Development of solution phase hybridisation PCR-ELISA for the detection and quantification of Enterococcus faecalis and Pediococcus pentosaceus in Nurmi-type cultures

Nurmi-type cultures (NTCs), derived from the fermentation of caecal contents of specifically pathogen-free (SPF) birds, have been used successfully to control salmonella colonisation in chicks. These cultures are undefined in nature and, consequently, it is difficult to obtain approval from regulatory agencies for their use as direct fed microbials (DFMs) for poultry. Progress towards the generation of effective defined probiotics requires further knowledge of the composition of these cultures. As such, species-specific, culture-independent quantification methodologies need to be developed to elucidate the concentration of specific bacterial constituents of NTCs. Quantification of specific bacterial species in such ill-defined complex cultures using conventional culturing methods is inaccurate due to low levels of sensitivity and reproducibility, in addition to slow turnaround times. Furthermore, these methods lack selectivity due to the nature of the accompanying microflora. This study describes the development of a rapid, sensitive, reliable, reproducible, and species-specific culture-independent, solution phase hybridisation PCR-ELISA procedure for the detection and quantification of Enterococcus faecalis and Pediococcus pentosaceus in NTCs. In this technique, biotin-labelled primers were designed to amplify a species-specific fragment of a marker gene of known copy number, in both species. Resulting amplicons were hybridised with a dinitrophenol (DNP)-labelled oligonucleotide probe in solution and were subsequently captured on a streptavidin-coated microtitre plate. The degree of binding was determined by the addition of IgG (anti-DNP)-horseradish peroxidase conjugate, which was subsequently visualised using a chromogenic substrate, tetramethylbenzidine. This novel quantitative method was capable of detecting E. faecalis and P. pentosaceus at levels as low as 5 CFU per PCR reaction.     

Caspase-mediated specific cleavage of BubR1 is a determinant of mitotic progression

The fidelity of chromosomal duplication is monitored by cell cycle checkpoints operational during mitosis. One such cell cycle delay is invoked by microtubule-targeting agents such as nocodazole or paclitaxel (Taxol) and is mediated by mitotic checkpoint proteins that include BubR1. Relatively little is known about the regulation of expression and stability of BubR1 (or other checkpoint proteins) and how these factors dictate the durability of the cell cycle delay. We report here that treatment of HeLa cells with spindle-disrupting agents resulted in caspase activation and precipitated the cleavage of BubR1. This mechanism ultimately leads to reduced levels of full-length protein, which are accompanied by abrogation of the mitotic block; the checkpoint abrogation is substantially accelerated by inhibition of de novo protein synthesis. In contrast, inhibition of caspase activity blocked BubR1 degradation and prolonged mitosis. To confirm a direct link between caspase activity and BubR1 protein expression, we identified by site-directed mutagenesis the specific caspase cleavage sites cleaved after exposure to paclitaxel. Surprisingly, BubR1 has two sites of cleavage: primarily at Asp607/Asp610 and secondarily at Asp576/Asp579. BubR1 mutated at both locations (BubR1Delta579Delta610) was resistant to paclitaxel-induced degradation. Expression of BubR1Delta579Delta610 augmented the mitotic delay induced by spindle disruption in transfected cells as well as in clones engineered to inducibly express the mutant protein upon exposure to doxycycline and ultimately led to increased aneuploidy. Underscoring the importance of these caspase cleavage sites, both tetrapeptide motifs are identified in the amino acid sequences of human, mouse, chicken, and Xenopus BubR1. These results are potentially the first to link the control of the stability of a key mitotic checkpoint protein to caspase activation, a regulatory pathway that may be involved in killing defective cells and that has been evolutionarily conserved.     

