bluuebunny/crossref_metadata_2025 huggingface dataset:
Perceptions of behaviour and patterns of exclusion: Gypsy Traveller students in English secondary schools
Authors: Derrington Chris
Published: May 2005

This paper draws on a recent and unique longitudinal study of Gypsy Traveller students in fifteen local education authorities in England. Gypsy Traveller students are less likely to complete secondary education than any other minority ethnic group and it is estimated that up to 12 000 young Travellers in England are not registered at any secondary school. The study tracked a sample of 44 students over a three‐year period, using a multi‐perspective, phenomenological design to explore the complex interplay of attitudes, expectations and relationships that might impact on secondary school engagement and retention. Findings indicate that, although the students’ behaviour was perceived to be good by their primary school teachers, problems began to emerge during the first year at secondary school and twenty‐four pupils had self‐excluded by the age of fourteen. Furthermore, almost one in three of the students in the study were temporarily excluded by their schools on at least one occasion during the study. The research suggests that patterns of resistance to secondary schooling should not be solely attributed to cultural influences. In many cases, parents expressed positive perceptions of their child's school until relationships broke down. Above all, parents and students want assurance that racism will be challenged and dealt with effectively.
The attributes of a successful Learning Support Teacher in Australian inclusive classrooms
Authors: Fielding‐Barnsley Ruth
Published: May 2005

This study used focus groups to investigate 18 general classroom teachers’ and 12 learning support teachers’ conceptions of the attributes of successful learning support teachers. Based on the attributes emerging from the focus groups, a 20‐item questionnaire was designed consisting of statements in four categories: Knowledge and Experience; Organisational Skills; Communication; and Personal Traits. The questionnaire was completed by 117 teachers. Results indicated that there was agreement between the focus groups and teachers who responded to the questionnaire. However, there was a mismatch between learning support teachers and classroom teachers as to what they regarded as being the most important attributes. Learning support teachers indicated that Knowledge and Experience was the most important attribute but this same category was not rated as highly by general classroom teachers. In the total sample of teachers, Personal Traits was rated significantly higher than the other three categories. Communication was ranked second which was significantly higher than Organisational Skills.
Behavioral Headache Treatment: History, Review of the Empirical Literature, and Methodological Critique
Authors: Rains Jeanetta C. , Penzien Donald B. , McCrory Douglas C. , Gray Rebecca N.
Published: May 2005

Theoretical developments and burgeoning research on stress and illness in the mid‐20th century yielded the foundations necessary to conceptualize headache as a psychophysiological disorder and eventually to develop and apply contemporary behavioral headache treatments. Over the past three decades, these behavioral headache treatments (relaxation training, biofeedback, cognitive‐behavioral therapy, and stress‐management training) have amassed a sizeable evidence base. Meta‐analytic reviews of the literature consistently have shown behavioral interventions to yield 35% to 55% improvements in migraine and tension‐type headache and that these outcomes are significantly superior to control conditions. The strength of the evidence has lead many professional practice organizations to recommend use of behavioral headache treatments alongside pharmacologic treatments for primary headache. The present overview was prepared as a companion article to and intended to provide a background for the Guidelines for Trials of Behavioral Treatments for Recurrent Headache also published within this journal supplement. This article begins with a synopsis of key historical developments leading to our current conceptualization of migraine and tension‐type headache as psychophysiological disorders amenable to behavioral intervention. The evolution of the behavioral headache literature is discussed, exemplified by publication trends in the journal Headache. Leading empirically‐based behavioral headache interventions are described, and meta‐analytic reviews examining the migraine and tension‐type headache literatures are summarized, compared, and contrasted. A critique of the methodological quality of the clinical trials literature is presented, highlighting the strengths and weaknesses in relation to recruitment and selection of patients, sample size and statistical power, the use of a credible control, and the reproducibility of the study interventions in clinical practice.
CULTURE, DIVORCE, AND FAMILY MEDIATION IN HONG KONG
Authors: Sullivan Patricia L.
Published: May 2005

Over the past three decades, Hong Kong has witnessed unprecedented economic prosperity, a decrease in family solidarity, an increase in social problems, and a dramatic rise in the incidence of divorce. Under colonial rule for 150 years, which ended with the return of sovereignty to China in 1997, the identity of the Hong Kong Chinese is a combination of traditional Chinese and modern Western culture. Despite Western influences, Chinese cultural heritage remains the major source of influence in all aspects of life. This article presents a summary of traditional Chinese culture, characteristics of contemporary Hong Kong society, factors believed to influence the high divorce rate, and the impact of divorce on Hong Kong families. The development, use, acceptability, and effectiveness of mediation in Hong Kong is reviewed, and suggestions for adapting it to Chinese families are proposed. Direction for future development of mediation in Hong Kong focuses on the need for further research to develop a scientifically sound knowledge base that will help to inform culturally competent mediation practice.
USING EXTERNSHIPS TO INTRODUCE FAMILY LAW STUDENTS TO NEW PROFESSIONAL ROLES
Authors: Steegh Nancy Ver
Published: May 2005

Law schools are seeking ways to familiarize family law students with exciting new professional roles arising in connection with family court reform. This article describes the family law externship program at William Mitchell College of Law which includes a classroom component allowing students placed in different practice settings to compare and contrast their experiences. The program assists students in making career choices and enhances their ability to counsel future clients.
SUPER‐SIZED KIDS:
Authors: Patel Deena
Published: May 2005

