Search PubMed⌕ Search

Biomedical subjects

W G Miller

Publications and source records attributed to W G Miller.

At least 19 recordsLinked to original sources
PubMed2005

Comparison of bacterial populations and chemical composition of dairy wastewater held in circulated and stagnant lagoons.

AIMS: This study compared the chemical, physical and bacterial composition of circulated and stagnant dairy wastewaters. METHODS AND RESULTS: Samples taken from circulated and stagnant wastewater lagoons, over a 1-year period, were analysed for 10 chemical (total N, NH3, NO3, NO2, Na, Ca, HCO3, Fe, P and K) and six physical (biological oxygen demand, chemical oxygen demand, dissolved solids, electrical conductivity, pH and sodium absorption ratio) parameters and were found to be similar. The 16S rDNA genes from the samples were amplified, cloned and BLAST analysed. In total, 996 stagnant and 1052 circulated wastewater derived sequences were obtained, comprising 294 and 362 operational taxonomic units (OTUs) from the circulated and stagnant wastewaters respectively. Coverage estimates of the OTUs identified were 72.1% for the stagnant, and 63.6% for the circulated wastewater libraries. The greatest difference between the two wastewaters was a c. sixfold greater number of sequences representative of the family Chromatiaceae in the circulated wastewater derived library and a c. fivefold greater number of sequences representative of the phylum Chloroflexi in the stagnant wastewater derived library. CONCLUSIONS: Circulation of dairy wastewater does not affect any of the chemical or physical parameters tested; however, circulation does alter the bacterial community structure. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides evidence that circulation of dairy wastewater promotes the growth of bacteria within the family Chromatiaceae and that stagnant systems promote the growth of the phylum Chloroflexi.

Absorption↗
PubMed2005

Production of autoinducer 2 in Salmonella enterica serovar Thompson contributes to its fitness in chickens but not on cilantro leaf surfaces.

Food-borne illness caused by Salmonella enterica has been linked traditionally to poultry products but is associated increasingly with fresh fruits and vegetables. We have investigated the role of the production of autoinducer 2 (AI-2) in the ability of S. enterica serovar Thompson to colonize the chicken intestine and the cilantro phyllosphere. A mutant of S. enterica serovar Thompson that is defective in AI-2 production was constructed by insertional mutagenesis of luxS. The population size of the S. enterica serovar Thompson parental strain was significantly higher than that of its LuxS(-) mutant in the intestine, spleen, and droppings of chicks 12 days after their oral inoculation with the strains in a ratio of 1:1. In contrast, no significant difference in the population dynamics of the parental and LuxS(-) strain was observed after their inoculation singly or in mixtures onto cilantro plants. Digital image analysis revealed that 54% of S. enterica serovar Thompson cells were present in large aggregates on cilantro leaves but that the frequency distributions of the size of aggregates formed by the parental strain and the LuxS(-) mutant were not significantly different. Carbon utilization profiles indicated that the AI-2-producing strain utilized a variety of amino and organic acids more efficiently than its LuxS(-) mutant but that most sugars were utilized similarly in both strains. Thus, inherent differences in the nutrients available to S. enterica in the phyllosphere and in the chicken intestine may underlie the differential contribution of AI-2 synthesis to the fitness of S. enterica in these environments.

Animals↗
PubMed2002

Assessing the role of Pseudomonas aeruginosa surface-active gene expression in hexadecane biodegradation in sand.

