bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular and Biochemical Characterization of the Selenocysteine Se-Methyltransferase Gene and Se-Methylselenocysteine Synthesis in Broccoli
Authors: Lyi Sangbom M. , Heller Laurence I. , Rutzke Michael , Welch Ross M. , Kochian Leon V. , Li Li
Published: April 2005

Selenium (Se) plays an indispensable role in human nutrition and has been implicated to have important health benefits, including being a cancer preventative agent. While different forms of Se vary in their anticarcinogenic efficacy, Se-methylselenocysteine (SeMSC) has been demonstrated to be one of the most effective chemopreventative compounds. Broccoli (Brassica oleracea var. italica) is known for its ability to accumulate high levels of Se with the majority of the selenoamino acids in the form of Se-methylselenocysteine. Therefore, it serves as a good model to study the regulation of SeMSC accumulation in plants. A cDNA encoding selenocysteine Se-methyltransferase, the key enzyme responsible for SeMSC formation, was cloned from broccoli using a homocysteine S-methyltransferase gene probe from Arabidopsis (Arabidopsis thaliana). This clone, designated as BoSMT, was functionally expressed in Escherichia coli, and its identity was confirmed by its substrate specificity in the methylation of selenocysteine. The BoSMT gene represents a single copy sequence in the broccoli genome. Examination of BoSMT gene expression and SeMSC accumulation in response to selenate, selenite, and sulfate treatments showed that the BoSMT transcript and SeMSC synthesis were significantly up-regulated in plants exposed to selenate but were low in plants supplied with selenite. Simultaneous treatment of selenate with selenite significantly reduced SeMSC production. In addition, high levels of sulfate suppressed selenate uptake, resulting in a dramatic reduction of BoSMT mRNA level and SeMSC accumulation. Our results reveal that SeMSC accumulation closely correlated with the BoSMT gene expression and the total Se status in tissues and provide important information for maximizing the SeMSC production in this beneficial vegetable plant.
Contribution of the cornea to cytokine levels in the whole eye induced during the early phase of Pseudomonas aeruginosa challenge
Authors: Cole Nerida , Hume Emma , Khan Shamila , Madigan Michele , Husband Alan J , Garthwaite Linda , Willcox Mark
Published: May 2005

Pseudomonas aeruginosa keratitis is one of the most destructive diseases of the cornea. The host response to this infection is critical to the outcome, and is regulated by cytokines produced in the ocular tissue. In this study, we assessed the relative contribution of the cytokines produced in the cornea to the inflammatory response of the whole eye to gain a better understanding of the inflammatory and regulatory processes in the ocular environment during localized corneal infection. C57BL/6 mice were challenged by topical application of P. aeruginosa to wounded corneas. Corneas and whole eyes were harvested 24 h post‐challenge and bacterial numbers, myeloperoxidase levels and the levels of cytokines known to be important in keratitis were determined. The site of production of IL‐6 and KC in the retina was determined by in situ hybridization. Before infection, 90% of macrophage inflammatory protein (MIP)‐2 and approximately 80% of all IFN‐γ and IL‐10 produced constitutively in the eye was found outside the cornea. Twenty‐four hours after infection, bacterial numbers, levels of myeloperoxidase, and levels of MIP‐2 and IL‐1 were not different, whether measured in cornea or whole eye. However, expression of IL‐6, KC, IFN‐γ and IL‐10 was significantly greater in whole eyes than in the corneas of infected eyes. The cells expressing IL‐6 and KC in the retina were identified by in situ hybridization. This study indicates that during corneal inflammation, the response of the whole eye as well as the cornea needs to be considered.
Upregulation of LPS‐induced chemokine KC expression by 15‐deoxy‐Δ12,14‐prostaglandin J2 in mouse peritoneal macrophages
Authors: Kim Hyo Y , Kim Hyun K , Kim Jae R , Kim Hee S
Published: May 2005

15‐Deoxy‐Δ12,14‐prostaglandin J2 (15d‐PGJ2) was initially identified as a high affinity natural ligand for the peroxisome proliferator‐activated receptor (PPAR)‐γ. Recent studies have shown that it has a potent anti‐inflammatory effect by attenuating the expression of proinflammatory mediators in activated macrophages, mainly through the inhibition of nuclear factor (NF)‐κB‐dependent transcription of inflammatory genes. In this study, we investigated the synergistic effect of 15d‐PGJ2 on the expression of LPS‐induced chemokine KC mRNA in mouse peritoneal macrophages. The time course of KC mRNA expression in cells stimulated with 15d‐PGJ2 plus LPS simultaneously (15d‐PGJ2/LPS) showed similar patterns to the cells treated with LPS alone, and 15d‐PGJ2 had no effect on the stability of LPS‐induced KC mRNA expression. Although NF‐κB activity in cells treated with LPS was augmented by 15d‐PGJ2, pyrrolidone dithiocarbamate (PDTC) did not block the synergistic effect of 15d‐PGJ2 on LPS‐induced KC mRNA expression. However, the synergistic effect of 15d‐PGJ2 was markedly inhibited when the macrophages were treated with a inhibitor of the mitogen‐activated protein kinase (MAPK) signalling pathway, 2′‐amino‐3′‐methoxyflavine (PD98059). Therefore, the mechanism of synergistic action of 15d‐PGJ2 on the expression of LPS‐induced KC mRNA in mouse peritoneal macrophages is possibly related to the MAPK signalling pathway, not to NF‐κB activation. These data may contribute to unravelling some of the different mechanisms contrary to the anti‐inflammatory effect of 15d‐PGJ2.
Immune responses to dsRNA: Implications for gene silencing technologies
Authors: Karpala Adam J , Doran Tim J , Bean Andrew GD
Published: May 2005

