Lateral Dispersal and Foraging Behavior of Entomopathogenic Nematodes in the Absence and Presence of Mobile and Non-Mobile Hosts

Entomopathogenic nematodes have been classified into cruisers (active searchers) and ambushers (sit and wait foragers). However, little is known about their dispersal and foraging behavior at population level in soil. We studied lateral dispersal of the ambush foraging Steinernema carpocapsae (ALL strain) and cruise foraging Heterorhabditis bacteriophora (GPS11 strain) from infected host cadavers in microcosms (0.05 m²) containing Wooster silt-loam soil (Oxyaquic fragiudalf) and vegetation in the presence or absence of non-mobile and mobile hosts. Results showed that the presence of a non-mobile host (Galleria mellonella larva in a wire mesh cage) enhanced H. bacteriophora dispersal for up to 24 hr compared with no-host treatment, but had no impact on S. carpocapsae dispersal. In contrast, presence of a mobile host (G. mellonella larvae) increased dispersal of S. carpocapsae compared with no host treatment, but had no effect on H. bacteriophora dispersal. Also H. bacteriophora was better at infecting non-mobile than mobile hosts released into the microcosms and S. carpocapsae was better at infecting mobile than non-mobile hosts, thus affirming the established cruiser-ambusher theory. However, results also revealed that a large proportion of infective juveniles (IJs) of both species stayed near (≤ 3.8 cm) the source cadaver (88-96% S. carpocapsae; 67–79% H. bacteriophora), and the proportion of IJs reaching the farthest distance (11.4 cm) was significantly higher for S. carpocapsae (1.4%) than H. bacteriophora (0.4%) in the presence of mobile hosts. S. carpocapsae also had higher average population displacement than H. bacteriophora in the presence of both the non-mobile (5.07 vs. 3.6 cm/day) and mobile (8.06 vs. 5.3 cm/day) hosts. We conclude that the two species differ in their dispersal and foraging behavior at the population level and this behavior is affected by both the presence and absence of hosts and by their mobility.     

High efficiency protocol of DNA extraction from Micromys minutus mandibles from owl pellets: a tool for molecular research of cryptic mammal species

Owl pellets have high potential as a source of DNA. However, this noninvasive method of collecting DNA is rarely used, and its methodological aspects are poorly understood. We investigated the methodology for DNA extraction and amplification from owl pellets containing the smallest European rodent—the Harvest mouse Micromys minutus—as an example. We used mandibles identified in owl pellets for mitochondrial and nuclear DNA amplification. For DNA extraction, we tested two commercial protocols and utilized a protocol being a combination of two commercial kits which ensured high efficiency of DNA extraction. Additionally, we recorded that the amount of DNA was five times higher in extracts from teeth as compared to DNA extracts from jawbones derived from the same mandible. The quantity of DNA was significantly positively correlated with biological sample weight; however, the age of the pellet remains had an impact on the level of inhibition. We recorded inhibition in 40 % of mtDNA extracts derived from pellets older than 150 months, whereas in DNA extracts from pellets younger than 80 months, we did not observe a negative impact of inhibition on PCR efficiency. The amplification success rate was 89.9 % for the mitochondrial fragment and 39.4 % in the case of the nuclear fragment. We observed partial degradation of DNA evidenced by the fact that the longest fragments that we were able to amplify in the case of mtDNA were 450 and 200 bp for nuDNA. The study shows that pellets can be considered as a source of DNA and have high potential for molecular research in the case of threatened species and species that are difficult to study using standard field techniques.     