Low pollutant substrate bioavailability limits hydrocarbon biodegradation in soils. Bacterially produced surface-active compounds, such as rhamnolipid biosurfactant and the PA bioemulsifying protein produced by Pseudomonas aeruginosa, can improve bioavailability and biodegradation in liquid culture, but their production and roles in soils are unknown. In this study, we asked if the genes for surface-active compounds are expressed in unsaturated porous media contaminated with hexadecane. Furthermore, if expression does occur, is biodegradation enhanced? To detect expression of genes for surface-active compounds, we fused the gfp reporter gene either to the promoter region of pra, which encodes for the emulsifying PA protein, or to the promoter of the transcriptional activator rhlR. We assessed green fluorescent protein (GFP) production conferred by these gene fusions in P. aeruginosa PG201. GFP was produced in sand culture, indicating that the rhlR and pra genes are both transcribed in unsaturated porous media. Confocal laser scanning microscopy of liquid drops revealed that gfp expression was localized at the hexadecane-water interface. Wild-type PG201 and its mutants that are deficient in either PA protein, rhamnolipid synthesis, or both were studied to determine if the genetic potential to make surface-active compounds confers an advantage to P. aeruginosa biodegrading hexadecane in sand. Hexadecane depletion rates and carbon utilization efficiency in sand culture were the same for wild-type and mutant strains, i.e., whether PG201 was proficient or deficient in surfactant or emulsifier production. Environmental scanning electron microscopy revealed that colonization of sand grains was sparse, with cells in small monolayer clusters instead of multilayered biofilms. Our findings suggest that P. aeruginosa likely produces surface-active compounds in sand culture. However, the ability to produce surface-active compounds did not enhance biodegradation in sand culture because well-distributed cells and well-distributed hexadecane favored direct contact to hexadecane for most cells. In contrast, surface-active compounds enable bacteria in liquid culture to adhere to the hexadecane-water interface when they otherwise would not, and thus production of surface-active compounds is an advantage for hexadecane biodegradation in well-dispersed liquid systems.

Aldose-Ketose Isomerases↗
PubMed2001

Biological sensor for sucrose availability: relative sensitivities of various reporter genes.

A set of three sucrose-regulated transcriptional fusions was constructed. Fusions p61RYTIR, p61RYlac, and p61RYice contain the scrR sucrose repressor gene and the promoterless gfp, lacZ, and inaZ reporter genes, respectively, fused to the scrY promoter from Salmonella enterica serovar Typhimurium. Cells of Erwinia herbicola containing these fusions are induced only in media amended with sucrose, fructose, or sorbose. While a large variation in sucrose-dependent reporter gene activity was observed in cells harboring all gene fusions, fusions to the inaZ reporter gene yielded a much wider range of activity and were responsive to lower levels of sucrose than either lacZ or gfp. The lacZ reporter gene was found to be more efficient than gfp, requiring approximately 300-fold fewer cells for a detectable response over all concentrations of sucrose. Similarly, inaZ was found to be more efficient than lacZ, requiring 30-fold fewer cells at 1.45 microM sucrose and 6,100-fold fewer cells at 29 mM sucrose for a quantifiable response. The fluorescence of individual cells containing p61RYTIR was quantified following epifluorescence microscopy in order to relate the fluorescence exhibited by populations of cells in batch cultures with that of individual cells in such cultures. While the mean fluorescence intensity of a population of individual cells increased with increasing concentrations of sucrose, a wide range of fluorescence intensity was seen among individual cells. For most cultures the distribution of fluorescence intensity among individual cells was log-normally distributed, but cells grown in intermediate concentrations of sucrose exhibited two distinct populations of cells, one having relatively low fluorescence and another with much higher fluorescence. When cells were inoculated onto bean leaves, whole-cell ice nucleation and gfp-based biological sensors for sucrose each indicated that the average concentration of sucrose on moist leaf surfaces was about 20 microM. Importantly, the variation in green fluorescent protein fluorescence of biosensor cells on leaves suggested that large spatial variations in sugar availability occur on leaves.

Bacterial Proteins↗
PubMed2000

Improved gfp and inaZ broad-host-range promoter-probe vectors.

A new set of broad-host-range promoter-probe vectors has been constructed. One subset contains the pVS1 and p15a replicons and confers resistance to either gentamicin or kanamycin. The other set contains the broad-host-range replicon from pBBR1 and confers resistance to kanamycin, tetracycline, ampicillin, or spectinomycin/streptomycin. Both plasmid sets are highly stable and are maintained without selection for more than 30 generations in several bacterial taxa. Each plasmid contains a promoter-probe cassette that consists of a multicloning site, containing several unique restriction sites, and gfp or inaZ as a reporter gene. The cassette is bound by transcriptional terminators to permit the insertion of strong promoters and to insulate the cassette from external transcription enabling the detection of weak or moderate promoters. The vector suite was augmented with derivatives of the kanamycin-resistant gfp promoter-probe plasmids that encode Gfp variants with different half-life times.

