PubMed Health⌕ Search

Biomedical subjects

David Shepherd

Publications and source records attributed to David Shepherd.

12 recordsLinked to original sources
PubMed2006

A rapid (one day), sensitive real-time polymerase chain reaction assay for detecting Escherichia coli O157:H7 in ground beef.

The purpose of this study was to apply our rapid, integrated double enrichment 5' nuclease real-time polymerase chain reaction assay for the detection of Escherichia coli O157:H7 and evaluate its efficacy. The assay targeted ground beef, an important vehicle in disease epidemiology. The assay reliably determined in 8 h the presence of E. coli O157:H7 in ground beef at the level of 1 colony-forming unit (CFU)/g. The sensitivity and specificity of the assay were compared with that of standard enrichment diagnostic techniques. A correlation of 100% in detection was achieved to the limit of 1 CFU/g. This assay can be used as a rapid, automatic process for identification of E. coli O157:H7 in ground beef or can be integrated with standard culture procedures, resulting in considerable cost and time savings.

Animals↗
PubMed2006

Immunolocalisation of the D. melanogaster Nramp homologue Malvolio to gut and Malpighian tubules provides evidence that Malvolio and Nramp2 are orthologous.

Nramp (Slc11a1) genes in mammals are associated with the transport of iron and other divalent cations; Nramp1 in macrophages involved in the innate immune response against intracellular pathogens, and Nramp2 with duodenal iron uptake and the transferrin-transferrin-receptor pathway of iron assimilation. The Drosophila melanogaster Nramp-related gene is known as Malvolio. The localisation of Malvolio protein was inferred from the enhancer trap line initially used to isolate Malvolio in a screen for mutants with defects in taste perception. Here we describe the generation of a Malvolio-reactive polyclonal antibody and apply it to evaluate Malvolio localisation during stages of D. melanogaster development, and compare the results with the localisation of the enhancer trap line identified with beta-galactosidase. All immunolocalisation studies have been confirmed to be specific with Malvolio-blocking peptides. Our results demonstrated expression within Malpighian tubules, testis, brain, the amnioserosa of embryos, the larval and adult alimentary canal. Expression within the gut was of significant interest, as mammalian Nramp2 in the gut plays a primary role in the acquisition of dietary iron. We confirm expression within the central nervous system and in cells of the haematopoietic system. By immunohistochemistry we showed that expression within cells was either punctuate, diffuse cytoplasmic or plasma membrane associated, or both. The staining within the gut indicates a degree of conservation of components for iron acquisition between flies and mammals, suggesting that a comparable mechanism has been retained during evolution.

Animals↗
PubMed2006

A rapid molecular-based assay for direct quantification of viable bacteria in slaughterhouses.

A rapid test for microbial quantification in carcass and environmental swabs that does not require enrichment and provides results in less than 4 h is described here. Steps in the assay include the rapid concentration of bacteria on sponge swabs by vacuum filtration followed by real-time PCR detection. The assay has been applied for the detection of coliforms, Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes on carcass swabs and environmental samples in a slaughterhouse-processing line. Comparison of this rapid method with standard culture techniques for coliform counts on beef and pork carcass swabs revealed higher numbers of bacteria (2- to 50-fold) by the rapid test compared with the plate counts. This was due to the detection of all bacteria (live, dead, and non-culturable forms) in the rapid assay. To allow detection of only viable bacteria, concentrated samples were treated with ethidium monoazide (EMA) prior to DNA extraction and real-time PCR detection, thereby preventing the amplification of DNA from bacteria with damaged cell walls and allowing only the DNA from bacteria with intact membranes to be detected. EMA treatment resulted in a significant reduction (P < 0.001) in the number of coliforms detected compared to real-time PCR without EMA treatment. In beef swabs, the counts obtained in EMA real-time PCR were not significantly different (P < 0.08) from the culture counts and the correlation coefficient between the two assays was 0.7385. A lower correlation coefficient (0.402) was obtained with pork swabs. The assay described herein has the potential to be applied on a routine basis to slaughterhouse lines for the detection of indicator organisms or specific pathogens.

Abattoirs↗
PubMed2005

Over-expression of tau results in defective synaptic transmission in Drosophila neuromuscular junctions.

We have shown that over-expression of human tau (0N3R) in Drosophila larval motor neurons causes significant morphological and functional disruption to the neuromuscular junctions (NMJs). Tau-expressing NMJs are reduced in size with irregular and abnormal bouton structure. Immunocytochemical analysis shows that the abnormal NMJs still retain synaptotagmin expression and form active zones. Functionally, the NMJs exhibit abnormal endo/exocytosis as revealed by incorporation of the styryl dye FM1-43. Electrophysiological studies showed that with low frequency stimulation (1 Hz), evoked synaptic potentials produced from tau over-expressing motor neurons were indistinguishable from wild type, however, following high frequency stimulation (50 Hz), evoked synaptic potentials were significantly decreased. Analysis of the number and distribution of mitochondria showed that motor neurons over-expressing tau had a significant reduction in functional mitochondria in the presynaptic terminal. Collapsing the mitochondrial membrane potential in wild type larvae phenocopied the effects of tau over-expression on synaptic transmission. Our results demonstrate that tau over-expression in vivo cause a synaptic dysfunction, which may be caused by a reduced complement of functional mitochondria.