Morbid obesity is an unfortunate problem that is only becoming worse everyday. The alarming aspect of it is that it is affecting people at a much earlier age; thus, young children are becoming morbidly obese and are experiencing the same health problems as middle‐aged adults. The first section of this note defines morbid obesity and its causes and trends. Section two describes child neglect in general and then distinguishes medical neglect. Section three discusses education and prevention, which are both crucial steps in the struggle with weight‐loss and weight‐gain. Section four summarizes the beneficial aspects of removing a morbidly obese child who is in a life‐threatening position from his or her parents. Section five highlights case‐studies from California, Iowa, Indiana, New Mexico, and Texas, where children have either died or were removed from their families because of morbid obesity. Section six briefly touches upon similar child neglect scenarios where removal is permissible and compares those situations with those of morbidly obese children. The seventh section considers some credible counterarguments to government intervention, and the final section suggests some recommendations on how to prevent the morbid obesity crisis from becoming worse than it already is. Too many people are dying from weight‐related problems already, yet morbid obesity is preventable! This severe state of obesity is something that can be controlled and prevented, but only if parents take an active role in their child's diet and exercise.
Nondaily Hormonal Contraception: Considerations in Contraceptive Choice and Patient Counseling
Authors: Freeman Sarah
Published: May 2005

Purpose

To review currently available choices for non‐daily hormonal contraception, considering efficacy, safety, patient counseling issues, and appropriate patient selection.

Data Sources

Worldwide medical literature and the individual products' prescribing information.

Conclusions

Patients and clinicians have many nondaily hormonal contraceptive options available—from Depo‐Provera quarterly injection, which has been available in the United States for over 10 years, to several new entries (Mirena 5‐year intrauterine system, Lunelle monthly injection, NuvaRing monthly intravaginal ring, and Ortho Evra weekly transdermal patch). All these options offer high efficacy and enhanced convenience for many patients over daily oral contraceptives (OCs). Barriers to use of these agents may include patients’ lack of information as well as fear or misconceptions regarding the hormones and methods. All of these can be addressed with adequate patient counseling and open dialogue. The clinician and patient need to be well‐informed regarding these options so that they can work together and identify the best contraceptive fit for the patient—with the ultimate goal being to increase patient satisfaction and adherence and, thus, avoid unintended pregnancy.

Implications for Practice

Despite the efficacy of OCs, missed pills are quite common and contribute to unintended pregnancy. Many women in all population categories would benefit from the convenience and reliability of nondaily hormonal contraceptives. The highest efficacy rates with typical use are associated with agents that require minimal user participation (i.e., Depo‐Provera, Mirena).Compared to daily regimens, all nondaily options offer increased convenience and may contribute to improved patient adherence.However, barriers to use may exist.Patient fears regarding use of hormones can be minimized by discussing the long‐term safety of hormonal contraceptives. (The data are predominantly derived from Depo‐Provera and OCs because these agents have been available in the United States and in the rest of the world for much longer than the newer nondaily options.) Patient counseling and appropriate expectations regarding changes in menstrual pattern have been demonstrated to further enhance patient adherence to therapy. Finally, patient lifestyle preferences must be considered. The finding that many women are comfortable with or even prefer amenorrhea, which is associated with options such as Depo‐Provera, highlights how important it is for clinicians to avoid making assumptions about a patient's contraceptive preferences. Rather, clinicians and patients should exchange information through an open dialogue. For the majority of patients, nondaily hormonal contraceptives should be considered and offered as first‐line options.
Identifying the Drug Seeker: The Advanced Practice Nurse's Role in Managing Prescription Drug Abuse
Authors: Gerhardt Annie M.
Published: May 2005

Purpose

To provide advanced practice nurses (APNs) with a definition of drug‐seeking behaviors, to identify the common signs and symptoms of drug‐seeking behaviors, to furnish tips for managing interactions with and treatment of the drug‐seeking patient, and to describe pre‐scription parameters that will help APNs avoid legal ramifications.

Data Sources

A review of the current scientific and clinical literature regarding interaction with and treatment of patients who abuse prescription drugs.

Conclusions

A basic understanding of drug‐seeking behaviors and adherence to appropriate procedures for prescribing controlled medications are essential for APNs to maintain their safe prescribing habits; carry on with quality, effective care; and continue the advancement of their profession.

Implications for Practice

As primary care providers, APNs must be aware of the potential complications resulting from interacting with patients who may be seeking prescription drugs for recreational or illicit use, rather than for legitimate reasons.
Down Syndrome With an Unusual Etiology: Case Report and Review
Authors: Pastva Mattie , Corwin Elizabeth J. , Morin Karen
Published: May 2005

Purpose

To present the genetic etiology of an unusual case of Down syndrome, arising from translocation of chromosome 21 to chromosome 9; to discuss advanced genetic diagnostic techniques, focusing on how pinpointing a specific genetic mistake can influence treatment and outcome;and to review the role of the nurse practitioner (NP) in caring for families of children with Down syndrome.

Data Sources

Case report, literature review, and advanced genetic techniques.

Conclusions

NPs often answer patient questions regarding genetic abnormalities and risk factors for transmission of genetic errors to offspring. Advanced genetic analysis allows for the diagnosis of unusual causes of common genetic disorders. The ability to identify a specific genetic mistake and to predict the scope of its effect on an individual offers health care providers the opportunity to develop a case‐by‐case plan of care.