Stimulus change dis-habituates operant responding supported by water reinforcers

The present experiment examined whether habituation contributes to within-session decreases in operant responding for water reinforcers. The experiment asked if this responding can be dishabituated, a fundamental property of habituated behavior. During baseline, rats’ lever pressing was reinforced by water on a variable interval 15-s schedule. During experimental conditions, rats responded on the same schedule and a new stimulus was introduced for 5 min at 15, 30 or 45 min into the 60-min session. The new stimulus was extinction, continuous reinforcement or flashing lights in different conditions. Rate of responding primarily decreased within the session during baseline. Introducing a new stimulus sometimes suppressed (extinction, continuous reinforcement) and sometimes increased (flashing lights) responding while it was in effect. The new stimulus increased responding after it ended and before it was presented in the session. The results are incompatible with the idea that non-habituation satiety factors (e.g., cellular hydration and blood volume) contributed to within-session changes in responding. These satiety factors should increase with increases in consumption, decrease with decreases in consumption and remain constant with constant consumption of water. Nevertheless, all stimulus changes increased operant responding for water. These results support the idea that habituation contributes to within-session decreases in responding for water reinforcers.     

An AFLP-based survey of genetic diversity among accessions of sea oats (Uniola paniculata, Poaceae) from the southeastern Atlantic and Gulf coast states of the United States

Uniola paniculata, commonly known as sea oats, is a C₄ perennial grass capable of stabilizing sand dunes. It is most abundant along the Gulf of Mexico and southeastern Atlantic coastal regions of the United States. The species exhibits low seed set and low rates of germination and seedling emergence, and so extensive clonal reproduction is achieved through production of rhizomes, which may contribute to a decline in genetic diversity. To date, there has been no systematic assessment of genetic variability and population structure in naturally occurring stands in the USA. This study was conducted to assess the genetic relationship and diversity among nineteen U. paniculata accessions representing eight states: Texas, Louisiana, Mississippi, Alabama, Florida, South Carolina, North Carolina, and Virginia, using amplified fragment length polymorphism (AFLP). Twelve AFLP EcoRI+MseI primer combinations generated a wide range of polymorphisms (42–81%) with a mean of 59%. Overall, the sea oats plants exhibited a low range of genetic similarity. Florida accessions, FL-33 and FL-39, were most genetically diverse and the accessions from both Carolinas and Virginia (NC-1, NC-11, SC-15, and VA-53) harbored less genetic variability. Cluster analysis using the UPGMA approach separated U. paniculata plants into four major clusters which were also confirmed by principal coordinate analysis (PCO). Further examination of the different components of genetic variation by analysis of molecular variance (AMOVA) indicated the largest proportion of variability at the state level (47.8%) followed by the variation due to the differences among the genotypes within an accession (34.4%), and the differences among the accessions within a state (17.8%). The relationship between genetic diversity and geographic source of sea oats populations of the United States as revealed through this comprehensive study will be helpful to resource managers and commercial nurseries in identifying suitable plant materials for restoration of new areas without compromising the adaptation and genetic diversity.     

Prevalence of Salmonella spp. in oysters in the United States

Food-borne diseases such as salmonellosis can be attributed, in part, to the consumption of raw oysters. To determine the prevalence of Salmonella spp. in oysters, oysters harvested from 36 U.S. bays (12 each from the West, East, and Gulf coasts in the summer of 2002, and 12 bays, four per coast, in the winter of 2002-2003) were tested. Salmonella was isolated from oysters from each coast of the United States, and 7.4% of all oysters tested contained Salmonella. Isolation tended to be bay specific, with some bays having a high prevalence of Salmonella, while other bays had none. Differences in the percentage of oysters from which Salmonella was isolated were observed between the summer and winter months, with winter numbers much lower probably due to a variety of weather-related events. The vast majority (78/101) of Salmonella isolates from oysters were Salmonella enterica serovar Newport, a major human pathogen, confirming the human health hazard of raw oyster consumption. Contrary to previous findings, no relationship was found between the isolation of fecal coliforms and Salmonella from oysters, indicating a necessity for specific monitoring for Salmonella and other pathogens rather than the current reliance on fecal coliform testing.     