Nucleic acid‐induced gene silencing, such as RNA interference (RNAi), induces a multitude of responses in addition to the knockdown of a gene. This is best understood in the context of the antiviral immune response, from which the processes of RNAi are thought to be derived. Viral challenge of a vertebrate host leads to an intricate series of responses that orchestrate antiviral immunity. The success of this multifaceted system in overcoming viral encounters hinges on complex pathogen–host interactions. One aspect of these interactions, the nucleic acid‐based immune response, is key to the successful resolution of a viral challenge. In particular, dsRNA, a nucleic acid associated with viral replication, is involved in numerous interactions contributing to induction, activation and regulation of antiviral mechanisms. Specifically, dsRNA is responsible for stimulating important protective responses, such as the activation of dicer‐related antiviral pathways, induction of type 1 IFN, and stimulation of dsRNA‐activated protein kinase and oligoadenylate synthetase. Furthermore, the modulation and shaping of this overall immune response is facilitated through nucleic acid interactions with pattern recognition receptors such as toll‐like receptor 3. These diverse dsRNA‐induced antiviral responses have implications for biotechnologies that use dsRNA to harness one arm of the host antiviral machinery for silencing a specific target gene. The interlinked nature of these response elements means that it may be difficult to completely isolate one element from the other arms of the antiviral response program of an organism. Thus, it is beneficial to understand all aspects of the immune response to dsRNA in order to manipulate these systems and minimize unwanted non‐specific effects.
Genome‐wide screening for gene function using RNAi in mammalian cells
Authors: Cullen Lara M , Arndt Greg M
Published: May 2005

Mammalian genome sequencing has identified numerous genes requiring functional annotation. The discovery that dsRNA can direct gene‐specific silencing in both model organisms and mammalian cells through RNA interference (RNAi) has provided a platform for dissecting the function of independent genes. The generation of large‐scale RNAi libraries targeting all predicted genes within mouse, rat and human cells, combined with the large number of cell‐based assays, provides a unique opportunity to perform high‐throughput genetics in these complex cell systems. Many different formats exist for the generation of genome‐wide RNAi libraries for use in mammalian cells. Furthermore, the use of these libraries in either genetic screens or genetic selections allows for the identification of known and novel genes involved in complex cellular phenotypes and biological processes, some of which underpin human disease. In this review, we examine genome‐wide RNAi libraries used in model organisms and mammalian cells and provide examples of how these information rich reagents can be used for determining gene function, discovering novel therapeutic targets and dissecting signalling pathways, cellular processes and complex phenotypes.
Effectiveness of short‐tongued bees as pollinators of apparently ornithophilous New Zealand mistletoes
Authors: ROBERTSON ALASTAIR W. , LADLEY JENNY J. , KELLY DAVE
Published: May 2005

Abstract  The flowers of two species of threatened New Zealand mistletoes (Peraxilla tetrapetala and Peraxilla colensoi, Loranthaceae) have explosive buds that do not open unless force is applied by birds or two species of native short‐tongued bees. Opened flowers are visited by a variety of birds and insects. Although both species of Peraxilla conform to a pollination syndrome of ornithophily, bees may be effective alternative pollinators. We investigated the effectiveness of bees and birds as pollinators of P. colensoi at one site and P. tetrapetala at two sites in the South Island. Bees and other insects outnumbered birds as flower visitors at all three sites. By excluding birds with wire cages, we showed that two bee species regularly open flowers of P. tetrapetala, but only rarely open flowers of P. colensoi. Few pollen grains were deposited when either birds or bees opened buds, so opening buds was not by itself sufficient for adequate pollination. Instead, pollen continued to accumulate over the next 6 or 7 days, even inside cages that excluded birds. Both populations of P. tetrapetala were regularly pollen‐limited, but in different ways. At Ohau, opened flowers gained enough pollen to produce seeds, but many buds were not opened and hence failed to set seed. In contrast, at Craigieburn, nearly all buds were opened, but many of these did not receive enough pollen. These results demonstrate that native bees can partially replace birds as pollinators of mistletoes, despite their apparent ornithophilous syndrome. Ongoing reductions in New Zealand forest bird numbers means that the service bees provide may be important for the long‐term future of these plants.
A Pilot Investigation of the Short‐Term Effects of an Interdisciplinary Intervention Program on Elderly Patients with Hip Fracture in Taiwan
Authors: Shyu Yea‐Ing Lotus , Liang Jersey , Wu Chi‐Chuan , Su Juin‐Yih , Cheng Huey‐Shinn , Chou Shih‐Wei , Yang Ching‐Tzu
Published: May 2005

Objectives: To evaluate an interdisciplinary intervention program for older people with hip fracture in Taiwan.

Design: Randomized experimental design.

Setting: A 3,800‐bed medical center in northern Taiwan.

Participants: Elderly patients with hip fracture (N=137) were randomly assigned to an experimental (n=68) or control (n=69) group.

Intervention: An interdisciplinary program of geriatric consultation, continuous rehabilitation, and discharge planning.

Measurements: Demographic and outcome variables were measured. Outcome variables included service utilization, clinical outcomes, self‐care abilities, health‐related quality‐of‐life (HRQOL) outcomes, and depressive symptoms.

Results: Subjects in the experimental group improved significantly more than those in the control group in the following outcomes: ratio of hip flexion 1 month after discharge (P=.02), recovery of previous walking ability at 1 month (P=.04) and 3 months (P=.001) after discharge, and activities of daily living at 1 month (P=.01) and 2 months (P=.001) after discharge. Three months after discharge, the experimental group showed significant improvement in peak force of the fractured limb's quadriceps (P=.04) and the following health outcomes: bodily pain (P=.03), vitality (P<.001), mental health (P=.02), physical function (P< .001), and role physical (P=.006). They also had fewer depressive symptoms (P=.008) 3 months after discharge.

