RBCs) travel through the vertebrates blood delivering O₂ to all tissues and organs and purging the unwanted CO₂, they represent an interesting model to investigate biological adaptations. We have used atomic force microscopy (AFM) to compare the shape and size of RBCs of the Pygoscelid penguins. A total of 18 landmarks were measured in AFM images. When analyzed individually, the parameters were not capable of discriminating the RBCs of each species. However, the simultaneous use of multiple parameters discriminated (74%) among the RBCs. In addition, the use of RBC measurements was sufficient to hierarchically cluster the species in accordance to other common and reliable phylogenetic strategies. In light of these results, the use of RBC characters could effectively benefit taxonomic inferences.     

Resveratrol and ascorbic acid prevent DNA damage induced by cryopreservation in human semen

Cryopreservation of human semen can cause DNA damages, which compromise the fertilization and normal embryo development. The present study showed that the antioxidant resveratrol prevents these damages both in fertile and infertile men. The addition of ascorbic acid before cryopreservation can reduce DNA damages only in infertile men. Although further studies are needed, the present work showed that resveratrol could be considered in human cryopreservation procedures to avoid/minimize DNA damages and preserve sperm integrity.