Gene-specific contributions to mumps virus neurovirulence and neuroattenuation

Mumps virus (MuV) is highly neurotropic and was the leading cause of aseptic meningitis in the Western Hemisphere prior to widespread use of live attenuated MuV vaccines. Due to the absence of markers of virus neuroattenuation and neurovirulence, ensuring mumps vaccine safety has proven problematic, as demonstrated by the occurrence of aseptic meningitis in recipients of certain vaccine strains. Here we examined the genetic basis of MuV neuroattenuation and neurovirulence by generating a series of recombinant viruses consisting of combinations of genes derived from a cDNA clone of the neurovirulent wild-type 88-1961 strain (r88) and from a cDNA clone of the highly attenuated Jeryl Lynn vaccine strain (rJL). Testing of these viruses in rats demonstrated the ability of several individual rJL genes and gene combinations to significantly neuroattenuate r88, with the greatest effect imparted by the rJL nucleoprotein/matrix protein combination. Interestingly, no tested combination of r88 genes, including the nucleoprotein/matrix protein combination, was able to convert rJL into a highly neurovirulent virus, highlighting mechanistic differences between processes involved in neuroattenuation and neurovirulence.     

Live-cell microarray surface coatings supporting reverse transduction by adeno-associated viruses

High-throughput live-cell microarray technologies that facilitate combinatorial screening of genes and RNA interference (RNAi) would be invaluable in the identification of key gene expression profiles involved in complex cellular behaviors. Each spot on such a microarray can comprise a unique combination of genes or RNAi packaged into gene delivery vectors. Live target cells seeded on top of the microarrays would express the combination of genetic factors, potentially leading to phenotypic changes within cells. Here, we investigate the feasibility of using adeno-associated virus (AAV) as a gene delivery agent for such live-cell genetic microarrays. A robotic spotter was used to deposit AAV onto gamma-amino propyl silane, amine silane, or nitrocellulose-coated glass slides. Virus deposition and reverse transduction of target cells were found to be surface coating-dependent with nitrocellulose coating yielding the best AAV deposition, while also producing discrete islands of highly transduced cells. Our results demonstrate the feasibility of using nitrocellulose-coated surfaces for the development of AAV-based genetic microarrays.     

Major phenolic compounds in olive oil: metabolism and health effects

It has been postulated that the components in olive oil in the Mediterranean diet, a diet which is largely vegetarian in nature, can contribute to the lower incidence of coronary heart disease and prostate and colon cancers. The Mediterranean diet includes the consumption of large amounts of olive oil. Olive oil is a source of at least 30 phenolic compounds. The major phenolic compounds in olive oil are oleuropein, hydroxytyrosol and tyrosol. Recently there has been a surge in the number of publications that has investigated their biological properties. The phenolic compounds present in olive oil are strong antioxidants and radical scavengers. Olive "waste water" also possesses compounds which are strong antioxidant and radical scavengers. Typically, hydroxytyrosol is a superior antioxidant and radical scavenger to oleuropein and tyrosol. Hydroxytyrosol and oleuropein have antimicrobial activity against ATTC bacterial strains and clinical bacterial strains. Recent syntheses of labeled and unlabelled hydroxytyrosol coupled with superior analytical techniques have enabled its absorption and metabolism to be studied. It has recently been found that hydroxytyosol is renally excreted unchanged and as the following metabolites as its glucuronide conjugate, sulfate conjugate, homovanillic acid, homovanillic alcohol, 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxyphenylacetaldehyde. Studies with tyrosol have shown that it is excreted unchanged and as its conjugates. This review summarizes the antioxidant abilities; the scavenging abilities and the biological fates of hydroxytyrosol, oleuropein and tyrosol which have been published in recent years.     

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the “gold standard” enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.     

pp60v-src association with the cytoskeleton induces actin reorganization without affecting polymerization status

The mechanism by which Rous sarcoma virus (RSV) induces a reorganization of actin and its associated proteins and a reduction in microfilament bundles is at present poorly understood. To examine the relationship between the organization of the microfilament system and the polymerization state of actin after transformation, we have investigated these changes in a Rat-1 cell line transformed by LA29, a temperature-sensitive (ts) mutant of RSV. Parallel immunofluorescence and biochemical analysis demonstrated that LA29 pp60v-src was ts for tyrosine kinase activity and cytoskeletal association. Changes in the distribution and organization of actin, alpha-actinin and vinculin were dependent on the association of a kinase-active pp60v-src molecule with the detergent-insoluble cytoskeleton. Whilst there was a transformation-dependent loss of microfilament bundles, biochemical quantitation demonstrated that the polymerization state of the actin in both detergent-soluble and insoluble fractions of these cells grown at temperatures either permissive or restrictive for transformation was quantitatively unchanged. These results indicate that the loss of microfilament bundles after transformation is not due to a net depolymerization of filamentous actin but rather to a reorganization of polymeric actin from microfilament bundles and stress fibers to other polymeric forms within the cell. The polymeric nature of the actin in these cells was confirmed by electron microscopy of cytoskeletons and substrate-adherent membranes.     

Translocation of pp60c-src to the cytoskeleton during platelet aggregation.

