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Biomedical subjects

J Stanley

Publications and source records attributed to J Stanley.

At least 37 records · Page 2Linked to original sources
PubMed2001

Rapid identification of Campylobacter spp. by melting peak analysis of biprobes in real-time PCR.

We describe rapid PCR-biprobe identification of Campylobacter spp. This is based on real-time PCR with product analysis in the same system. The assay identifies enteropathogenic campylobacters to the species level on the basis of their degree of hybridization to three 16S ribosomal DNA (rDNA) biprobes. First-round symmetric PCR is performed with genus-specific primers which selectively target and amplify a portion of the 16S rRNA gene common to all Campylobacter species. Second-round asymmetric PCR is performed in a LightCycler in the presence of one of three biprobes; the identity of an amplified DNA-biprobe duplex is established after determination of the species-specific melting peak temperature. The biprobe specificities were determined by testing 37 reference strains of Campylobacter, Helicobacter, and Arcobacter spp. and 59 Penner serotype reference strains of Campylobacter jejuni and C. coli. From the combination of melting peak profiles for each probe, an identification scheme was devised which accurately detected the five taxa pathogenic for humans (C. jejuni/C. coli, C. lari, C. upsaliensis, C. hyointestinalis, and C. fetus), as well as C. helveticus and C. lanienae. The assay was evaluated with 110 blind-tested field isolates; when the code was broken their previous phenotypic species identification was confirmed in every case. The PCR-biprobe assay also identified campylobacters directly from fecal DNA. PCR-biprobe testing of stools from 38 diarrheic subjects was 100% concordant with PCR-enzyme-linked immunosorbent assay identification (13, 20) and thus more sensitive than phenotypic identification following microaerobic culture.

Campylobacter↗
PubMed2001

Integrating evidence-based practices and the recovery model.

Consumer advocacy has emerged as an important factor in mental health policy during the past few decades. Winning consumer support for evidence-based practices requires recognition that consumers' desires and needs for various types of treatments and services differ significantly. The authors suggest that the degree of support for evidence-based practices by consumer advocates depends largely on the degree of disability of the persons for whom they are advocating. Advocates such as members of the National Alliance for the Mentally Ill, who focus on the needs of the most seriously disabled consumers, are most likely to be highly supportive of research that is grounded in evidence-based practices. On the other hand, advocates who focus more on the needs of consumers who are further along their road to recovery are more likely to be attracted to the recovery model. Garnering the support of this latter group entails ensuring that consumers, as they recover, are given increasing autonomy and greater input about the types of treatments and services they receive. The authors suggest ways to integrate evidence-based practices with the recovery model and then suggest a hybrid theory that maximizes the virtues and minimizes the weaknesses of each model.

Evidence-Based Medicine↗
PubMed2000

A unique virus complex causes Ageratum yellow vein disease.

Ageratum conyzoides L., a weed species widely distributed throughout southeast Asia, frequently exhibits striking yellow vein symptoms associated with infection by Ageratum yellow vein virus (AYVV), a member of the Geminiviridae (genus Begomovirus). Most begomoviruses have bipartite genomes (DNAs A and B), but only a DNA A has been identified for AYVV. We demonstrate that yellow vein disease of A. conyzoides results from co-infection by AYVV DNA A (2,741 nt) and a circular DNA that is approximately half its size (1,347 nt) that we designate DNA beta. Apart from the sequence TAATATTAC, common to all geminiviruses and containing the initiation site of rolling circle replication, DNA beta shows negligible sequence homology either to AYVV DNA A or to DNA B associated with bipartite begomoviruses. DNA beta depends on DNA A for replication and is encapsidated by DNA A-encoded coat protein and so has characteristics of a DNA satellite. However, systemic infection of A. conyzoides by DNA A alone is sporadic and asymptomatic, and DNA A accumulation is reduced to 5% or less of its accumulation in the presence of DNA beta. Therefore, DNA A and DNA beta together form a previously unrecognized disease-inducing complex. Our data also demonstrate that the nanovirus-like DNA 1 component associated with infected A. conyzoides plays no essential role in the disease and represents a satellite-like DNA. Furthermore, the satellite DNA previously found associated with tomato leaf curl virus is probably a defective DNA beta homologue.

Base Sequence↗
PubMed2000

Fluorescent amplified-fragment length polymorphism analysis of the MRSA epidemic.