Protective effects of N-acetylserotonin against 6-hydroxydopamine-induced neurotoxicity

The present work studied in vivo neuroprotective effects of n-acetylserotonin (NAS), the immediate precursor of melatonin, on the dopaminergic system, in rats lesioned with the unilateral intrastriatal injection of the neurotoxin 6-hydroxydopamine (6-OHDA). Two weeks after the lesion, the dopamine receptor agonist, apomorphine, produced rotational asymmetry, and the NAS treatment significantly reduced the motor deficit following the apomorphine challenge. The apomorphine-induced rotational behavior was blocked by 84, 86 and 53% after NAS, at doses of 2, 5 and 10 mg/kg, i.p., respectively. The injection of 6-OHDA significantly decreased DA, DOPAC and HVA levels in the rat striatum. In contrast, the NAS (2, 5 and 10 mg/kg, i.p., daily for 7 days) treatment partially reversed the decreases caused by 6-OHDA, and the neurotransmitter levels were brought to approximately 50% of that observed in the contralateral sides. NAS was more efficient at the smaller doses. NAS (5 mg/kg) produced an up-regulation of D1 (37%) and D2 (37%) receptors associated with a decrease in Kd values.     

Infection of nonhuman primates with recombinant human metapneumovirus lacking the SH, G, or M2-2 protein categorizes each as a nonessential accessory protein and identifies vaccine candidates

Recombinant human metapneumovirus (HMPV) in which the SH, G, or M2 gene or open reading frame was deleted by reverse genetics was evaluated for replication and vaccine efficacy following topical administration to the respiratory tract of African green monkeys, a permissive primate host. Replication of the deltaSH virus was only marginally less efficient than that of wild-type HMPV, whereas the deltaG and deltaM2-2 viruses were reduced sixfold and 160-fold in the upper respiratory tract and 3,200-fold and 4,000-fold in the lower respiratory tract, respectively. Even with the highly attenuated mutants, there was unequivocal HMPV replication at each anatomical site in each animal. Thus, none of these three proteins is essential for HMPV replication in a primate host, although G and M2-2 increased the efficiency of replication. Each gene-deletion virus was highly immunogenic and protective against wild-type HMPV challenge. The deltaG and deltaM2-2 viruses are promising vaccine candidates that are based on independent mechanisms of attenuation and are appropriate for clinical evaluation.     

Phylogenetic congruence of <Emphasis Type="Italic">Sarcocystis neurona</Emphasis> Dubey et al., 1991 (Apicomplexa: Sarcocystidae) in the United States based on sequence analysis and restriction fragment length polymorphism (RFLP)

The objectives of the present study were to assess the genetic diversity, phylogeny and phylogeographical relationships of available Sarcocystis neurona isolates from different localities in the United States. All 13 Sarcocystis isolates from different hosts were subjected to polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analyses using two published DNA markers (25/396 and 33/54). The 334 bp sequence of the 25/396 marker of these isolates and Besnoitia darlingi, B. bennetti, Toxoplasma gondii and Neospora caninum were sequenced and compared. Phylogenetic analysis was performed using neighbour-joining (NJ), maximum parsimony (MP) and minimum evolution (ME) methods based on the sequences of the 25/396 marker of the 13 Sarcocystis isolates obtained in this study and sequences of 10 related isolates from GenBank. Phylogenetic trees revealed a close relatedness among S. neurona isolates in the US (nucleotide sequence diversity <5.0%). US isolates formed a monophyletic group and appeared more closely related to each other than to the South American isolates, which formed a separate lineage. NJ and ME trees with Kimura 2-parameter model separated S. neurona into two separate groups: a northern US group and a Southern US group. These findings suggest a correlation between grouping of the isolates and geographical segregation and were consistent with a genetic bottleneck hypothesis during opossum colonisation of North America. These data do not support either the view of S. neurona as a single super-species or its division into multiple subspecies.