Conclusion: This intervention program may benefit older people with hip fractures in Taiwan by improving their clinical outcomes, self‐care abilities, and HRQOL and by decreasing depressive symptoms within 3 months after discharge.
The Effect of Hospitalization with Medical Illnesses on the Suicide Risk in the Oldest Old: A Population‐Based Register Study
Authors: Erlangsen Annette , Vach Werner , Jeune Bernard
Published: May 2005

Objectives: To compare the effect of hospitalization with medical illnesses on the suicide risk in the oldest old (≥80) with that in the old (65–79) and middle‐aged (52–64) using nationwide data.

Design: Event‐history analysis using time‐varying covariates based on prospective individual‐level register data.

Setting: Population‐based record linkage.

Participants: All persons aged 52 and older living in Denmark during 1996 to 1998 (N=1,684,205).

Measurements: The studied event is completed suicide. The following time‐varying variables are included in the analysis: current age, hospitalization with medical illnesses within previous 2 years, and number of different medical diagnoses given at hospitalizations within previous 2 years. Relative suicide risks were calculated using event‐history analysis. The proportional attributable risk was calculated to assess to which extent hospitalization can explain the increased suicide risk in the oldest old.

Results: During the 3‐year study period, 1,184 persons committed suicide. Hospitalization with medical illnesses was associated with an increased suicide risk in the study population. The highest suicide rates were found in the oldest‐old men who had been hospitalized during the previous 2 years: 113 per 100,000 versus 80 per 100,000 in the general population of men aged 80 and older. Also, the oldest‐old women with a history of hospitalization had a higher suicide rate than their peer group. Nevertheless, the oldest old experienced a lower increase in risk after hospitalization than the middle‐aged. Experiencing three or more different diagnoses increased the risk further. Almost two‐thirds of the oldest old who committed suicide had experienced a medical hospitalization during a 2‐year period preceding the suicide. A substantial part of the greater suicide rate in the oldest old than in the middle‐aged can be attributed to the increased prevalence of medical hospitalization.

Conclusion: The oldest old who have been hospitalized with medical illnesses have a significantly higher suicide risk than people in the same age range with no hospitalization, although they experience a lower increase in risk after hospitalization than the middle‐aged. The increased prevalence of medical illnesses explains a part of the greater suicide risk with age, especially for the oldest old women. Considering that hospitalization with medical illness often precedes suicide in the oldest old, hospitalization may play an important role in identification of suicidal ideation in older people.
Timing of Hospice Referral and Families' Perceptions of Services: Are Earlier Hospice Referrals Better?
Authors: Rickerson Elizabeth , Harrold Joan , Kapo Jennifer , Carroll Janet T. , Casarett David
Published: May 2005

Objectives: To determine whether families of patients who enroll in hospice near the end of life believe that they receive less benefit from hospice services than families of patients who enroll earlier.

Design: Semistructured interviews at the time of hospice enrollment and 1 month after the patient's death.

Setting: This study was conducted at the Hospice of Lancaster County, Lancaster, PA, over a 12‐month period spanning 2003–2004.

Participants: First‐time hospice admissions.

Measurements: Interviews assessed the anticipated helpfulness of six hospice services (enrollment interviews) and the actual helpfulness of the same services (follow‐up interviews).

Results: Length of stay in hospice was associated with families' reports of the number of services provided (Spearman rho=0.34, P<.001), and with the mean helpfulness ratings for the services that were provided (Spearman rho=0.34, P<.001), although even with short lengths of stay, most families reported that they received the services that they anticipated and that those services were helpful.

Conclusion: These results indicate that families feel they receive greater benefits from longer lengths of stay in hospice. Future efforts to define an “optimal” length of stay in hospice should consider patients' and families' perceptions of the benefits that hospice offers.
Newly Reported Chronic Conditions and Onset of Functional Dependency
Authors: Wolff Jennifer L. , Boult Chad , Boyd Cynthia , Anderson Gerard
Published: May 2005

Objectives: To examine the relationship between newly reported chronic conditions and subsequent functional dependency in older adults.

Design: Three‐year cohort study.

Setting: United States.

Participants: A national sample of Americans age 65 and older (N=4,968) who participated in the Medicare Current Beneficiary Survey.

Measurements: Self‐reports of new physician diagnoses between baseline and 12 months and functional dependency onset at 12‐, 24‐, and 36‐month follow‐up. Functional dependency is defined as needing help with or being unable to perform one or more activities of daily living or residence in a long‐term care facility.

Results: After 12 months of follow‐up, 29.8% of participants reported one or more newly diagnosed conditions, increasing to 48.7% at 24 months and 61.3% at 36 months. Number of newly reported conditions was associated with greater probability of functional dependency; this association was strongest at 12 months. The odds of becoming functionally dependent were nearly twice as great in participants who reported one new chronic condition (odds ratio (OR)=1.9, 95% confidence interval (CI)=1.3–2.8), more than four times as great in those who reported two new chronic conditions (OR=4.3, 95%=CI 2.7–6.9), and 13 times as great in those who reported three or more new chronic conditions (OR=13.0, 95%=CI 6.5–26.3) as in those who reported no new chronic conditions. Newly reported dementia, stroke, psychological disorders, low body mass index, and obesity were significantly and consistently related to functional dependency throughout all 3 years of observation (OR=2.1–14.1).