Genotypes and putative genetic relationships were characterised for epidemic methicillin-resistant Staphylococcus aureus (EMRSA) strains and isolates from England and Wales, using a high resolution DNA fingerprinting technique, fluorescent amplified-fragment length polymorphism (FAFLP). Each of the phage types of EMRSA had a distinct FAFLP profile. The technique revealed clusters of strains and isolates, and could distinguish isolates belonging to the same phage type. FAFLP provides a new approach to the epidemiological study and control of MRSA.

DNA Fingerprinting↗
PubMed2000

Campylobacter lanienae sp. nov., a new species isolated from workers in an abattoir.

Campylobacter-like organisms were isolated from the faeces of healthy individuals during a hygiene survey of abattoir workers. The strains, which exhibited characteristics of Campylobacter, being non-glucose-fermenting, oxidase- and catalase-positive, Gram-negative, motile rods, were identified to the genus level by a PCR assay. Nucleotide sequence analysis of the 16S rRNA gene, DNA homology experiments and determination of G + C content demonstrated that they constituted a previously undescribed species, whose nearest phylogenetic neighbours were Campylobacter hyointestinalis subsp. hyointestinalis, Campylobacter fetus and Campylobacter mucosalis. The name Campylobacter lanienae sp. nov. is proposed for this taxon and species-specific PCR primers were evaluated which will find use in the study of its epidemiology, prevalence and pathogenicity.

Abattoirs↗
PubMed2000

Fluorescent amplified-fragment length polymorphism genotyping of Neisseria meningitidis identifies clones associated with invasive disease.

Fluorescent amplified-fragment length polymorphism (FAFLP), a genotyping technique with phylogenetic significance, was applied to 123 isolates of Neisseria meningitidis. Nine of these were from an outbreak in a British university; 9 were from a recent outbreak in Pontypridd, Glamorgan; 15 were from sporadic cases of meningococcal disease; 26 were from the National Collection of Type Cultures; 58 were carrier isolates from Ironville, Derbyshire; 1 was a disease isolate from Ironville; and five were representatives of invasive clones of N. meningitidis. FAFLP analysis results were compared with previously published multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) results. FAFLP was able to identify hypervirulent, hyperendemic lineages (invasive clones) of N. meningitidis as well as did MLST. PFGE did not discriminate between two strains from the outbreak that were classified as similar but distinct by FAFLP. The results suggest that high resolution of N. meningitidis for outbreak and other epidemiological analyses is more cost efficient by FAFLP than by sequencing procedures.

Bacterial Typing Techniques↗
PubMed2000

Genotyping of verocytotoxin-producing Escherichia coli O157: comparison of isolates of a prevalent phage type by fluorescent amplified-fragment length polymorphism and pulsed-field gel electrophoresis analyses.

We applied the high-resolution genotyping technique fluorescent amplified-fragment length polymorphism (FAFLP) analysis to 71 isolates of a single phage type (PT8) of pulsed-field gel electrophoresis (PFGE)-characterized verocytotoxin-producing Escherichia coli O157. Twenty-seven similar, but not identical, groupings were defined by both FAFLP analysis and the PFGE profiles. Given the FAFLP analysis conditions described here, these two methods exhibited equivalent discriminatory powers.

Bacteriophage Typing↗
PubMed2000

Genome-sequence-based fluorescent amplified-fragment length polymorphism analysis of Mycobacterium tuberculosis.

The whole-genome fingerprinting technique, fluorescent amplified-fragment length polymorphism (FAFLP) analysis, was applied to Mycobacterium tuberculosis. Sixty-five clinical isolates were analyzed to determine the value of FAFLP as a stand-alone genotyping technique and to compare it with the well-established IS6110 typing system. The genome sequence of M. tuberculosis strain H37Rv (S. T. Cole et al., Nature 393:537-544, 1998) was used to model computer-generated informative primer combination(s), and the precision and reproducibility of FAFLP were evaluated by comparing the results of in vitro and computer-generated experiments. Multiplex FAFLP was used to increase resolving power in a predictable and systematic fashion. FAFLP analysis was broadly congruent with IS6110 typing for those strains with multiple IS6110 copies. It was also able to resolve an epidemiologically unlinked group of strains with only one copy of IS6110; up to 10% of clinical isolates may fall into this category. For certain epidemiological investigations, it was concluded that a combination of FAFLP and IS6110 typing would give higher resolution than would either alone. FAFLP data were digital, precise, reproducible, and suitable for rapid electronic dissemination, manipulation, interlaboratory comparison, and storage in national or international epidemiological databases. Because FAFLP samples and analyzes base substitution across the genome as a whole, FAFLP could generate new information about the microevolution of the M. tuberculosis complex.