The norepinephrine transporter (NET) radioligand (S,S)-[18F]FMeNER-D2 shows significant decreases in NET density in the human brain in Alzheimer's disease: A post-mortem autoradiographic study

Earlier post-mortem histological and autoradiographic studies have indicated a reduction of cell numbers in the locus coeruleus (LC) and a corresponding decrease in norepinephrine transporter (NET) in brains obtained from Alzheimer's disease (AD) patients as compared to age-matched healthy controls. In order to test the hypothesis that the regional decrease of NET is a disease specific biomarker in AD and as such, it can be used in PET imaging studies for diagnostic considerations, regional differences in the density of NET in various anatomical structures were measured in whole hemisphere human brain slices obtained from AD patients and age-matched control subjects in a series of autoradiographic experiments using the novel selective PET radioligand for NET (S,S)-[¹⁸F]FMeNER-D₂. (S,S)-[¹⁸F]FMeNER-D₂ appears to be a useful imaging biomarker for quantifying the density of NET in various brain structures, including the LC and the thalamus wherein the highest densities are found in physiological conditions. In AD significant decreases of NET densities can be demonstrated with the radioligand in both structures as compared to age-matched controls. The decreases in AD correlate with the progress of the disease as indicated by Braak grades. As the size of the LC is below the spatial resolution of the PET scanners, but the size of the thalamus can be detected with appropriate spatial accuracy in advanced scanners, the present findings confirm our earlier observations with PET that the in vivo imaging of NET with (S,S)-[¹⁸F]FMeNER-D₂ in the thalamus is viable. Nevertheless, further studies are warranted to assess the usefulness of such an imaging approach for the early detection of changes in thalamic NET densities as a disease-specific biomarker and the possible use of (S,S)-[¹⁸F]FMeNER-D₂ as a molecular imaging biomarker in AD.     

Antimicrobial susceptibilities of Coagulase-Negative Staphylococci (CNS) and Streptococci from bovine subclinical mastitis cases

The prevalence and antimicrobial susceptibilities of Staphylococci and Streptococci were assessed from subclinical mastitis cases. One hundred Coagulase-Negative Staphylococci (CNS) and 34 Streptoccocci were identified. The most frequently isolated species were Staphylococcus haemolyticus (27%) and Staphylococcus simulans (24%). Susceptible CNS species revealed the highest resistance to penicillin G (58%), ampicillin (48%), neomycin (20%), and oleandomycin (14%). CNS methicillin resistance rates within 82 isolates were 21.95% and 1.22% by disk diffusion and PCR methods, respectively. These results suggested the disk diffusion method was more prone to yield false positives. Partial sequencing of the 16S rRNA region from the mecA carrying isolate (S. haemolyticus) was homologous with S. haemolyticus sequences/accessions obtained from GenBank. However, the mecA gene sequence from this isolate was more closely allied with the S. aureus mecA gene of human origins. Identical sequence data was acquired from the National Center for Biotechnology Information (NCBI) database, suggesting horizontal gene transfer between the two species. CNS β-lactamase activity within 81 isolates was 29.63%. The most frequently isolated Streptococcus species were S. uberis (52%) and S. agalactiae (15%). Oleandomycin was the least effective antimicrobial agent on these isolates with 59% susceptibility. Results indicated that CNS and Streptococci exhibited various antimicrobial resistance responses. Consequently, isolation and identification of udder pathogens in herds suffering from subclinical agents is essential to select the most effective antimicrobial agent. Moreover, multiple resistance features of methicillin resistant (MR) isolates should be considered during antimicrobial susceptibility tests.     

The Occurrence of Multidrug-Resistant Pseudomonas aeruginosa on Hydrocarbon-Contaminated Sites