Implications for Practice

NPs have a responsibility to be informed about the processes involved in the transmission of genetic errors and about the subtleties of various expressions of a genetic disorder. The availability of new genetic‐testing techniques allows NPs to individualize patient care and family teaching.
Nurse Practitioners' and Physicians' Care Activities and Clinical Outcomes With an Inpatient Geriatric Population
Authors: Lambing Angela Y. , Adams Denise L. C. , Fox Dorothy H. , Divine George
Published: May 2005

Objective

Less is known about nurse practitioners' (NPs') effectiveness in acute care than about their effectiveness in outpatient settings. This study investigated care activities and clinical outcomes for hospitalized geriatric patients treated by NPs compared with those treated by intern and resident physicians.

Data Sources

A descriptive comparative research design involved random selection of 100 inpatient geriatric patients and a convenience sample of 17 professional providers who staffed three hospital units. A 1‐month study period produced retrospective and prospective data for analysis.

Self‐reports concerning 10 primary activity categories indicated that NPs spent a higher percentage of time doing progress notes and care planning than did physicians (28% versus 15%, p=.011) and that physicians spent more time on literature reviews (5% versus 1%, p=.008). When prioritizing care activities, NPs ranked advance directive discussion higher than did physicians (2nd versus 7th, p=.036), a difference confirmed by medical record documentation. Physicians were more attentive to functional status (1st versus 3rd, p=.023), but medical record documentation showed NPs to be more attentive to physical and occupational therapy referrals (p=.001). Analysis of 13 independent organ areas revealed that NPs cared for more musculoskeletal (p=.036) and psychiatric (p=.005) problems. Physicians cared for more cardiac patients (p=.001). NPs’ patients were older (p=.022) and sicker at admission (p <. 001) and discharge (p <. 001). Charges per length of stay were lower (p <. 001) for the physician provider group, and patients in that group had shorter stays (p <. 001). Readmission and mortality rates were similar.

Implications for Practice

NPs provide effective care to hospitalized geriatric patients, particularly to those who are older and sicker.
Clinical Evaluation and Patterns of Chemotherapy‐Induced Peripheral Neuropathy
Authors: Visovsky Constance , Daly Barbara J.
Published: May 2005

Purpose

To determine the pattern of change in peripheral nerve function (as measured by vision, hearing, deep tendon reflexes, vibratory sense, cutaneous sensation, gait and balance, muscle strength, and orthostatic blood pressure) occurring among individuals receiving cancer treatment with known neurotoxic agents.

Data Sources

A convenience sample of 16 participants with cancer who were receiving chemotherapy had their peripheral nerve function assessed at baseline and at 4 weeks and 12 weeks of treatment. Data were analyzed using plots and regression slopes to determine change over time in clinical measures of peripheral nerve function. Outcome variables were vision, hearing, deep tendon reflexes, vibratory sense, cutaneous sensation, gait and balance, muscle strength, and changes in orthostatic blood pressure.

Conclusions

This is the first prospective study to use comprehensive clinical measures of peripheral nerve and muscle changes resulting from combination chemotherapy or a biotherapy regimen. Alterations in vision, hearing, deep tendon reflexes, vibratory sense, cutaneous sensation, balance, muscle strength, and orthostatic blood pressure were noted, but gait remained unchanged.

Implications for Practice

This study provides beginning evidence of the need for careful, ongoing assessment of treatment‐induced peripheral neuropathy. Standardized clinical practice procedures that incorporate patient evaluation for peripheral neuropathy must still be developed.In addition, we must educate our patients about the functional changes they may expect, and we must develop strategies to assist them in managing limitations that they experience as a result of peripheral neuropathy.
Attitudes of Registered Nurses Toward Nurse Practitioners
Authors: Gooden Janet M. , Jackson Elaine
Published: May 2005

Purpose

Registered nurses (RNs)work closely with nurse practitioners (NPs) in all facets of patient care and often have a direct influence on the patient's perception of the professional who is directing the care, and a patient's perceptions may affect clinical outcomes. However, few studies have been conducted to measure RN acceptance of the NP. The authors surveyed RNs in southern Illinois to assess their attitudes concerning the level of care provided by NPs. Southern Illinois was chosen because of the limited number of NPs in the area and because of concerns that limited exposure might negatively influence RN attitudes about the NP role.

Data Collection

A 26‐item research questionnaire, previously used with school nurses, was modified to reflect RNs in general. Seven items related to demographic information, and 19 items created a five‐response Likert‐formatted scale (r=.93). The questionnaire was mailed to 500 randomly selected RNs from the 11 southernmost counties of Illinois.

Conclusions

The results showed support of the NP role. RNs believed that NPs were knowledgeable, competent health care providers. RNs were also comfortable working with NPs and often consulted them for advice and information. They saw the role of the NP as a positive addition to the health care team.

Implications for Practice

One identified concern was that scores indicated RNs felt that they received limited respect from NPs. Although the scores were high in this category, some RNs had concerns that NPs might not respect or understand the difficulty of the RN role in providing patient care. NPs need to recognize their collegial and professional ties to RNs and should take time to provide positive feedback to RNs as they collaborate to provide health care. Such behavior would only solidify the health team approach.
Impingement Heat Transfer: Correlations and Numerical Modeling
Authors: Zuckerman Neil , Lior Noam
Published: May 2005

Uses of impinging jet devices for heat transfer are described, with a focus on cooling applications within turbine systems. Numerical simulation techniques and results are described, and the relative strengths and drawbacks of the k-ε,k-ω, Reynolds stress model, algebraic stress models, shear stress transport, and v2f turbulence models for impinging jet flow and heat transfer are compared. Select model equations are provided as well as quantitative assessments of model errors and judgments of model suitability.
Turbulent Transport in Film Cooling Flows
Authors: Kohli Atul , Bogard David G.
Published: May 2005