Bacterial Typing Techniques↗
PubMed1999

A nanovirus-like DNA component associated with yellow vein disease of Ageratum conyzoides: evidence for interfamilial recombination between plant DNA viruses.

Yellow vein disease of Ageratum conyzoides, a weed species that is widely distributed throughout Asia, has been attributed to infection by the geminivirus Ageratum yellow vein virus (AYVV). In addition to a single AYVV genomic component (DNA A), we have previously demonstrated that infected plants contain chimeric defective viral components, comprising DNA A and nongeminiviral sequences, that act as defective interfering DNAs. A database search has revealed that the nongeminiviral sequences of one such defective component (def19) show significant homology with sequences of nanovirus components that encode replication-associated proteins (Reps). Primers designed to hybridise to the nongeminiviral DNA were used to PCR-amplify a full-length nanovirus-like component, referred to as DNA 1, from an extract of infected A. conyzoides. DNA 1 is unrelated to AYVV DNA A but resembles nanovirus components that encode Reps and is most closely related (73% identity) to a nanovirus-like DNA recently isolated from geminivirus-infected cotton. DNA 1 is dependent on AYVV DNA A for systemic infection of A. conyzoides and Nicotiana benthamiana and can systemically infect N. benthamiana in the presence of the bipartite geminivirus African cassava mosaic virus. A. conyzoides plants coinfected with AYVV DNA A and DNA 1 remain asymptomatic, indicating that additional factors are required to elicit yellow vein disease. Our results provide direct evidence for recombination between distinct families of plant single-stranded DNA viruses and suggest that coinfection by geminivirus and nanovirus-like pathogens may be a widespread phenomenon. The ability of plant DNA viruses to recombine in this way may greatly increase their scope for diversification.

Amino Acid Sequence↗
PubMed1999

Identification of a novel circular single-stranded DNA associated with cotton leaf curl disease in Pakistan.

Recent reports have suggested that cotton leaf curl virus (CLCuV), a geminivirus of the genus Begomovirus, may be responsible for cotton leaf curl disease in Pakistan. However, the causal agent of the disease remains unclear as CLCuV genomic components resembling begomovirus DNA A are unable to induce typical disease symptoms when reintroduced into plants. All attempts to isolate a genomic component equivalent to begomovirus DNA B have been unsuccessful. Here, we describe the isolation and characterisation of a novel circular single-stranded (ss) DNA associated with naturally infected cotton plants. In addition to a component resembling DNA A, purified geminate particles contain a smaller unrelated ssDNA that we refer to as DNA 1. DNA 1 was cloned from double-stranded replicative form of the viral DNA isolated from infected cotton plants. Blot hybridisation using probes specific for either CLCuV DNA or DNA 1 was used to demonstrate that both DNAs co-infect naturally infected cotton plants from different geographical locations. DNA 1 was detected in viruliferous Bemisia tabaci and in tobacco plants infected under laboratory conditions using B. tabaci, indicating that it is transmitted by whiteflies. Sequence analysis showed that DNA 1 is approximately half the size of CLCuV DNA but shares no homology, indicating that it is not a defective geminivirus component. DNA 1 has some homology to a genomic component of members of Nanoviridae, a family of DNA viruses that are normally transmitted by aphids or planthoppers. DNA 1 encodes a homologue of the nanovirus replication-associated protein (Rep) and has the capacity to autonomously replicate in tobacco. The data suggest that a nanovirus-like DNA has become whitefly-transmissible as a result of its association with a geminivirus and that cotton leaf curl disease may result from a mutually dependent relationship that has developed between members of two distinct DNA virus families that share a similar replication strategy.

Amino Acid Sequence↗
PubMed1999

Bean yellow dwarf virus RepA, but not rep, binds to maize retinoblastoma protein, and the virus tolerates mutations in the consensus binding motif.