Animals↗
PubMed2004

CTIP1 and CTIP2 are differentially expressed during mouse embryogenesis.

Chicken ovalbumin upstream promoter transcription factor-interacting proteins 1 and 2 (CTIP1 and CTIP2) are related transcriptional regulatory proteins. While overexpression of both of these proteins has been linked to the development of several lymphoid malignancies, lack of CTIP1 and CTIP2 expression results in defective lymphopoiesis and abnormal thymocyte development, respectively. Here, we describe the expression patterns of CTIP1 and CTIP2 during mouse embryogenesis and in the post-natal brain. Both CTIP1 and CTIP2 were expressed diffusely in the embryo at 10.5 days post-coitum (d.p.c.). However, the expression of both genes became increasingly restricted to the central nervous system (CNS) during the course of fetal development, culminating with high, but differential, expression levels throughout the hippocampal subregions, olfactory bulb and cortex, limbic system, basal ganglia and frontal cortex of the developing brain, and in dorsal cells of the spinal cord. The brain expression domains of CTIP1 and CTIP2 were maintained into adulthood. Outside the CNS, both genes exhibited differential expression within the facial mesenchyme at 12.5 d.p.c., and CTIP2 was selectively expressed from day 12.5 onwards in the olfactory epithelium and developing thymus, and to a lesser extent in oral and gut epithelia. Strong CTIP2 expression was maintained in the thymus at 18.5 d.p.c. These results support the selective contributions of both CTIP1 and CTIP2 in the development and function of both the central nervous and immune systems and the importance of future investigations to define the function(s) of both proteins.

Animals↗
PubMed2004

Developmental architecture of adult-specific lineages in the ventral CNS of Drosophila.

In Drosophila most thoracic neuroblasts have two neurogenic periods: an initial brief period during embryogenesis and a second prolonged phase during larval growth. This study focuses on the adult-specific neurons that are born primarily during the second phase of neurogenesis. The fasciculated neurites arising from each cluster of adult-specific neurons express the cell-adhesion protein Neurotactin and they make a complex scaffold of neurite bundles within the thoracic neuropils. Using MARCM clones, we identified the 24 lineages that make up the scaffold of a thoracic hemineuromere. Unlike the early-born neurons that are strikingly diverse in both form and function, the adult specific cells in a given lineage are remarkably similar and typically project to only one or two initial targets, which appear to be the bundled neurites from other lineages. Correlated changes in the contacts between the lineages in different segments suggest that these initial contacts have functional significance in terms of future synaptic partners. This paper provides an overall view of the initial connections that eventually lead to the complex connectivity of the bulk of the thoracic neurons.

Animals↗
PubMed2002

Inhibition of growth and metastasis of orthotopic human prostate cancer in athymic mice by combination therapy with pegylated interferon-alpha-2b and docetaxel.

We evaluated whether treatment of orthotopic human prostate cancer in nude mice with pegylated IFN-alpha-2b (PEG-IFN-alpha-2b) and docetaxel could represent a two-compartment targeting of primary tumor (tumor cells and tumor-associated endothelial cells) and inhibition of regional lymph node metastasis. The antiangiogenic properties of IFN were combined with the cytotoxic properties of docetaxel, resulting in apoptosis of both tumor cells and endothelium and hence significant inhibition of primary tumor growth. We first determined the optimal biological dose of PEG-IFN-alpha-2b (70,000 IU/week) necessary to down-regulate the expression of basic fibroblast growth factor, matrix metalloprotease-9, and matrix metalloprotease-2. The therapeutic dose of docetaxel (10 mg/kg/week) was determined by efficacy and minimal body weight loss. Therapy beginning 3 days after orthotopic implantation of PC3-MM2 prostate cancer cells reduced tumor weight by 37% in mice treated with PEG-IFN-alpha-2b, by 60% in mice treated with docetaxel, and by 83% in those given both drugs. PEG-IFN-alpha-2b also induced apoptosis of tumor-associated endothelial cells and hence a significant decrease in microvessel density. Our data indicate that the combination of PEG-IFN-alpha and docetaxel inhibits neoplastic angiogenesis by inducing a decrease in the local production of proangiogenic molecules by tumor cells, resulting in increased apoptosis of tumor-associated endothelial cells.

Angiogenesis Inhibitors↗
PubMed2002

Persistent larval sensory neurones are required for the normal development of the adult sensory afferent projections in Drosophila.

We have tested the hypothesis that larval neurones guide growth of adult sensory axons in Drosophila. We show that ablation of larval sensory neurones causes defects in the central projections of adult sensory neurones. Spiralling axons and ectopic projections indicate failure in axon growth guidance. We show that larval sensory neurones are required for peripheral pathfinding, entry into the CNS and growth guidance within the CNS. Ablation of subsets of neurones shows that larval sensory neurones serve specific guidance roles. Dorsal neurones are required for axon guidance across the midline, whereas lateral neurones are required for posterior growth. We conclude that larval sensory neurones pioneer the assembly of sensory arrays in adults.

Animals↗