Bacterial Outer Membrane Proteins↗
PubMed2000

Detection on surfaces and in Caco-2 cells of Campylobacter jejuni cells transformed with new gfp, yfp, and cfp marker plasmids.

We have developed two sets of Campylobacter shuttle vectors containing either the gfp (green fluorescent protein), yfp (yellow fluorescent protein), or cfp (cyan fluorescent protein) reporter gene. In one set, the reporter gene is fused to a consensus Campylobacter promoter sequence (P(c)). The other set contains a pUC18 multicloning site upstream of the reporter gene, allowing the construction of transcriptional fusions using known promoters or random genomic fragments. C. jejuni cells transformed with the P(c) fusion plasmids are strongly fluorescent and easily visualized on chicken skin, on plant tissue, and within infected Caco-2 cells. In each C. jejuni strain tested, these plasmids were maintained over several passages in the absence of antibiotic selection. Also, in many C. jejuni strains, >91% of the cells transformed with the P(c) fusion plasmids remained fluorescent after several days. Experiments with yellow fluorescent and cyan fluorescent C. jejuni transformants suggest that aggregates containing two or more strains of C. jejuni may be present in an enrichment broth culture. Colonies arising from these aggregates would be heterologous in nature; therefore, isolation of a pure culture of C. jejuni, by selecting single colonies, from an environmental sample may not always yield a single strain.

Animals↗
PubMed1999

Genomic fingerprinting and development of a dendrogram for Brucella spp. isolated from seals, porpoises, and dolphins.

Genomic DNA from reference strains and biovars of the genus Brucella was analyzed using pulsed-field gel electrophoresis (PFGE). Fingerprints were compared to estimate genetic relatedness among the strains and to obtain information on evolutionary relationships. Electrophoresis of DNA digested with the restriction endonuclease XbaI produced fragment profiles for the reference type strains that distinguished these strains to the level of species. Included in this study were strains isolated from marine mammals. The PFGE profiles from these strains were compared with those obtained from the reference strains and biovars. Isolates from dolphins had similar profiles that were distinct from profiles of Brucella isolates from seals and porpoises. Distance matrix analyses were used to produce a dendrogram. Biovars of B. abortus were clustered together in the dendrogram; similar clusters were shown for biovars of B. melitensis and for biovars of B. suis. Brucella ovis, B. canis, and B. neotomae differed from each other and from B. abortus, B. melitensis, and B. suis. The relationship between B. abortus strain RB51 and other Brucella biovars was compared because this strain has replaced B. abortus strain 19 for use as a live vaccine in cattle and possibly in bison and elk. These results support the current taxonomy of Brucella species and the designation of an additional genomic group(s) of Brucella. The PFGE analysis in conjunction with distance matrix analysis was a useful tool for calculating genetic relatedness among the Brucella species.

Animals↗
PubMed1999

Brucella-induced abortions and infection in bottlenose dolphins (Tursiops truncatus).

Two bottlenose dolphins (Tursiops truncatus) aborted fetuses that died as a result of Brucella infection. Brucella placentitis occurred in both cases. Infected placenta and vaginal/uterine fluids may transmit Brucella species to other cetaceans. In a third case, an identical organism was cultured from lung necropsy tissue of an adult female T. truncatus. Microbiology, specific polymerase chain reaction, and pulsed-field gel electrophoresis results supported the designation of an additional genomic group(s), Brucella delphini, for isolates adapted to T. truncatus. Current serologic diagnostic tests reliable for known Brucella species are unreliable in detecting dolphin brucellosis. Our findings, together with previous reports, suggest that dolphin brucellosis is a naturally occurring disease that can adversely impact reproduction in cetaceans. The zoonotic significance of cetacean brucellosis is unknown, although the disease has not been reported in people who have frequent contact with dolphins. Further studies on the zoonotic aspects, distribution, prevalence, virulence, and impact of this disease in cetaceans and other marine mammal species are needed.

Abortion, Veterinary↗
PubMed1999

Pushing proficiency testing results to their limits.