The main aim of this paper was the comprehensive estimation of the occurrence rate and the antibiotic-resistance conditions of opportunistic pathogen Pseudomonas aeruginosa in hydrocarbon-contaminated environments. From 2002 to 2007, 26 hydrocarbon-contaminated sites of Hungary were screened for the detection of environmental isolates. Altogether, 156 samples were collected and examined for the determination of appearance, representative cell counts, and antibiotic-resistance features of P. aeruginosa. The detected levels of minimal inhibitory concentrations of ten different drugs against 36 environmental strains were compared to the results of a widely used reference strain ATCC 27853 and four other clinical isolates of P. aeruginosa. Based on our long-term experiment, it can be established that species P. aeruginosa was detectable in case of 61.5% of the investigated hydrocarbon-contaminated sites and 35.2% of the examined samples that shows its widespread occurrence in polluted soil–groundwater systems. In the course of the antibiotic-resistance assay, our results determined that 11 of the examined 36 environmental strains had multiple drug-resistance against several clinically effective antimicrobial classes: cephalosporins, wide spectrum penicillins, carbapenems, fluoroquinolones, and aminoglycosides. The fact that these multiresistant strains were isolated from 8 different hydrocarbon-contaminated sites, mainly from outskirts, confirms that multiple drug-resistance of P. aeruginosa is widespread not only in clinical, but also in natural surroundings as well.     

Endopeptidase Activities of Botulinum Neurotoxin Type B Complex,Holotoxin, and Light Chain

Botulinum neurotoxin (BoNT) serotype B (BoNT/B) is one of the serotypes of BoNT that causes deadly human botulism, though it is used clinically for treatment of many neuromuscular diseases. BoNT/B is produced by Clostridium botulinum, and it is secreted along with a group of neurotoxin-associated proteins (NAPs) in the form of a BoNT/B complex. The complex dissociates into a 150-kDa holotoxin and NAPs at alkaline pHs. The 150-kDa BoNT/B holotoxin can be nicked to produce a 50-kDa domain referred to as the light chain (LC) and a 100-kDa heavy chain, with the former possessing a unique endopeptidase activity. The two chains remain linked through a disulfide bond that can be reduced to separate the two chains. The endopeptidase activity is present in all three forms of the toxin (complex, purified BoNT/B holotoxin, and separated light chain), which are used by different researchers to develop detection methods and screen for inhibitors. In this research, the endopeptidase activities of the three forms, for the first time, were compared under the same conditions. The results show that enzyme activities of the three forms differ significantly and are largely dependent on nicking and disulfide reduction conditions. Under the conditions used, LC had the highest level of activity, and the complex had the lowest. The activity was enhanced by nicking of BoNT/B holotoxin and was enhanced even more by dithiothreitol (DTT) reduction after nicking. This information is useful for understanding the properties of BoNT endopeptidases and for comparing the efficacies of different inhibitors when they are tested with different forms of BoNT endopeptidase.Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most toxic substances known to humans and block the release of neurotransmitters, resulting in flaccid muscle paralysis. There are seven serotypes of BoNT, designated A to G, which are serologically distinct. An antitoxin against one serotype does not work on other serotypes. Different BoNT serotypes differ in their amino acid sequences, their substrates, or cleavage sites on the same substrate. Of the seven serotypes, BoNT type A (BoNT/A), BoNT/B, BoNT/E, and BoNT/F are known to cause human botulism (9). The extreme lethality of BoNTs makes them potent bioterror agents. BoNT/A and BoNT/B are two serotypes which have been approved by the Food and Drug Administration (FDA) for cosmetic purposes and for treatment of a wide range of neuromuscular diseases, including cervical dystonia (3).Like other BoNT serotypes, BoNT/B is secreted by the bacteria as a complex of the holotoxin and several nontoxic proteins called neurotoxin-associated proteins (NAPs). The NAPs protect the holotoxin from harsh environmental conditions, such as the high temperature, low pH, and multiple proteases present in the gastrointestinal tract (14, 17). The holotoxin, of about 150 kDa, can be obtained by removing the non-covalently bound accessory proteins with ion-exchange chromatography. The 150-kDa polypeptide chain consists of a 100-kDa heavy chain (HC) and a 50-kDa light chain (LC), which are synthesized as a single polypeptide chain but nicked by endogenous or exogenous proteases and remain linked through a disulfide bond (Fig. ​(Fig.1).1). The HC binds the receptors on neuronal cells and helps translocate the LC into the cell. The BoNT/B LC cleaves the vesicle-associated membrane protein (VAMP), also called synaptobrevin. VAMP is necessary for the docking and fusion of synaptic vesicles to plasma membrane at the neuromuscular junctions for neurotransmitter release. Once the VAMP is cleaved, the neurotransmitters in synaptic vesicles cannot be released, resulting in flaccid paralysis that can be fatal.Open in a separate windowFIG. 1.Schematic diagram of BoNT/B pure toxin. Dark gray, light chain; light gray, heavy chain; hatch-marked box, the active site of the toxin. The 50-kDa light chain and 100-kDa heavy chain are linked through a disulfide bridge as well as a covalent bond. The latter is partially nicked by bacterial proteases before the toxin is secreted.Strains producing BoNT/B can be nonproteolytic or proteolytic (4). BoNT/B from nonproteolytic strains occurs as a single polypeptide chain of 150 kDa. BoNT/B secreted by proteolytic strains is a mixture of the single polypeptide chain and a dichain in which the peptide bond linking the HC and LC has been nicked by proteases produced by the bacteria (Fig. ​(Fig.1).1). The single polypeptide chain in both nonproteolytic and proteolytic cultures can be converted to the dichain form through in vitro trypsinization. The HC and LC in the dichain can be further separated by breaking the disulfide bond with a reducing agent such as dithiothreitol (DTT) and treating it with chaotropic reagents such as urea (10).The complex, holotoxin, and LC are three different forms of BoNT/B with endopeptidase activity, although LC is the only active unit in all three forms. The complex is the native form of the toxin, which causes botulism. It is also the main component of the only licensed drug with BoNT/B currently available (2). The complex, holotoxin, and LC of BoNT/B have all been extensively used to develop methods to detect this serotype or to screen for inhibitors against the toxin (1, 5, 7, 8, 13, 15, 16). Since different forms of the toxin were used by different researchers, it is difficult to compare the sensitivities of different detection methods or the efficacies of different inhibitors. Therefore, in this study, the activities of BoNT/B complex, holotoxin, and LC were compared under the same conditions for the first time. The results suggest that the endopeptidase activity with a peptide substrate varies substantially depending on whether BoNT/B is used in its native complex form, its isolated holotoxin form, or a separated LC form. The LC form was the most active form of the endopeptidase under the conditions used.     