Conclusion: Findings from this study demonstrate the strong relationship between newly diagnosed chronic conditions and functional dependency and highlight the potential benefit of prevention in older adults.
Cost Analysis of Nursing Home Registered Nurse Staffing Times
Authors: Dorr David A. , Horn Susan D. , Smout Randall J.
Published: May 2005

Objectives: To examine potential cost savings from decreased adverse resident outcomes versus additional wages of nurses when nursing homes have adequate staffing.

Design: A retrospective cost study using differences in adverse outcome rates of pressure ulcers (PUs), urinary tract infections (UTIs), and hospitalizations per resident per day from low staffing and adequate staffing nursing homes. Cost savings from reductions in these events are calculated in dollars and compared with costs of increasing nurse staffing.

Setting: Eighty‐two nursing homes throughout the United States.

Participants: One thousand three hundred seventy‐six frail elderly long‐term care residents at risk of PU development.

Measurements: Event rates are from the National Pressure Ulcer Long‐Term Care Study. Hospital costs are estimated from Medicare statistics and from charges in the Healthcare Cost and Utilization Project. UTI costs and PU costs are from cost‐identification studies. Time horizon is 1 year; perspectives are societal and institutional.

Results: Analyses showed an annual net societal benefit of $3,191 per resident per year in a high‐risk, long‐stay nursing home unit that employs sufficient nurses to achieve 30 to 40 minutes of registered nurse direct care time per resident per day versus nursing homes that have nursing time of less than 10 minutes. Sensitivity analyses revealed a robust set of estimates, with no single or paired elements reaching the cost/benefit equality threshold.

Conclusion: Increasing nurse staffing in nursing homes may create significant societal cost savings from reduction in adverse outcomes. Challenges in increasing nurse staffing are discussed.
Consensus Sequence-Based Scheme for Epidemiological Typing of Clinical and Environmental Isolates ofLegionella pneumophila
Authors: Gaia Valeria , Fry Norman K. , Afshar Baharak , Lück P. Christian , Meugnier Hélène , Etienne Jerome , Peduzzi Raffaele , Harrison Timothy G.
Published: May 2005

A previously described sequence-based epidemiological typing method for clinical and environmental isolates ofLegionella pneumophilaserogroup 1 was extended by the investigation of three additional gene targets and modification of one of the previous targets. Excellent typeability, reproducibility, and epidemiological concordance were determined for isolates belonging to both serogroup 1 and the other serogroups investigated. Gene fragments were amplified from genomic DNA, and PCR amplicons were sequenced by using forward and reverse primers. Consensus sequences are entered into an online database, which allows the assignment of individual allele numbers. The resulting sequence-based type or allelic profile comprises a string of the individual allele numbers separated by commas, e.g., 1,4,3,1,1,1, in a predetermined order, i.e.,flaA,pilE,asd,mip,mompS, andproA. The index of discrimination (D) obtained with these six loci was calculated following analysis of a panel of 79 unrelated clinical isolates. ADvalue of >0.94 was obtained, and this value appears to be sufficient for use in the epidemiological investigation of outbreaks caused byL. pneumophila. TheDvalue rose to 0.98 when the results of the analysis were combined with those of monoclonal antibody subgrouping. Sequence-based typing ofL. pneumophilais epidemiologically concordant and discriminatory, and the data are easily transportable. This consensus method will assist in the epidemiological investigation ofL. pneumophilainfections, especially travel-associated cases, by which it will allow a rapid comparison of isolates obtained in more than one country.
Antifungal Susceptibilities of
Candida
Species Causing Vulvovaginitis and Epidemiology of Recurrent Cases
Authors: Richter Sandra S. , Galask Rudolph P. , Messer Shawn A. , Hollis Richard J. , Diekema Daniel J. , Pfaller Michael A.
Published: May 2005

There are limited data regarding the antifungal susceptibility of yeast causing vulvovaginal candidiasis, since cultures are rarely performed. Susceptibility testing was performed on vaginal yeast isolates collected from January 1998 to March 2001 from 429 patients with suspected vulvovaginal candidiasis. The charts of 84 patients with multiple positive cultures were reviewed. The 593 yeast isolates were
Candida albicans
(
n
= 420),
Candida glabrata
(
n
= 112),
Candida parapsilosis
(
n
= 30),
Candida krusei
(
n
= 12),
Saccharomyces cerevisiae
(
n
= 9),
Candida tropicalis
(
n
= 8),
Candida lusitaniae
(
n
= 1), and
Trichosporon
sp. (
n
= 1). Multiple species suggesting mixed infection were isolated from 27 cultures. Resistance to fluconazole and flucytosine was observed infrequently (3.7% and 3.0%); 16.2% of isolates were resistant to itraconazole (MIC ≥ 1 μg/ml). The four imidazoles (econazole, clotrimazole, miconazole, and ketoconazole) were active: 94.3 to 98.5% were susceptible at ≤1 μg/ml. Among different species, elevated fluconazole MICs (≥16 μg/ml) were only observed in
C. glabrata
(15.2% resistant [R], 51.8% susceptible-dose dependent [S-DD]),
C. parapsilosis
(3.3% S-DD),
S. cerevisiae
(11.1% S-DD), and
C. krusei
(50% S-DD, 41.7% R, considered intrinsically fluconazole resistant). Resistance to itraconazole was observed among
C. glabrata
(74.1%),
C. krusei
(58.3%),
S. cerevisiae
(55.6%), and
C. parapsilosis
(3.4%). Among 84 patients with recurrent episodes, non-albicans species were more common (42% versus 20%). A ≥4-fold rise in fluconazole MIC was observed in only one patient with
C. parapsilosis
. These results support the use of azoles for empirical therapy of uncomplicated candidal vulvovaginitis. Recurrent episodes are more often caused by non-albicans species, for which azole agents are less likely to be effective.
Comparison ofBorrelia burgdorferiSensu Lato Strains Isolated from Specimens Obtained Simultaneously from Two Different Sites of Infection in Individual Patients
Authors: Ružić-Sabljić Eva , Arnež Maja , Logar Mateja , Maraspin Vera , Lotrič-Furlan Stanka , Cimperman Jože , Strle Franc
Published: May 2005