This experimental study was performed on a single row of round holes with a 35° surface angle, representing film cooling geometry commonly used in turbine engines. Simultaneous velocity and temperature measurements were made using a cold-wire in conjunction with a LDV. The experimentally determined cross correlations provide a direct indication of the extent of turbulent transport of heat and momentum in the flow, which in turn governs dispersion of the film cooling jet. Actual engine environments have elevated mainstream turbulence levels that can severely reduce the cooling capability of film cooling jets. Clearly, the turbulent transport for very high mainstream turbulence is expected to be markedly different than that with low mainstream turbulence, and would improve our understanding of the mechanisms involved in the dispersion of film cooling jets. Experimental cross-correlation data was obtained for two vastly different freestream turbulence levels (0.6% and 20%) in this study. For this purpose, eddy diffusivities for momentum and heat transport were estimated from the measured data. These results will help develop new turbulence models and also explain why gradient diffusion based models do not give good predictions relative to experimental results.
A Fully Implicit Hybrid Solution Method for a Two-Phase Thermal-Hydraulic Model
Authors: Mousseau Vincent A.
Published: May 2005

This paper will present a hybrid solution algorithm for the two-phase flow equations coupled to wall heat conduction. The partial differential equations in the physical model are the same as in RELAP5. The hybrid solution algorithm couples two solution methods, the solution method currently employed by RELAP5 and an implicitly balanced solution method. The resulting hybrid solution method is both fast and accurate. Results will be presented that show when accuracy and CPU time are considered simultaneously that there are ranges when the hybrid solution algorithm is preferred over the RELAP5 solution method.
Film-Cooling Effectiveness on a Gas Turbine Blade Tip Using Pressure-Sensitive Paint
Authors: Ahn Jaeyong , Mhetras Shantanu , Han Je-Chin
Published: May 2005

Effects of the presence of squealer, the locations of the film-cooling holes, and the tip-gap clearance on the film-cooling effectiveness were studied and compared to those for a plane (flat) tip. The film-cooling effectiveness distributions were measured on the blade tip using the pressure-sensitive paint technique. Air and nitrogen gas were used as the film-cooling gases, and the oxygen concentration distribution for each case was measured. The film-cooling effectiveness information was obtained from the difference of the oxygen concentration between air and nitrogen gas cases by applying the mass transfer analogy. Plane tip and squealer tip blades were used while the film-cooling holes were located (a) along the camber line on the tip or (b) along the tip of the pressure side. The average blowing ratio of the cooling gas was 0.5, 1.0, and 2.0. Tests were conducted with a stationary, five-bladed linear cascade in a blow-down facility. The free-stream Reynolds number, based on the axial chord length and the exit velocity, was 1,138,000, and the inlet and the exit Mach numbers were 0.25 and 0.6, respectively. Turbulence intensity level at the cascade inlet was 9.7%. All measurements were made at three different tip-gap clearances of 1%, 1.5%, and 2.5% of blade span. Results show that the locations of the film-cooling holes and the presence of squealer have significant effects on surface static pressure and film-cooling effectiveness, with film-cooling effectiveness increasing with increasing blowing ratio.
IMP Dehydrogenase from the Protozoan ParasiteToxoplasma gondii
Authors: Sullivan William J. , Dixon Stacy E. , Li Catherine , Striepen Boris , Queener Sherry F.
Published: May 2005

The opportunistic apicomplexan parasiteToxoplasma gondiidamages fetuses in utero and threatens immunocompromised individuals. The toxicity associated with standard antitoxoplasmal therapies, which target the folate pathway, underscores the importance of examining alternative pharmacological strategies. Parasitic protozoa cannot synthesize purines de novo; consequently, targeting purine salvage enzymes is a plausible pharmacological strategy. Several enzymes critical to purine metabolism have been studied inT. gondii, but IMP dehydrogenase (IMPDH), which catalyzes the conversion of IMP to XMP, has yet to be characterized. Thus, we have cloned the gene encoding this enzyme inT. gondii. Northern blot analysis shows that two IMPDH transcripts are present inT. gondiitachyzoites. The larger transcript contains an open reading frame of 1,656 nucleotides whose deduced protein sequence consists of 551 amino acids (TgIMPDH). The shorter transcript is an alternative splice product that generates a 371-amino-acid protein lacking the active-site flap (TgIMPDH-S). When TgIMPDH is expressed as a recombinant protein fused to a FLAG tag, the fusion protein localizes to the parasite cytoplasm. Immunoprecipitation with anti-FLAG was employed to purify recombinant TgIMPDH, which converts IMP to XMP as expected. Mycophenolic acid is an uncompetitive inhibitor relative to NAD+, with a intercept inhibition constant (Kii) of 0.03 ± 0.004 μM. Tiazofurin and its seleno analog were not inhibitory to the purified enzyme, but adenine dinucleotide analogs such as TAD and the nonhydrolyzable β-methylene derivatives of TAD or SAD were inhibitory, withKiivalues 13- to 60-fold higher than that of mycophenolic acid.
Pharmacodynamic Modeling of the Evolution of Levofloxacin Resistance in
Staphylococcus aureus
Authors: Campion Jeffrey J. , Chung Philip , McNamara Patrick J. , Titlow William B. , Evans Martin E.
Published: May 2005