It has previously been reported that complementary-sense gene products of wheat dwarf virus (WDV), a geminivirus of the genus Mastrevirus that infects monocotyledonous plants, bind to human and maize retinoblastoma (Rb) protein. Rb proteins control cell-cycle progression by sequestering transcription factors required for entry into S-phase, suggesting that the virus modifies the cellular environment to produce conditions suitable for viral DNA replication. Using a yeast two-hybrid assay, we have investigated whether the complementary-sense gene products of bean yellow dwarf virus, a mastrevirus that is adapted to dicotyledonous plants, also bind maize Rb protein. We demonstrate that whereas RepA binds to Rb protein, Rep does not, suggesting that RepA alone regulates host gene expression and progression of cells to S-phase. RepA mutants containing L --> I, C --> S, C --> G, and E --> Q mutations within the consensus Rb protein binding motif LXCXE retained the ability to bind to Rb, but with reduced efficiency. Most notably, the E --> Q mutation reduced binding by approximately 95%. Nonetheless, all LXCXE mutants were able to replicate in tobacco protoplasts and to systemically infect Nicotiana benthamiana and bean, in which they produced wild-type symptoms.

Binding Sites↗
PubMed1999

Automatic karyotyping of metaphase cells with overlapping chromosomes.

Image analysis research aimed at analyzing images of banded, human chromosomes is described. An experimental automated karyotyping system (AKS) has been assembled. The goal of the system is to label the chromosomes from a metaphase image with minimal human intervention. The AKS is unique in that it is designed to automatically process cells containing overlapping chromosomes. It also has four novel algorithmic features: the use of cross section sequence graphs, an over-segmentation-based segmentation strategy, the use of the 'pale path' concept for banded chromosomes and the application of mathematical programming for cell-level chromosome classification in cells with overlapping chromosomes. System performance was analyzed using a variety of metaphase images exhibiting various levels of overlapping chromosomes.

Algorithms↗
PubMed1999

Campylobacter coli strains with enlarged flagellin genes isolated from river water.

A group of campylobacters isolated from river water were found to possess unusually large flagellin genes. Both phenotype and serology were consistent with identification as Campylobacter coli. Phylogenetic analysis of small (16S, rrs) and large subunit (23S, rrl) rRNA genes of a representative strain, NCTC 13006, demonstrated high levels of relatedness with C. jejuni and C. coli (99.1 and 98.3% similarity for 16S; 99.3 and 99.4% similarity for 23S). Large flagellin proteins were demonstrated by SDS-PAGE analysis. The flaA and flaB genes were sequenced and aligned with known campylobacter flagellin amino acid sequences. The encoded FlaA protein of the new group exhibited a high degree of divergence from other Campylobacter species. Within the central variable region of FlaA, a further hypervariable domain was identified containing characteristic repeated motifs. Separate pairwise alignments performed for the variable regions of the polypeptide indicated these large fla genes were more closely related to those of C. upsaliensis than to those of C. coli or C. jejuni.

Amino Acid Sequence↗
PubMed1999

Tumour-specific arginine vasopressin promoter activation in small-cell lung cancer.

Small-cell lung cancer (SCLC) can produce numerous mitogenic neuropeptides, which are not found in normal respiratory epithelium. Arginine vasopressin is detected in up to two-thirds of SCLC tumours whereas normal physiological expression is essentially restricted to the hypothalamus. This presents the opportunity to identify elements of the gene promoter which could be exploited for SCLC-specific targeting. A series of human vasopressin 5' promoter fragments (1048 bp, 468 bp and 199 bp) were isolated and cloned upstream of a reporter gene. These were transfected into a panel of ten cell lines, including SCLC with high or low endogenous vasopressin transcription, non-SCLC and bronchial epithelium. All these fragments directed reporter gene expression in the five SCLC cell lines, but had negligible activity in the control lines. The level of reporter gene expression reflected the level of endogenous vasopressin production, with up to 4.9-fold (s.d. 0.34) higher activity than an SV40 promoter. The elements required for this strong, restricted, SCLC-specific promoter activity are contained within the 199-bp fragment. Further analysis of this region indicated involvement of E-box transcription factor binding sites, although tumour-specificity was retained by a 65-bp minimal promoter fragment. These data show that a short region of the vasopressin promoter will drive strong expression in SCLC in vitro and raise the possibility of targeting gene therapy to these tumours.

Arginine Vasopressin↗
PubMed1999

Adaptation of the geminivirus bean yellow dwarf virus to dicotyledonous hosts involves both virion-sense and complementary-sense genes.