The aim of the present study was to analyze and compareBorreliastrains isolated from two different specimens obtained simultaneously from individual patients with Lyme borreliosis. Fifty such patients and 50 corresponding pairs ofBorreliaisolates (100 low-propagated strains) were subjected to genotypic and phenotypic analysis, including pulsed-field gel electrophoresis for species identification and plasmid profile determination and protein profile electrophoresis for the assessment of the presence and molecular masses of separated proteins. The strains were isolated from two distinct skin lesions (12 patients), skin and blood (28 patients), skin and cerebrospinal fluid (8 patients), and blood and cerebrospinal fluid (2 patients). Out of 100 isolates, 63 were typed asB. afzeliiand 37 asB. garinii. From each individual specimen only a singleBorreliaspecies was cultured. Comparison of 50Borreliastrain pairs isolated from two different specimens of an individual patient revealed that 12/50 (24%) patients were simultaneously infected with two differentBorreliastrains; in 3/50 (6%) patients strains differed at the species level, in 4 out of the remaining 47 (9%) patients a strain difference in plasmid profile was established, while 5 out of the remaining 43 (11%) patient strain pairs differed in regard to the protein profiles of the two concurrently isolated strains. The results of the present study indicate that human patients with Lyme borreliosis may simultaneously harbor differentB. burgdorferisensu lato strains.
Phylogeny and Identification of Enterococci by
atpA
Gene Sequence Analysis
Authors: Naser S. , Thompson F. L. , Hoste B. , Gevers D. , Vandemeulebroecke K. , Cleenwerck I. , Thompson C. C. , Vancanneyt M. , Swings J.
Published: May 2005

The relatedness among 91
Enterococcus
strains representing all validly described species was investigated by comparing a 1,102-bp fragment of
atpA
, the gene encoding the alpha subunit of ATP synthase. The relationships observed were in agreement with the phylogeny inferred from 16S rRNA gene sequence analysis. However,
atpA
gene sequences were much more discriminatory than 16S rRNA for species differentiation. All species were differentiated on the basis of
atpA
sequences with, at a maximum, 92% similarity. Six members of the
Enterococcus faecium
species group (
E. faecium
,
E. hirae
,
E. durans
,
E. villorum
,
E. mundtii
, and
E. ratti
) showed >99% 16S rRNA gene sequence similarity, but the highest value of
atpA
gene sequence similarity was only 89.9%. The intraspecies
atpA
sequence similarities for all species except
E. faecium
strains varied from 98.6 to 100%; the
E. faecium
strains had a lower
atpA
sequence similarity of 96.3%. Our data clearly show that
atpA
provides an alternative tool for the phylogenetic study and identification of enterococci.
Evaluation of 12 Commercial Tests and the Complement Fixation Test for
Mycoplasma pneumoniae
-Specific Immunoglobulin G (IgG) and IgM Antibodies, with PCR Used as the “Gold Standard”
Authors: Beersma Matthias F. C. , Dirven Kristien , van Dam Alje P. , Templeton Kate E. , Claas Eric C. J. , Goossens Herman
Published: May 2005

Serology and nucleic acid amplification are the main diagnostic tools for the diagnosis of
Mycoplasma pneumoniae
infection. Since no reference standard is generally accepted, serologic assays for
M. pneumoniae
have not been evaluated on a broad scale. In this study, 12 commercially available serologic assays (for immunoglobulin G [IgG] and IgM) and the complement fixation test (CFT) were evaluated by using
M. pneumoniae
DNA detection by real-time PCR as the “gold standard.” The assays tested were Platelia EIA (Bio-Rad), SeroMP EIA (Savyon), Serion classic EIA (Virion/Serion), Biotest EIA (Biotest), Ridascreen EIA (r-Biopharm), AniLabsystems EIA (Labsystems), Novum EIA (Novum Diagnostica), Diagnosys EIA (MP products), Genzyme/Virotech EIA, ImmunoWell EIA (Genbio), ImmunoCard EIA (Meridian), and SerodiaMycoII microparticle agglutination (Fujirebio). Serum samples (
n
= 46) from 27 PCR-positive patients with a known first day of disease and sera (
n
= 33) from PCR-negative controls were obtained from prospective studies of acute lower respiratory tract infections. Additionally, control sera (
n
= 63) from patients with acute viral or bacterial respiratory infections other than those caused by
M. pneumoniae
were tested. The results showed low specificities for both the Novum and the ImmunoCard IgM assays. The IgM assays with the best performances in terms of sensitivity and specificity were AniLabsystems (77% and 92%, respectively), SeroMP (71% and 88%, respectively), and CFT (65% and 97%, respectively). Good receiver operating characteristic areas under the curve were found for CFT (0.94), the Platelia assay (0.87), and the AniLabsystems assay (0.85). We conclude that there are few commercial serologic assays for the detection of
M. pneumoniae
infections with appropriate performances in terms of sensitivity and specificity and that PCR has become increasingly important for the diagnosis of
M. pneumoniae
infections in defined groups of patients.
Microarray-Based Detection of 90 Antibiotic Resistance Genes of Gram-Positive Bacteria
Authors: Perreten Vincent , Vorlet-Fawer Lorianne , Slickers Peter , Ehricht Ralf , Kuhnert Peter , Frey Joachim
Published: May 2005