Previously, we demonstrated the importance of low-level-resistant variants to the evolution of resistance in
Staphylococcus aureus
exposed to ciprofloxacin in an in vitro system and developed a pharmacodynamic model which predicted the emergence of resistance. Here, we examine and model the evolution of resistance to levofloxacin in
S. aureus
exposed to simulated levofloxacin pharmacokinetic profiles. Enrichment of subpopulations with mutations in
grlA
and low-level resistance varied with levofloxacin exposure. A regimen producing average steady-state concentrations (
C
avg ss
) just above the MIC selected
grlA
mutants with up to 16-fold increases in the MIC and often additional mutations in
grlA/grlB
and
gyrA
. A regimen providing
C
avg ss
between the MIC and the mutant prevention concentration (MPC) suppressed bacterial numbers to the limit of detection and prevented the appearance of bacteria with additional mutations or high-level resistance. Regimens producing
C
avg ss
above the MPC appeared to eradicate low-level-resistant variants in the cultures and prevent the emergence of resistance. There was no relationship between the time concentrations remained between the MIC and the MPC and the degree of resistance or the presence or type of mutations that appeared in
grlA/B
or
gyrA
. Our pharmacodynamic model described the growth and levofloxacin killing of the parent strains and the most resistant
grlA
mutants in the starting cultures and correctly predicted conditions that enrich subpopulations with low-level resistance. These findings suggest that the pharmacodynamic model has general applicability for describing fluoroquinolone resistance in
S. aureus
and further demonstrate the importance of low-level-resistant variants to the evolution of resistance.
Detection of
rpoB
Mutations Associated with Rifampin Resistance in
Mycobacterium tuberculosis
Using Denaturing Gradient Gel Electrophoresis
Authors: McCammon Mark T. , Gillette John S. , Thomas Derek P. , Ramaswamy Srinivas V. , Graviss Edward A. , Kreiswirth Barry N. , Vijg Jan , Quitugua Teresa N.
Published: May 2005

Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with rifampin (RIF) resistance in the
rpoB
gene of
Mycobacterium tuberculosis
. DGGE scans for mutations across large regions of DNA and is comparable to DNA sequencing in detecting DNA alterations. Specific mutations are often recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint. Five DGGE primer sets that scanned for DNA alterations across 775 bp of
rpoB
were developed. These primer sets were used to scan
rpoB
for DNA alterations in 296
M. tuberculosis
patient isolates from the United States-Mexico border states of Texas and Tamaulipas. The most useful primer set scanned for mutations in the rifampin resistance-determining region (RRDR) and detected mutations in 95% of the RIF-resistant isolates compared to 2% of RIF-susceptible isolates. Thirty-four different alterations were observed within the RRDR by DGGE. In addition, isolates harboring mixtures of DNA within
rpoB
were readily detected by DGGE. A second PCR primer set was used to detect the V146A mutation in 5 to 7% of RIF-resistant isolates. A third primer set was used to detect mutations in 3% of RIF-resistant isolates, some of which also harbored mutations in the RRDR. Only 1 of 153 RIF-resistant isolates did not have a detectable
rpoB
mutation as determined by DGGE and DNA sequencing. These results demonstrate the power and usefulness of DGGE in detecting mutations associated with drug resistance in
M. tuberculosis
.
Detection by Denaturing Gradient Gel Electrophoresis of
pncA
Mutations Associated with Pyrazinamide Resistance in
Mycobacterium tuberculosis
Isolates from the United States-Mexico Border Region
Authors: McCammon Mark T. , Gillette John S. , Thomas Derek P. , Ramaswamy Srinivas V. , Rosas Ishmael I. , Graviss Edward A. , Vijg Jan , Quitugua Teresa N.
Published: May 2005

Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with pyrazinamide (PZA) resistance in the
pncA
gene of
Mycobacterium tuberculosis
. DGGE scans for mutations across large regions of DNA and rivals sequencing in its ability to detect DNA alterations. Specific mutations can often be recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint. Five PCR target fragments were designed to scan for DNA alterations across 600 bp of
pncA
in 181
M. tuberculosis
isolates from patients residing in the U.S-Mexico border states of Texas and Tamaulipas, respectively. A region of
pncA
was observed with a high GC content and a melting temperature approaching 90°C that was initially refractory to denaturation, and a DGGE target fragment was specifically designed to detect mutations in this region. DGGE detected
pncA
mutations in 82 of 83 PZA-resistant isolates. By contrast, only 1 of 98 PZA-susceptible isolates harbored a detectable DNA alteration. The
pncA
gene was sequenced from 41 isolates, and 32 DNA alterations in 32 PZA-resistant isolates were identified, including 11 new mutations. DGGE also detected nine isolates whose susceptibility to PZA appeared to be incorrect, and DNA sequencing confirmed these apparent errors in drug susceptibility testing. These results demonstrate the power and usefulness of DGGE in detecting mutations associated with PZA resistance in
M. tuberculosis
.
In Vitro Antiviral Activity and Single-Dose Pharmacokinetics in Humans of a Novel, Orally Bioavailable Inhibitor of Human Rhinovirus 3C Protease
Authors: Patick Amy K. , Brothers Mary A. , Maldonado Fausto , Binford Susan , Maldonado Oscar , Fuhrman Shella , Petersen Annkatrin , Smith George J. , Zalman Leora S. , Burns-Naas Leigh Ann , Tran Jonathan Q.
Published: May 2005