Bean yellow dwarf virus (BeYDV) and maize streak virus (MSV) belong to the geminivirus genus Mastrevirus and have host ranges confined to dicotyledonous and monocotyledonous species, respectively. To investigate viral determinants of host range specificity, chimeras were constructed by exchanging their coding and non-coding regions. BeYDV chimeras containing MSV ORF V1, ORF V2 or small intergenic region sequences, either individually or in various sequential combinations, replicated and produced virus particles in Nicotiana tabacum protoplasts. BeYDV chimeras containing MSV ORFs C1 and C2 and/or the large intergenic region were unable to replicate. None of the chimeras was able to systemically infect either N. benthamiana or maize. Complementation experiments using BeYDV chimeras containing MSV ORF V1 and/or ORF V2 suggest that expression of MSV movement protein and/or coat protein prevents BeYDV movement. The results demonstrate that factors involved in both viral DNA replication and virus movement are exclusively adapted to either monocotyledonous or dicotyledonous host backgrounds.

Adaptation, Physiological↗
PubMed1999

Genotyping of epidemic methicillin-resistant Staphylococcus aureus phage type 15 isolates by fluorescent amplified-fragment length polymorphism analysis.

Fluorescent amplified-fragment length polymorphism (FAFLP) analysis was investigated for its ability to identify and subtype isolates of an epidemic methicillin-resistant phage type of Staphylococcus aureus, EMRSA-15. These isolates were also characterized by PCR-restriction fragment length polymorphism (PCR-RFLP) of the coagulase gene and pulsed-field gel electrophoresis (PFGE). For FAFLP, DNA was double digested with restriction enzymes ApaI plus TaqI or EcoRI plus MseI. Site-specific adaptors were ligated to one or the other set of restriction fragments, and PCR amplification was carried out with adaptor-specific primers. Amplified fragments separated on an ABI 377 automated sequencer and analyzed with Genescan version 2.1 software generated FAFLP profiles for all the isolates. The presence or absence of fragments was scored, similarity coefficients were calculated, and UPGMA (unweighted pair group method using arithmatic averages) cluster analysis was performed. Either enzyme-primer combination readily differentiated EMRSA-15 from other methicillin-resistant S. aureus (MRSA) isolates and also revealed heterogeneity within the phage type. The discriminatory power of FAFLP was high. By combining both enzyme-primer data sets, 24 isolates were divided into 11 profiles. PCR-RFLP did not discriminate among these EMRSA-15 isolates. PFGE could discriminate well between isolates but was not as reproducible as FAFLP. All S. aureus and MRSA isolates in this study were typeable by FAFLP, which was easy to perform, robust, and reproducible, with evident potential to subtype MRSA for purposes of hospital infection control.

Bacteriophage Typing↗
PubMed1999

Large-scale survey of Campylobacter species in human gastroenteritis by PCR and PCR-enzyme-linked immunosorbent assay.

A PCR-based study of the incidence of enteropathogenic campylobacter infection in humans was done on the basis of a detection and identification algorithm consisting of screening PCRs and species identification by PCR-enzyme-linked immunosorbent assay. This was applied to DNA extracted from 3,738 fecal samples from patients with sporadic cases of acute gastroenteritis, submitted by seven regional Public Health Laboratories in England and Wales over a 2-year period. The sending laboratories had cultured "Campylobacter spp." from 464 samples. The PCR methodologies detected 492 Campylobacter-positive samples, and the combination of culture and PCR yielded 543 Campylobacter-positive samples. There was identity (overlap) for 413 samples, but 79 PCR-positive samples were culture negative, and 51 culture-positive samples were PCR negative. While there was no statistically significant difference between PCR and culture in detection of C. jejuni-C. coli (PCR, 478 samples; culture, 461 samples), PCR provided unique data about mixed infections and non-C. jejuni and non- C. coli campylobacters. Mixed infections with C. jejuni and C. coli were found in 19 samples, and mixed infection with C. jejuni and C. upsaliensis was found in one sample; this was not apparent from culture. Eleven cases of gastroenteritis were attributed to C. upsaliensis by PCR, three cases were attributed to C. hyointestinalis, and one case was attributed to C. lari. This represents the highest incidence of C. hyointestinalis yet reported from human gastroenteritis, while the low incidence of C. lari suggests that it is less important in this context.

Campylobacter↗