Transcriptome analysis of germinating maize kernels exposed to smoke-water and the active compound KAR<Subscript>1</Subscript>

Background  

Smoke released from burning vegetation functions as an important environmental signal promoting the germination of many plant species following a fire. It not only promotes the germination of species from fire-prone habitats, but several species from non-fire-prone areas also respond, including some crops. The germination stimulatory activity can largely be attributed to the presence of a highly active butenolide compound, 3-methyl-2H-furo[2,3-c]pyran-2-one (referred to as karrikin 1 or KAR₁), that has previously been isolated from plant-derived smoke. Several hypotheses have arisen regarding the molecular background of smoke and KAR₁ action.     

MptpB, a virulence factor from Mycobacterium tuberculosis, exhibits triple-specificity phosphatase activity

Bacterial pathogens have developed sophisticated mechanisms of evading the immune system to survive in infected host cells. Central to the pathogenesis of Mycobacterium tuberculosis is the arrest of phagosome maturation, partly through interference with PtdIns signalling. The protein phosphatase MptpB is an essential secreted virulence factor in M. tuberculosis. A combination of bioinformatics analysis, enzyme kinetics and substrate-specificity characterization revealed that MptpB exhibits both dual-specificity protein phosphatase activity and, importantly, phosphoinositide phosphatase activity. Mutagenesis of conserved residues in the active site signature indicates a cysteine-based mechanism of dephosphorylation and identifies two new catalytic residues, Asp165, essential in catalysis, and Lys164, apparently involved in substrate specificity. Sequence similarities with mammalian lipid phosphatases and a preference for phosphoinositide substrates suggests a potential novel role of MptpB in PtdIns metabolism in the host and reveals new perspectives for the role of this phosphatase in mycobacteria pathogenicity.