A disposable microarray was developed for detection of up to 90 antibiotic resistance genes in gram-positive bacteria by hybridization. Each antibiotic resistance gene is represented by two specific oligonucleotides chosen from consensus sequences of gene families, except for nine genes for which only one specific oligonucleotide could be developed. A total of 137 oligonucleotides (26 to 33 nucleotides in length with similar physicochemical parameters) were spotted onto the microarray. The microarrays (ArrayTubes) were hybridized with 36 strains carrying specific antibiotic resistance genes that allowed testing of the sensitivity and specificity of 125 oligonucleotides. Among these were well-characterized multidrug-resistant strains of
Enterococcus faecalis
,
Enterococcus faecium
, and
Lactococcus lactis
and an avirulent strain of
Bacillus anthracis
harboring the broad-host-range resistance plasmid pRE25. Analysis of two multidrug-resistant field strains allowed the detection of 12 different antibiotic resistance genes in a
Staphylococcus haemolyticus
strain isolated from mastitis milk and 6 resistance genes in a
Clostridium perfringens
strain isolated from a calf. In both cases, the microarray genotyping corresponded to the phenotype of the strains. The ArrayTube platform presents the advantage of rapidly screening bacteria for the presence of antibiotic resistance genes known in gram-positive bacteria. This technology has a large potential for applications in basic research, food safety, and surveillance programs for antimicrobial resistance.
Genetic Background Affects Stability of
mecA
in
Staphylococcus aureus
Authors: Katayama Yuki , Robinson D. Ashley , Enright Mark C. , Chambers Henry F.
Published: May 2005

The staphylococcal methicillin resistance determinant,
mecA
, resides on a mobile genetic element, staphylococcus chromosomal cassette
mec
(SCC
mec
). The distribution of SCC
mec
in nature is limited to relatively few clonal complexes of related methicillin-resistant
Staphylococcus aureus
(MRSA). We have previously reported that some genetic backgrounds are restrictive of
mecA
and penicillin-binding protein 2a expression, which could account for the restricted clonal distribution of SCC
mec
in nature. In this study, we investigate the potential role of the host chromosome in the transformability and expression of
mecA
in 103 naturally occurring methicillin-susceptible
S. aureus
clinical isolates. The isolates, which had been genotyped previously by multilocus sequence typing, were classified into one of two mutually exclusive categories based on whether the isolates belonged to “major” MRSA lineages or to “other” lineages that are never or occasionally MRSA. We introduced
mecA
expressed on the low-copy-number plasmid pYK20 into each MSSA strain and assayed the phenotype of resistance to nafcillin by population analysis to assess the relationship between the stability of
mecA
expression and genetic background. Strains from the major MRSA lineages were more transformable with pYK20 and better able to maintain the plasmid and express resistance in comparison to strains from other lineages. These data support the hypothesis that the presence of
mecA
within relatively few clonal complexes is partly due to genetic factors that are permissive of
mecA
and its gene product.
Effect of Sequence Polymorphisms on Performance of Two Real-Time PCR Assays for Detection of Herpes Simplex Virus
Authors: Stevenson Jeffery , Hymas Weston , Hillyard David
Published: May 2005

Herpes simplex virus (HSV) is the most common cause of acquired, sporadic encephalitis in the United States. PCR identification of HSV in spinal fluid has become the diagnostic gold standard due to its sensitivity and potential for speed, replacing other methods such as culture. We developed a real-time PCR assay to detect HSV, using a new type of hybridization probe, the Eclipse probe. In this study, we ran 323 samples (171 positives and 152 negatives) with the Eclipse real-time PCR assay and compared these results with another PCR assay using gel detection. The real-time assay agreed with our reference method for 319 out of the 323 samples tested (99%). Using two different real-time PCR platforms, we discovered that SNPs within the amplicon's probe binding region that are used to distinguish HSV-1 from HSV-2 can decrease assay sensitivity. This problem is potentially a general one for assays using fluorescent probes to detect target amplification in a real-time format. While real-time PCR can be a powerful tool in the field of infectious disease, careful sequence evaluation and clinical validation are essential in creating an effective assay.
Prevalence of Pathogenicity Island II
CFT073
Genes among Extraintestinal Clinical Isolates of
Escherichia coli
Authors: Parham Nick J. , Pollard Samantha J. , Chaudhuri Roy R. , Beatson Scott A. , Desvaux Mickaël , Russell Michael A. , Ruiz Joaquim , Fivian Amanda , Vila Jordi , Henderson Ian R.
Published: May 2005

Uropathogenic
Escherichia coli
is the most common cause of urinary tract infection (UTI). Cystitis in women is by far the most common UTI; pyelonephritis in both sexes and prostatitis in men are more severe but are less frequent complaints. The ability of
E. coli
to cause UTI is associated with specific virulence determinants, some of which are encoded on pathogenicity islands (PAI). One such PAI (PAI II
CFT073
), of the prototypical uropathogenic
E. coli
strain CFT073, contains 116 open reading frames, including iron-regulated genes, carbohydrate biosynthetic genes, the serine protease autotransporter
picU
, a two-partner secretion system, a type I secretion system, mobility genes, and a large number of hypothetical genes. To determine the association of PAI II
CFT073
with UTI, PCR was used to examine the prevalence of the five virulence-associated loci among the ECOR collection and a collection of
E. coli
isolated from patients with cystitis, pyelonephritis, prostatitis, or septicemia. All PAI II
CFT073
loci were found to be more prevalent among the B2 phylogenetic group than any other group within the ECOR collection and among invasive prostatitis strains than were cystitis or pyelonephritis strains. These data support the theory that clinical isolates causing prostatitis are more virulent than those producing cystitis or pyelonephritis in women.
Real-Time PCR for Detection and Identification of
Plasmodium
spp
Authors: Mangold Kathy A. , Manson Rebecca U. , Koay Evelyn S. C. , Stephens Lindsey , Regner MaryAnn , Thomson Richard B. , Peterson Lance R. , Kaul Karen L.
Published: May 2005