The high amount of pp60c-src in platelets has led to speculation that this kinase is responsible for tyrosine-specific phosphorylation of cellular proteins during platelet activation by different agonists, and is, therefore, implicated in signal transduction of these cells. Unlike pp60v-src, the association of which with the cytoskeleton appears to be a prerequisite for transformation, pp60c-src is detergent-soluble in fibroblasts overexpressing the c-src gene, and its role in normal cellular function remains elusive. To gain a better understanding of the function of pp60c-src we have investigated the subcellular distribution of pp60c-src and its relationship to the cytoskeleton during platelet activation. Quantitative immunoblotting and immunoprecipitation have revealed that pp60c-src is detergent-soluble in resting platelets, while 40% of total platelet pp60c-src becomes associated with the cytoskeletal fraction upon platelet activation. We have also shown that a small pool of pp60c-src is associated with the membrane skeletal fraction which remains unchanged during the activation process. The interaction of pp60c-src with cytoskeletal proteins strongly correlates with aggregation and is mediated by GPIIb/IIIa receptor-fibrinogen binding. We suggest that the translocation of pp60c-src to the cytoskeleton and its association with cytoskeletal proteins may regulate tyrosine phosphorylation in platelets.     

Repurposing environmental DNA samples—detecting the western pearlshell (Margaritifera falcata) as a proof of concept

Information on the distribution of multiple species in a common landscape is fundamental to effective conservation and management. However, distribution data are expensive to obtain and often limited to high‐profile species in a system. A recently developed technique, environmental DNA (eDNA) sampling, has been shown to be more sensitive than traditional detection methods for many aquatic species. A second and perhaps underappreciated benefit of eDNA sampling is that a sample originally collected to determine the presence of one species can be re‐analyzed to detect additional taxa without additional field effort. We developed an eDNA assay for the western pearlshell mussel (Margaritifera falcata) and evaluated its effectiveness by analyzing previously collected eDNA samples that were annotated with information including sample location and deposited in a central repository. The eDNA samples were initially collected to determine habitat occupancy by nonbenthic fish species at sites that were in the vicinity of locations recently occupied by western pearlshell. These repurposed eDNA samples produced results congruent with historical western pearlshell surveys and permitted a more precise delineation of the extent of local populations. That a sampling protocol designed to detect fish was also successful for detecting a freshwater mussel suggests that rapidly accumulating collections of eDNA samples can be repurposed to enhance the efficiency and cost‐effectiveness of aquatic biodiversity monitoring.     

The secondary structure of a pyrimidine-guanine sequence-specific ribonuclease possessing cytotoxic activity from the oocytes of Rana catesbeiana

Summary RC-RNase is a pyrimidine-guanine sequence-specific ribonuclease and a sialic-acid-binding lectin purified from Rana catesbeiana (bullfrog) oocytes. This 111-amino acid protein exhibits cytotoxicity toward several tumor cell lines. In this paper we report the assignments of proton NMR resonances and the identification of the secondary structure deduced from NOE constraints, chemical shift index, ³J_NH and amide proton exchange rates. The protein was directly isolated from bullfrog oocytes; we were able to assign all but five of the amino acid backbone protons of the unlabeled protein by analyzing a large set of two-dimensional proton NMR spectra obtained at several temperatures and pH conditions. Our results indicate that the structure of RC-RNase is dominated by the presence of two triple-stranded antiparallel -sheets and three -helices, similar to those of the pyrimidine family ribonucleases. Two sets of resonances were observed for 11 amide protons and 8 -protons located in the loop-1 region, an 2 helix, and three -strands (1, 3 and 4), suggesting the presence of nonlocalized multiple conformations for RC-RNase.Abbreviations DQF-COSY double-quantum-filtered correlation spectroscopy - DTT dithiothreitol - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy - PE-1 N-terminal pyroglutamate - RC-RNase ribonuclease from the oocyte of Rana catesbeiana - TOCSY total correlation spectroscopy - TPPI time-proportional phase incrementation - TSP sodium 3-trimethylsilylpropionate-2,2,3,3-d ₄     

Sarcoptes scabiei: The Mange Mite with Mighty Effects on the Common Wombat (Vombatus ursinus)

Parasitism has both direct and indirect effects on hosts. Indirect effects (such as behavioural changes) may be common, although are often poorly described. This study examined sarcoptic mange (caused by the mite Sarcoptes scabiei) in the common wombat (Vombatus ursinus), a species that shows severe symptoms of infection and often causes mortality. Wombats showed alterations to above ground behaviours associated with mange. Infected wombats were shown to be active outside of the burrow for longer than healthy individuals. Additionally, they spent more time scratching and drinking, and less time walking as a proportion of time spent above ground when compared with healthy individuals. They did not spend a higher proportion of time feeding, but did have a slower feeding rate and were in poorer body condition. Thermal images showed that wombats with mange lost considerably more heat to the environment due to a diminished insulation layer. Infection status did not have an effect on burrow emergence time, although this was strongly dependent on maximum daily temperature. This study, through the most detailed behavioural observations of wombats to date, contributes to a broader understanding of how mange affects wombat health and abundance, and also to our understanding of the evolution of host responses to this parasite. Despite being globally dispersed and impacting over 100 species with diverse intrinsic host traits, the effects of mange on hosts are relatively poorly understood, and it is possible that similar effects of this disease are conserved in other host species. The indirect effects that we observed may extend to other pathogen types.