PT is a valuable component of laboratory quality control and management practice. PT results are useful to determine over time that a method is performing in a laboratory according to the manufacturer's design parameters, as exhibited by the performance characteristic of a large group of peer laboratories. PT peer group results give a good measure of the interlaboratory imprecision of a method, and summary reports document the state-of-the-art in user preference. PT results typically cannot be used to validate the accuracy of a method or its clinical utility or to compare the accuracy of various methods. These limitations are caused by the common and unpredictable matrix interferences with PT materials and most routine laboratory methods. In situations where a laboratory adjusts the calibration of one method to make patient results agree with those of another method, PT specimens results must be reported without calibration adjustments to allow those results to be compared to an appropriate peer group mean target value.

Humans↗
PubMed1998

Effect of serum-clot contact time on clinical chemistry laboratory results.

The effect of serum-clot contact time on laboratory results was studied by dividing each blood specimen into four blood collection tubes. The control sera were separated from the clot within 30 min of the collection. The other tubes were incubated at 32 degrees C, and the sera were separated at 3, 6, and 24 h. The sera were stored at 4 degrees C and analyzed at the same time. The stability of the tests was determined by comparing the results of the 3-, 6-, and 24-h samples with the values from the 30-min samples. The acceptable limits around the 30-min values were derived from the analytical and intraindividual biological variation of the tests. A total of 63 analytes were studied. Potassium, phosphorous, and glucose were the least stable, and the serum should be separated from the clot within 3 h for these analytes. Albumin, bicarbonate, chloride, C-peptide, HDL-cholesterol, iron, LDL-cholesterol, and total protein should be separated within 6 h. The other analytes were stable for 24 h of serum-clot contact.

Analysis of Variance↗
PubMed1998

The accuracy of laboratory measurements in clinical chemistry: a study of 11 routine chemistry analytes in the College of American Pathologists Chemistry Survey with fresh frozen serum, definitive methods, and reference methods.

CONTEXT: Better procedures are needed whereby national proficiency testing survey providers can assess and improve the accuracy of laboratory measurements in clinical chemistry. SETTING: The 1994 College of American Pathologists Comprehensive Chemistry Survey. DESIGN: This study of matrix effects and the accuracy of laboratory measurements for 11 analytes linked the logistics of the Survey to definitive methods at the National Institutes of Standards and Technology, reference methods at the Centers for Disease Control and Prevention, proficiency testing materials, and a fresh frozen serum sample. The data were analyzed with a statistical model of laboratory measurements. RESULTS: (1) Matrix biases affected the results reported from 69% of the 644 peer group/survey specimen pairs evaluated. (2) Because of matrix biases, the reference value was the correct target value only 32% of the time; thus, the traceability established by definitive method and reference method value assignments on Chemistry Survey specimens did not assure accuracy on patient samples. (3) In contrast to matrix biases, the error caused by random matrix effects with proficiency testing samples was about the same as that caused by random specimen effects with fresh frozen serum, and both were less than within-run random analytic error. (4) Calibration biases occurred in 73% of the 180 peer groups evaluated, and, after matrix biases were removed, the total variance of interlaboratory measurements was due to peer group calibration bias (48%), within-peer-group random calibration error (31 %), within-run random error (14%), and random specimen effects (7%). CONCLUSIONS: An opportunity exists to improve method calibration accuracy in clinical chemistry. With improved design, national proficiency testing surveys can monitor and help reduce method calibration error by converting reported survey results to a true accuracy base that predicts accuracy on patient samples. For medical purposes, the correct target values on artificial (matrix-modified) chemistry materials are reference values adjusted for the matrix bias of each peer group. Matrix biases estimated by the use of fresh frozen serum can be used as factors to transfer the accuracy of definitive methods from artificial reference materials to patient samples.

Bias↗
PubMed1997

An improved GFP cloning cassette designed for prokaryotic transcriptional fusions.