Rapid and accurate detection of malaria parasites in blood is needed to institute proper therapy. We developed and used a real-time PCR assay to detect and distinguish four
Plasmodium
spp. that cause human disease by using a single amplification reaction and melting curve analysis. Consensus primers were used to amplify a species-specific region of the multicopy 18S rRNA gene, and SYBR Green was used for detection in a LightCycler instrument. Patient specimens infected at 0.01 to 0.02% parasitemia densities were detected, and analytical sensitivity was estimated to be 0.2 genome equivalent per reaction. Melting curve analysis based on nucleotide variations within the amplicons provided a basis for accurate differentiation of
Plasmodium falciparum
,
P. vivax
,
P. ovale
, and
P. malariae
. For assay validation, 358 patient blood samples from the National University Hospital in Singapore and Evanston Northwestern Healthcare in Illinois were analyzed. Of 76 blinded patient samples with a microscopic diagnosis of
P. falciparum
,
P. vivax
, or
P. ovale
infection, 74 (97.4%) were detected by real-time PCR, including three specimens containing mixed
P. falciparum
-
P. vivax
infections. No
Plasmodium
DNA was amplified in any of the 82 specimens sent for malaria testing but that were microscopically negative for
Plasmodium
infection. In addition, 200 blood samples from patients whose blood was collected for reasons other than malaria testing were also determined to be negative by real-time PCR. Real-time PCR with melting curve analysis could be a rapid and objective supplement to the examination of Giemsa-stained blood smears and may replace microscopy following further validation.
Sequencing of the
Chlamydophila psittaci ompA
Gene Reveals a New Genotype, E/B, and the Need for a Rapid Discriminatory Genotyping Method
Authors: Geens Tom , Desplanques Ann , Van Loock Marnix , Bönner Brigitte M. , Kaleta Erhard F. , Magnino Simone , Andersen Arthur A. , Everett Karin D. E. , Vanrompay Daisy
Published: May 2005

Twenty-one avian
Chlamydophila psittaci
isolates from different European countries were characterized using
ompA
restriction fragment length polymorphism,
ompA
sequencing, and major outer membrane protein serotyping. Results reveal the presence of a new genotype, E/B, in several European countries and stress the need for a discriminatory rapid genotyping method.
Distribution of IS1311and IS1245inMycobacterium aviumSubspecies Revisited
Authors: Johansen Tone Bjordal , Djønne Berit , Jensen Merete R. , Olsen Ingrid
Published: May 2005

We demonstrated that IS1245is not present inMycobacterium aviumsubsp.paratuberculosisby restriction fragment length polymorphism and that the designated three-banded bird pattern of IS1245inM. aviumsubsp.aviumconsists of one copy of IS1245and two copies of IS1311.Cross hybridization between the two elements can be avoided by using more specific probes.
Human
Cytomegalovirus
Infection of a Severe-Burn Patient: Evidence for Productive Self-Limited Viral Replication in Blood and Lung
Authors: Hamprecht Klaus , Pfau Mathias , Schaller Hans-Eberhard , Jahn Gerhard , Middeldorp Jaap M. , Rennekampff Hans-Oliver
Published: May 2005

To date, only seroepidemiological data are available on the role of human cytomegalovirus (HCMV) in patients with severe burns. We present the first longitudinal analysis of disseminated HCMV infection with a demonstration of self-limited productive viral replication identified in both the blood and lung of a burn patient.
BAXTER ALGEBRAS, STIRLING NUMBERS AND PARTITIONS
Authors: GUO LI
Published: May 2005

Recent developments of Baxter algebras have led to applications to combinatorics, number theory and mathematical physics. We relate Baxter algebras to Stirling numbers of the first and the second kinds, partitions and multinomial coefficients. This allows us to apply congruences from number theory to obtain congruences in Baxter algebras.
ANALYSIS ON THE INFLUENCE OF MODELING ERRORS ON SENSOR DYNAMIC COMPENSATION
Authors: WANG YONG , LIU ZHENGSHI , HE KAI , CHEN ENWEI , GE YUNJIAN
Published: May 2005

The relationship between modeling error of the system and widening multiple of the working frequency band was analyzed quantitatively and its spectrums were obtained. They offered a criterion to estimate the feasibility and effect in engineering practice of the compensating method to improve the sensor's dynamic characteristics, and proved that the compensating method has good reliability. Conclusions were drawn clearly: For a first-order system, the compensation effect is conspicuous, which only relates to modeling error but is independent of time constant; for a second-order system, the compensation effect is determined by modeling error and damping ratio. Within a wide range, if the damping ratio is larger, the modeling precision required to obtain the same compensation effect can be lower. Through quantitatively analysis, we found that the modeling precision in dynamic compensation with analogy or digital filter should not be so high as one is taken for granted currently.
Dynamic cerebral autoregulation during brain activation paradigms
Authors: Panerai Ronney B. , Moody Michelle , Eames Penelope J. , Potter John F.
Published: April 2005