(E)-(S)-4-((S)-2-{3-[(5-methyl-isoxazole-3-carbonyl)-amino]-2-oxo-2H-pyridin-1-yl}-pent-4-ynoylamino)-5-((S)-2-oxo-pyrrolidin-3-yl)-pent-2-enoic acid ethyl ester (Compound 1) is a novel, irreversible inhibitor of human rhinovirus (HRV) 3C protease {inactivation rate constant (K
obs
/[I]) of 223,000 M
−1
s
−1
}. In cell-based assays, Compound 1 was active against all HRV serotypes (35 of 35), HRV clinical isolates (5 of 5), and related picornaviruses (8 of 8) tested with mean 50% effective concentration (EC
5
0
) values of 50 nM (range, 14 to 122 nM), 77 nM (range, 72 to 89 nM), and 75 nM (range, 7 to 249 nM), respectively. Compound 1 inhibited HRV 3C-mediated polyprotein processing in infected cells in a concentration-dependent manner, providing direct confirmation that the cell-based antiviral activity is due to inhibition of 3C protease. In vitro and in vivo nonclinical safety studies showed Compound 1 to be without adverse effects at maximum achievable doses. Single oral doses of Compound 1 up to 2,000 mg in healthy volunteers were found to be safe and well tolerated in a phase I-ascending, single-dose study. Compound 1 estimated free observed maximum concentration in plasma (
C
ma
x
) for 500-, 1,000-, and 2,000-mg doses were higher than the protein binding-corrected EC
50
required to inhibit 80% of the HRV serotypes tested. Treatment of HRV 52-infected cells with one to five 2-h pulses of 150 nM Compound 1 (corresponding to the
C
max
at the 500-mg dose) was sufficient to effect a significant reduction in viral replication. These experiments highlight Compound 1 as a potent, orally bioavailable, irreversible inhibitor of HRV 3C protease and provide data that suggest that
C
max
rather than the
C
min
might be the key variable predicting clinical efficacy.
Occurrence of HypermutablePseudomonas aeruginosain Cystic Fibrosis Patients Is Associated with the Oxidative Stress Caused by Chronic Lung Inflammation
Authors: Ciofu Oana , Riis Bente , Pressler Tacjana , Poulsen Henrik Enghusen , Høiby Niels
Published: May 2005

Oxidative stress caused by chronic lung inflammation in patients with cystic fibrosis (CF) and chronic lung infection withPseudomonas aeruginosais characterized by the reactive oxygen species (ROS) liberated by polymorphonuclear leukocytes (PMNs). We formulated the hypothesis that oxidation of the bacterial DNA by ROS presents an increased risk for the occurrence of hypermutableP. aeruginosa. The occurrence of hypermutableP. aeruginosaisolates was investigated directly in the sputum of 79 CF patients and among 141 isolates collected from 11 CF patients (10 to 15 isolates/patient) collected from the 1st and up to the 25th year of their chronic lung infection. The level of oxidized guanine moiety 8-oxo-2′-deoxyguanosine (8-oxodG), which is a frequently investigated DNA oxidative lesion, was measured. HypermutableP. aeruginosaisolates were found in the sputum bacterial population of 54.4% of the CF patients. The earliest mutatorP. aeruginosaisolates were found after 5 years from the onset of the chronic lung infection, and once they were present in the CF lung, the prevalence increased with time. The hypermutable isolates were significantly more resistant to antipseudomonal antibiotics than nonhypermutable isolates (P≤ 0.001). The level of 8-oxodG/106deoxyguanosine (dG) was significantly higher in hypermutableP. aeruginosaisolates (87 ± 38) than in nonhypermutableP. aeruginosaisolates (59.4 ± 17) (P= 0.02), and an increase to 86.84 from 21.65 8-oxodG/106dG was found after exposure of the reference strain PAO1 to activated PMNs. Our results suggest that the chronic PMN inflammation in the CF lung promotes oxidative stress and is associated with the occurrence of hypermutable bacteria in the lung. The hypermutable phenotype can associate with mutations that confer adaptation of the bacteria in the lung and persistence of the infection.
Preclinical Testing of the Nitroimidazopyran PA-824 for Activity against
Mycobacterium tuberculosis
in a Series of In Vitro and In Vivo Models
Authors: Lenaerts Anne J. , Gruppo Veronica , Marietta Karen S. , Johnson Christine M. , Driscoll Diane K. , Tompkins Nicholas M. , Rose Jerry D. , Reynolds Robert C. , Orme Ian M.
Published: May 2005