A new gfp cloning cassette designed for prokaryotic transcriptional fusions has been constructed. This cassette consists of gfp (containing the S65T 'red-shift' [Heim et al. (1995) Nature 373, 663-664] and F64L 'protein solubility' [Cormack et al. (1996) Gene 173, 33-38] mutations) flanked by convenient restriction sites, a translational enhancer, and a consensus ribosome binding site with an optimized spacer region. gfp fusion strains containing this cassette demonstrate from 40- to 80-fold greater fluorescence intensity than wild-type gfp fusion strains. Additionally, this cassette confers sufficient fluorescence to recipient cells to be used in low copy-number plasmids, with promoters conferring low levels of transcription, and in bacterial taxa other than Escherichia, such as Pseudomonas.

Cloning, Molecular↗
PubMed1997

Use of the combination of myoglobin and CK-MB mass for the rapid diagnosis of acute myocardial infarction.

Early identification of patients presenting with myocardial infarction (MI) is necessary for rapid initiation of treatment. Currently, MI has been diagnosed using the combination of the history, electrocardiogram (ECG), and biochemical markers of myocardial necrosis. Unfortunately, all lack sufficient sensitivity and specificity to confidently identify most patients with MI in a timely enough fashion to influence early intervention. Development of newer immunochemical assays for CK-MB mass and myoglobin have allowed for earlier, more rapid diagnosis; however, each has important limitations. The diagnostic sensitivity of CK-MB mass, myoglobin, and the combination of both were analyzed at the time of presentation (0 hours) and again 4 hours later in 101 patients admitted from the emergency department (ED) with possible MI. Twenty patients were subsequently diagnosed as having MI. The sensitivity of the initial ECG was 60%, compared with the sensitivities of the initial myoglobin and CK-MB mass of 70% and 30%, respectively. By 4 hours the sensitivity of myoglobin had increased to 85% and CK-MB mass to 90%. The combination of the initial myoglobin and CK-MB mass had a sensitivity of 85%. Combining these two markers, using both the initial and 4-hour samples, raised the sensitivity to 100%, with a specificity of 100% and negative predictive value of 100%. When patients with diagnostic ECGs were excluded, the sensitivity of the combination at 0 hours was 80% with a specificity of 84%, while the use of the 0- and 4-hour markers had a sensitivity and specificity of 100% and 100%, respectively. We conclude that the combination of CK-MB mass and myoglobin can rapidly diagnose or exclude MI in as short as 4 hours after ED presentation, and accuracy is not different in patients without diagnostic ECGs. Application of this strategy could potentially lead to more rapid intervention in patients with MI, while also allowing early identification of lower risk patients.

Biomarkers↗
PubMed1997

Comparison of manual versus automated blood pressure measurements in treated hypertensive patients.

This study assesses differences in blood pressure (BP) levels prospectively between office (manual) measurement and ambulatory blood pressure monitoring (ABPM) in 70 treated, essential, hypertensive patients. The objective was to determine whether ABPM is superior to office measurement for assessing adequacy of therapy. Twenty-four patients received monotherapy and 46 received multiple therapy. Thirty-five patients were administered medication in the morning only, whereas 33 were administered medication in the morning and evening both. Mean systolic BP by manual method was identical to that obtained by ABPM (141.98 +/- 14.98 mm Hg versus 141.46 +/- 16.33 mm Hg, respectively). However, mean diastolic BP by manual method was significantly higher than that obtained by ABPM. (90.38 +/- 9.01 mm Hg versus 86.69 +/- 10.65 mm Hg, respectively; P < 0.001). Significant correlations (P < 0.01) were found between the BP levels measured by these two methods, although individual readings differed by 10 mm Hg or more systolic and by 5 mm or more diastolic in many subjects. No significant differences were noted in BP levels measured by either method for patients treated by monotherapy or multiple therapy, and none were noted whether they were taking medication in the morning or in both the morning and evening. In addition, no differences were noted in BP levels using either method for race. Thus, this study shows that the office measurement is grossly similar to ABPM for assessment of adequacy of therapy in treated hypertensive patients whose blood pressure is controlled adequately. However, ABPM is found to be superior to office measurement in identifying hypertensive patients whose blood pressure is not controlled adequately or is uncontrolled.

Adrenergic beta-Antagonists↗
PubMed1997

Performance of predispensed reagent and noncentrifugation methods for high-density lipoprotein cholesterol measurement.