Dynamic cerebral autoregulation (CA) describes the transient response of cerebral blood flow (CBF) to rapid changes in arterial blood pressure (ABP). We tested the hypothesis that the efficiency of dynamic CA is increased by brain activation paradigms designed to induce hemispheric lateralization. CBF velocity [CBFV; bilateral, middle cerebral artery (MCA)], ABP, ECG, and end-tidal Pco2 were continuously recorded in 14 right-handed healthy subjects (21–43 yr of age), in the seated position, at rest and during 10 repeated presentations (30 s on-off) of a word generation test and a constructional puzzle. Nonstationarities were not found during rest or activation. Transfer function analysis of the ABP-CBFV (i.e., input-output) relation was performed for the 10 separate 51.2-s segments of data during activation and compared with baseline data. During activation, the coherence function below 0.05 Hz was significantly increased for the right MCA recordings for the puzzle tasks compared with baseline values (0.36 ± 0.16 vs. 0.26 ± 0.13, P < 0.05) and for the left MCA recordings for the word paradigm (0.48 ± 0.23 vs. 0.29 ± 0.16, P < 0.05). In the same frequency range, significant increases in gain were observed during the puzzle paradigm for the right (0.69 ± 0.37 vs. 0.46 ± 0.32 cm·s−1·mmHg−1, P < 0.05) and left (0.61 ± 0.29 vs. 0.45 ± 0.24 cm·s−1·mmHg−1, P < 0.05) hemispheres and during the word tasks for the left hemisphere (0.66 ± 0.31 vs. 0.39 ± 0.15 cm·s−1·mmHg−1, P < 0.01). Significant reductions in phase were observed during activation with the puzzle task for the right (−0.04 ± 1.01 vs. 0.80 ± 0.86 rad, P < 0.01) and left (0.11 ± 0.81 vs. 0.57 ± 0.51 rad, P < 0.05) hemispheres and with the word paradigm for the right hemisphere (0.05 ± 0.87 vs. 0.64 ± 0.59 rad, P < 0.05). Brain activation also led to changes in the temporal pattern of the CBFV step response. We conclude that transfer function analysis suggests important changes in dynamic CA during mental activation tasks.
Proinflammatory phenotype of vascular smooth muscle cells: role of efficient Toll-like receptor 4 signaling
Authors: Yang Xin , Coriolan Daniel , Murthy Vanishree , Schultz Kelly , Golenbock Douglas T. , Beasley Debbie
Published: April 2005

Recent evidence supports a role of Toll-like receptor (TLR) signaling in the development of atherosclerotic lesions. In this study, we tested whether TLR4 signaling promotes a proinflammatory phenotype in human and mouse arterial smooth muscle cells (SMC), characterized by increased cytokine and chemokine synthesis and increased TLR expression. Human arterial SMC were found to express mRNA encoding TLR4 and the TLR4-associated molecules MD-2 and CD14 but not TLR2 mRNA. Mouse aortic SMC, on the other hand, expressed both TLR2 and TLR4 mRNA constitutively. Human SMC derived from the coronary artery, but not those from the pulmonary artery, were found to express cell surface-associated CD14. Low concentrations (ng/ml) of Escherichia coli LPS, the prototypical TLR4 agonist, markedly stimulated extracellular regulated kinase 1/2 (ERK1/2) activity, induced release of monocyte-chemoattractant protein-1 (MCP-1) and interleukin (IL)-6, and stimulated IL-1α expression in human aortic SMC, and exogenous CD14 enhanced these effects. Expression of a dominant negative form of TLR4 in human SMC attenuated LPS -induced ERK1/2 and MCP-1 release. LPS was a potent inducer of NF-κB activity, ERK1/2 phosphorylation, MCP-1 release, and TLR2 mRNA expression in wild-type mice but not in TLR4-signaling deficient mouse aortic SMC. These studies show that TLR4 signaling promotes a proinflammatory phenotype in vascular smooth muscle cells (VSMC) and suggest that VSMC may potentially play an active role in vascular inflammation via the release of chemokines, proinflammatory cytokines, and increased expression of TLR2.
Baroreflex control of muscle sympathetic nerve activity in postural orthostatic tachycardia syndrome
Authors: Muenter Swift N. , Charkoudian N. , Dotson R. M. , Suarez G. A. , Low P. A.
Published: April 2005

Postural orthostatic tachycardia syndrome (POTS) is characterized by excessive tachycardia during orthostasis. To test the hypothesis that patients with POTS have decreased sympathetic neural responses to baroreflex stimuli, we measured heart rate (HR) and muscle sympathetic nerve activity (MSNA) responses to three baroreflex stimuli including vasoactive drug boluses (modified Oxford technique), Valsalva maneuver, and head-up tilt (HUT) in POTS patients and healthy control subjects. The MSNA response to the Valsalva maneuver was significantly greater in the POTS group (controls, 26 ± 7 vs. POTS, 48 ± 6% of baseline MSNA/mmHg; P = 0.03). POTS patients also had an exaggerated MSNA response to 30° HUT (controls, 123 ± 24 vs. POTS, 208 ± 30% of baseline MSNA; P = 0.03) and tended to have an exaggerated response to 45° HUT (controls, 137 ± 27 vs. POTS, 248 ± 58% of baseline MSNA; P = 0.10). Sympathetic baroreflex sensitivity calculated during administration of the vasoactive drug boluses also tended to be greater in the POTS patients; however, this did not reach statistical significance ( P = 0.15). Baseline MSNA values during supine rest were not different between the groups (controls, 23 ± 4 vs. POTS, 16 ± 5 bursts/100 heartbeats; P = 0.30); however, resting HR was significantly higher in the POTS group (controls, 58 ± 3 vs. POTS, 82 ± 4 beats/min; P = 0.0001). Our results suggest that POTS patients have exaggerated MSNA responses to baroreflex challenges compared with healthy control subjects, although resting supine MSNA values did not differ between the groups.