This study extends earlier reports regarding the in vitro and in vivo efficacies of the nitroimidazopyran PA-824 against
Mycobacterium tuberculosis
. PA-824 was tested in vitro against a broad panel of multidrug-resistant clinical isolates and was found to be highly active against all isolates (MIC < 1 μg/ml). The activity of PA-824 against
M. tuberculosis
was also assessed grown under conditions of oxygen depletion. PA-824 showed significant activity at 2, 10, and 50 μg/ml, similar to that of metronidazole, in a dose-dependent manner. In a short-course mouse infection model, the efficacy of PA-824 at 50, 100, and 300 mg/kg of body weight formulated in methylcellulose or cyclodextrin/lecithin after nine oral treatments was compared with those of isoniazid, rifampin, and moxifloxacin. PA-824 at 100 mg/kg in cyclodextrin/lecithin was as active as moxifloxacin at 100 mg/kg and isoniazid at 25 mg/kg and was slightly more active than rifampin at 20 mg/kg. Long-term treatment with PA-824 at 100 mg/kg in cyclodextrin/lecithin reduced the bacterial load below 500 CFU in the lungs and spleen. No significant differences in activity between PA-824 and the other single drug treatments tested (isoniazid at 25 mg/kg, rifampin at 10 mg/kg, gatifloxacin at 100 mg/kg, and moxifloxacin at 100 mg/kg) could be observed. In summary, its good activity in in vivo models, as well as its activity against multidrug-resistant
M. tuberculosis
and against
M. tuberculosis
isolates in a potentially latent state, makes PA-824 an attractive drug candidate for the therapy of tuberculosis. These data indicate that there is significant potential for effective oral delivery of PA-824 for the treatment of tuberculosis.
Emergence of Macrolide-Resistant
Mycoplasma pneumoniae
with a 23S rRNA Gene Mutation
Authors: Morozumi Miyuki , Hasegawa Keiko , Kobayashi Reiko , Inoue Nagako , Iwata Satoshi , Kuroki Haruo , Kawamura Naohisa , Nakayama Eiichi , Tajima Takeshi , Shimizu Kouichi , Ubukata Kimiko
Published: May 2005

A total of 195
Mycoplasma pneumoniae
strains were isolated from 2,462 clinical specimens collected between April 2002 and March 2004 from pediatric outpatients with respiratory tract infections. Susceptibilities to six macrolide antibiotics (ML), telithromycin, minocycline, levofloxacin, and sitafloxacin were determined by the microdilution method using PPLO broth. A total of 183
M. pneumoniae
isolates were susceptible to all agents and had excellent MIC
90
s in the following order: 0.00195 μg/ml for azithromycin and telithromycin, 0.0078 μg/ml for clarithromycin, 0.0156 μg/ml for erythromycin, 0.0625 μg/ml for sitafloxacin, 0.5 μg/ml for minocycline, and 1 μg/ml for levofloxacin. Notably, 12 ML-resistant
M. pneumoniae
strains were isolated from patients with pneumonia (10 strains) or acute bronchitis (2 strains). These strains showed resistance to ML with MICs of ≥1 μg/ml, except to rokitamycin. Transition mutations of A2063G or A2064G, which correspond to A2058 and A2059 in
Escherichia coli
, in domain V on the 23S rRNA gene in 11 ML-resistant strains were identified. By pulsed-field gel electrophoresis typing, these strains were classified into groups I and Vb, as described previously (A. Cousin-Allery, A. Charron, B. D. Barbeyrac, G. Fremy, J. S. Jensen, H. Renaudin, and C. Bebear, Epidemiol. Infect.
124:
103-111, 2000). These findings suggest that excessive usage of MLs acts as a trigger to select mutations on the corresponding 23S rRNA gene with the resultant occurrence of ML-resistant
M. pneumoniae
. Monitoring ML susceptibilities for
M. pneumoniae
is necessary in the future.
Combination of Antimicrobial and Endotoxin-Neutralizing Activities of Novel Oleoylamines
Authors: Zorko Mateja , Majerle Andreja , Šarlah David , Keber Mateja Manček , Mohar Barbara , Jerala Roman
Published: May 2005

A combination of antimicrobial and endotoxin-neutralizing activities is desired in order to prevent progression from infection to sepsis due to the release of lipopolysaccharide from dying gram-negative bacteria. Lipopolyamines have emerged as a new type of endotoxin-neutralizing compound, but their antimicrobial activity has not been investigated. We synthesized a series of 10 oleoylamines differing in the polyamino head group, particularly in the number and separation between nitrogen atoms and the position of the oleoyl moiety. Compounds showed activity against both gram-negative and gram-positive bacteria in the micromolar range. Compounds were able to provide penetration of ethidium bromide into bacteria, indicating effects on the bacterial membrane. Oleoylamines neutralized endotoxin inLimulusamoebocyte lysate assays and by neutralization of tumor necrosis factor alpha release in human blood. Comparison of biological activities of compounds identified structural properties responsible for antimicrobial activity, and quantitative structure-property relationship analysis provided a quantitative model for prediction of activity of oleoylamines.
Effect of Cell-Photosensitizer Binding and Cell Density on Microbial Photoinactivation
Authors: Demidova Tatiana N. , Hamblin Michael R.
Published: May 2005

Photodynamic therapy involves the use of nontoxic dyes called photosensitizers and visible light to produce reactive oxygen species and cell killing. It is being studied as an alternative method of killing pathogens in localized infections due to the increasing problem of multiantibiotic resistance. Although much has been learned about the mechanisms of microbial killing, there is still uncertainty about whether dyes must bind to and penetrate various classes of microbe in order to produce effective killing after illumination. In this report, we compare the interactions of three antimicrobial photosensitizers: rose bengal (RB), toluidine blue O (TBO), and a poly-
l
-lysine chlorin(e6) conjugate (pL-ce6) with representative members of three classes of pathogens;
Escherichia coli
(gram-negative bacteria),
Staphylococcus aureus
(gram-positive bacteria),
Candida albicans
(yeast). We compared fluence-dependent cell survival after illumination with the appropriate wavelengths of light before and after extracellular dye had been washed out and used three 10-fold dilutions of cell concentration. pL-ce6 was overall the most powerful photosensitizer, was equally effective with and without washing, and showed a strong dependence on cell concentration. TBO was less effective in all cases after washing, and the dependence on cell concentration was less pronounced. RB was ineffective after washing (except for
S. aureus
) but still showed a dependence on cell concentration. The overall order of susceptibility was
S. aureus
>
E. coli
>
C. albicans
, but
C. albicans
cells were 10 to 50 times bigger than the bacteria. We conclude that the number and mass of the cells compete both for available dye binding and for extracellularly generated reactive oxygen species.
Human Salivary Mucin MUC7 12-Mer-land 12-Mer-dPeptides: Antifungal Activity in Saliva, Enhancement of Activity with Protease Inhibitor Cocktail or EDTA, and Cytotoxicity to Human Cells
Authors: Wei Guo-Xian , Bobek Libuse A.
Published: May 2005