OBJECTIVE: To evaluate the performance of high-density lipoprotein cholesterol methods with predispensed reagent (SPINPRO, ISO Spin, Vitros, and One Shots) and noncentrifugation separation (Magnetic-HDL) compared with traditional high-density lipoprotein cholesterol methods (PBI Plus, Boehringer-Mannheim, and Abbott). DESIGN: Precision was evaluated by running two concentrations of frozen human sera and two concentrations of lyophilized quality control material according to National Committee for Clinical Laboratory Standards document EP5-T2. Accuracy was evaluated by comparing sample results achieved by each method with those by the Cholesterol Reference Method Laboratory Network Designated Comparison Method and the Lipid Research Clinics' heparin-Mn++ method. Sera from donors with high triglyceride levels were used to challenge the ability of each method to measure lipemic samples for each method. SETTING: Outpatient clinic and university medical center. PATIENTS: Forty-two ambulatory donors with high-density lipoprotein cholesterol levels ranging from 30 to 102 mg/dL, cholesterol levels ranging from 107 to 679 mg/dL, and triglyceride levels ranging from 20 to 2450 mg/dL. MAIN OUTCOME MEASURE: Precision, accuracy, linearity, and ability to measure lipemic samples. RESULTS: Imprecision varied from CV 1.4% to 8.6%. The mean absolute bias for each method versus the Designated Comparison Method ranged from 2.6% to 13.3%. Deming regression analysis of all methods versus the heparin-Mn++ method gave slopes from 0.88 to 1.08 and intercepts from -4.5 to 0.0 mg/dL. All methods met the current 22% total error for individual specimens recommended by the National Cholesterol Education Program. Not all methods could measure high-density lipoprotein cholesterol in the presence of high concentrations of triglycerides. CONCLUSIONS: Performance of all methods met the current recommendations of the National Cholesterol Education Program. Not all methods met the 1998 performance goals.

Blood Donors↗
PubMed1997

Fiber-optic immunosensor for measurement of myoglobin.

A self-contained fiber-optic immunosensor was developed to measure the 16,500-Da protein myoglobin. The sensing element was constructed by entrapment of Cascade Blue-labeled antibody within polyacrylamide gel at the distal face of an optical fiber 300 microns in core diameter. The polyacrylamide gel composition was optimized to allow diffusion of myoglobin but to exclude hemoglobin and higher-molecular-mass proteins from the sensing area. The analytical signal was derived from fluorescence energy transfer between Cascade Blue and the heme group of myoglobin. Fluorescence quenching occurred when myoglobin bound to labeled antibody. The total amount of fluorescence quench was dependent on the antibody labeling conditions and the amount of antibody incorporated in the sensor gel matrix. Myoglobin concentrations > 5 nmol/L (83 micrograms/L) were measurable with response times of 15 to 130 min limited by diffusion into the sensing element. This report demonstrates the technical feasibility for a self-contained immunosensor to measure a protein analyte.

Acrylic Resins↗
PubMed1996

Antibody characteristics for a continuous response fiber optic immunosensor for theophylline.

A self-contained fiber optic immunosensor was developed to measure continuously theophylline concentrations. The analytical signal was derived from the non-radiative energy transfer quench of fluorescence following binding of a fluorescence donor, B-phycoerythrin labeled theophylline, to an energy acceptor, Texas Red labeled antibody. Increases in free theophylline analyte concentrations resulted in a shift in antibody binding equilibrium between labeled and unlabeled theophylline that elicited a proportional increase in the fluorescence. The selection criteria for a monoclonal antibody as a molecular recognition element, and the optimization of labeling conditions to maximize the dynamic range and minimize sensor response time are described. Under one or more Texas Red labeling conditions, five antibody clones exhibited significant quenching when mixed with labeled analyte and also demonstrated 95% or greater reversible binding to labeled analyte. Two clones failed to exhibit fluorescence quenching when mixed with labeled analyte. The response time of the indicator chemistry system was dependent on the dissociation rate constant of the antibody. The equilibrium response time of intact sensors was limited by analyte diffusion across the containment membrane.

Antigen-Antibody Reactions↗