MUC7 12-mer-lexhibits potent in vitro antifungal activity in low-ionic-strength buffers. In this study, we investigated the anticandidal activity and stability of MUC7 12-mer-land its all-d-amino-acid isomer, along with Hsn5 12-mer (P113) and magainin-II, in human clarified and unclarified saliva in the absence or presence of protease inhibitor cocktail (PIC, which includes EDTA) or EDTA alone. In the absence of PIC or EDTA in saliva, only MUC7 peptides showed significant candidacidal activity. At a 100 μM concentration in clarified saliva and unclarified saliva, MUC7 12-mer-ddemonstrated 94 versus 64% killing, respectively; MUC7 12-mer-lshowed 57 versus 32% killing; Hsn5 12-mer showed 16 versus 0% killing; and magainin-II showed no killing. Addition of PIC or EDTA to either saliva caused the enhancement of antifungal activities of all peptides, although to different degrees. Taken together, the results suggest that EDTA (a metal-dependent protease inhibitor and/or divalent cation chelator) enhanced the antifungal activity of all four peptides mainly by chelation of divalent cations present in saliva (known to inhibit peptide antifungal activity), and PIC enhanced the activity of the threelpeptides above that achievable by EDTA alone through inhibition of all classes of proteases. Peptide stability in saliva monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed no degradation of MUC7 12-mer-dand 23, 60, and 75% degradation of MUC7 12-mer-l, Hsn5 12-mer, and magainin-II, respectively. Cytotoxicity assays determined that, at 100 μM peptide concentrations, MUC7 12-mer-dand 12-mer-lcaused 3.5 and 4.3% hemolysis in phosphate-buffered saline and no toxicity to the HOK-16B cell line (derived from normal human oral keratinocytes). In summary, MUC7 12-mer peptides appear to be excellent candidates for investigation of antifungal activity in in vivo models of oral candidiasis.
Microdialysis Study of Imipenem Distribution in Skeletal Muscle and Lung Extracellular Fluids of Noninfected Rats
Authors: Marchand Sandrine , Dahyot Claire , Lamarche Isabelle , Mimoz Olivier , Couet William
Published: May 2005

The aim of this study was to investigate the imipenem distribution in muscle and lung interstitial fluids by microdialysis in rats and to compare the free concentrations in tissue with the free concentrations in blood. Microdialysis probes were inserted into the jugular vein, hind leg muscle, and lung. Imipenem recoveries in these three media were determined in each rat by retrodialysis by drug period before drug administration. Imipenem was infused intravenously at a dose of 120 mg · kg
−1
over 30 min, and microdialysis samples were collected for 150 min. The whole study was conducted with nonhydrated rats (
n
= 4) and hydrated rats (
n
= 6) while the animals were under isoflurane anesthesia. The decay of free concentrations in blood, muscle, and lung with time were monoexponential; and the concentration profiles in these three media were virtually superimposed in both groups. Accordingly, the ratios of the area under the curve (AUC) for tissue (muscle or lung) to the AUC for blood were always virtually equal to 1. Compared to values previously determined with awake rats, clearance was reduced by 2 and 1.5 in nonhydrated and hydrated rats, respectively, but the volume of distribution was unchanged. By combining microdialysis in blood and tissues, it was possible to demonstrate that free imipenem concentrations were virtually identical in blood, muscle, and lung.
Penetration of Linezolid into Soft Tissues of Healthy Volunteers after Single and Multiple Doses
Authors: Dehghanyar Pejman , Bürger Cornelia , Zeitlinger Markus , Islinger Florian , Kovar Florian , Müller Markus , Kloft Charlotte , Joukhadar Christian
Published: May 2005

The present study tested the ability of linezolid to penetrate soft tissues in healthy volunteers. Ten healthy volunteers were subjected to linezolid drug intake at a dose of 600 mg twice a day for 3 to 5 days. The first dose was administered intravenously. All following doses were self-administered orally. The tissue penetration of linezolid was assessed by use of in vivo microdialysis. In the single-dose experiments the ratios of the area under the concentration-time curve from 0 to 8 h (AUC
0-8
) for tissue to the AUC
0-8
for free plasma were 1.4 ± 0.3 (mean ± standard deviation) and 1.3 ± 0.4 for subcutaneous adipose and muscle tissue, respectively. After multiple doses, the corresponding mean ratios were 0.9 ± 0.2 and 1.0 ± 0.5, respectively. The ratios of the AUC from 0 to 24 h (AUC
0-24
) for free linezolid in tissues to the MIC were between 50 and 100 for target pathogens with MICs between 2 and 4 mg/liter. In conclusion, the present study showed that linezolid penetrates rapidly into the interstitial space fluid of subcutaneous adipose and skeletal muscle tissues in healthy volunteers. On the basis of pharmacokinetic-pharmacodynamic calculations, we suggest that linezolid concentrations in soft tissues can be considered sufficient to inhibit the growth of many clinically relevant bacteria.