Developments in Pb-210 methodologies to provide chronologies for environmental change.

Chronologies generated from core profiles to apply dates to environmental changes commonly use the measurement of the activity of radionuclides deposited and stratified with physical environmental material. The most commonly reported nuclide to define chronologies covering the last 150 years is Pb-210, for which accepted data processing methodologies in the literature have focussed on the constant rate of supply (CRS) model and the more recently published Bayesian Plum model. This short communication describes a validation approach using defined sediment layers referred to as 'varve' counting, which provide known points of reference to account for uncertainty between generated dates from each model using published Pb-210 measurements. A significant improvement in the chronologies was observed when applying reference date corrections to the models. This was shown to be essential in providing confidence in reported datasets and accuracy of predicted chronologies, which will better inform the interpretation of environmental change, e.g. sedimentation rates, climate change, pollution pathways and land degradation. Generated chronologies from both the CRS and Plum methods showed good agreement with the established varve dates (typically < 4-year difference).

Investigating G protein signalling bias at the glucagon-like peptide-1 receptor in yeast.

BACKGROUND AND PURPOSE: The glucagon-like peptide 1 (GLP-1) receptor performs an important role in glycaemic control, stimulating the release of insulin. It is an attractive target for treating type 2 diabetes. Recently, several reports of adverse side effects following prolonged use of GLP-1 receptor therapies have emerged: most likely due to an incomplete understanding of signalling complexities. EXPERIMENTAL APPROACH: We describe the expression of the GLP-1 receptor in a panel of modified yeast strains that couple receptor activation to cell growth via single Gα/yeast chimeras. This assay enables the study of individual ligand-receptor G protein coupling preferences and the quantification of the effect of GLP-1 receptor ligands on G protein selectivity. KEY RESULTS: The GLP-1 receptor functionally coupled to the chimeras representing the human Gαs, Gαi and Gαq subunits. Calculation of the dissociation constant for a receptor antagonist, exendin-3 revealed no significant difference between the two systems. We obtained previously unobserved differences in G protein signalling bias for clinically relevant therapeutic agents, liraglutide and exenatide; the latter displaying significant bias for the Gαi pathway. We extended the use of the system to investigate small-molecule allosteric compounds and the closely related glucagon receptor. CONCLUSIONS AND IMPLICATIONS: These results provide a better understanding of the molecular events involved in GLP-1 receptor pleiotropic signalling and establish the yeast platform as a robust tool to screen for more selective, efficacious compounds acting at this important class of receptors in the future.

Incidence of respiratory pathogens in persons hospitalized with pneumonia in two provinces in Thailand.

Although pneumonia is a leading cause of death from infectious disease worldwide, comprehensive information about its causes and incidence in low- and middle-income countries is lacking. Active surveillance of hospitalized patients with pneumonia is ongoing in Thailand. Consenting patients are tested for seven bacterial and 14 viral respiratory pathogens by PCR and viral culture on nasopharyngeal swab specimens, serology on acute/convalescent sera, sputum smears and antigen detection tests on urine. Between September 2003 and December 2005, there were 1730 episodes of radiographically confirmed pneumonia (34·6% in children aged <5 years); 66 patients (3·8%) died. A recognized pathogen was identified in 42·5% of episodes. Respiratory syncytial virus (RSV) infection was associated with 16·7% of all pneumonias, 41·2% in children. The viral pathogen with the highest incidence in children aged <5 years was RSV (417·1/100,000 per year) and in persons aged ≥50 years, influenza virus A (38·8/100,000 per year). These data can help guide health policy towards effective prevention strategies.

Glycemic control and implant stabilization in type 2 diabetes mellitus.

Diabetes mellitus is considered a relative contra-indication for implant therapy. However, the effect of glycemic level on implant integration in persons with diabetes remains poorly understood. The hypothesis of this research was that poor glycemic control is directly related to short-term-impairment implant stabilization. This prospective clinical study evaluated 10 non-diabetic individuals (12 implants) and 20 persons with type 2 diabetes (30 implants). Glycated hemoglobin (HbA1c) levels ranged from 4.7-12.6%. Implant stability was assessed by resonance frequency analysis over 4 months following placement. Minimum stability levels were observed 2-6 weeks following placement for all 42 implants. Persons with HbA1c > or = 8.1% had a greater maximum decrease in stability from baseline and required a longer time for healing, as indicated by return of stability level to baseline. This study demonstrates alterations in implant stability consistent with impaired implant integration for persons with type 2 diabetes mellitus in direct relation to hyperglycemic conditions.

The novel endocannabinoid receptor GPR55 is activated by atypical cannabinoids but does not mediate their vasodilator effects.

BACKGROUND AND PURPOSE: Atypical cannabinoids are thought to cause vasodilatation through an as-yet unidentified 'CBx' receptor. Recent reports suggest GPR55 is an atypical cannabinoid receptor, making it a candidate for the vasodilator 'CBx' receptor. The purpose of the present study was to test the hypothesis that human recombinant GPR55 is activated by atypical cannabinoids and mediates vasodilator responses to these agents. EXPERIMENTAL APPROACH: Human recombinant GPR55 was expressed in HEK293T cells and specific GTPgammaS activity was monitored as an index of receptor activation. In GPR55-deficient and wild-type littermate control mice, in vivo blood pressure measurement and isolated resistance artery myography were used to determine GPR55 dependence of atypical cannabinoid-induced haemodynamic and vasodilator responses. KEY RESULTS: Atypical cannabinoids O-1602 and abnormal cannabidiol both stimulated GPR55-dependent GTPgammaS activity (EC50 approximately 2 nM), whereas the CB1 and CB2-selective agonist WIN 55,212-2 showed no effect in GPR55-expressing HEK293T cell membranes. Baseline mean arterial pressure and heart rate were not different between WT and GPR55 KO mice. The blood pressure-lowering response to abnormal cannabidiol was not different between WT and KO mice (WT 20+/-2%, KO 26+/-5% change from baseline), nor was the vasodilator response to abnormal cannabidiol in isolated mesenteric arteries (IC50 approximately 3 micro M for WT and KO). The abnormal cannabidiol vasodilator response was antagonized equivalently by O-1918 in both strains. CONCLUSIONS: These results demonstrate that while GPR55 is activated by atypical cannabinoids, it does not appear to mediate the vasodilator effects of these agents.

Complex chimeras to map ligand binding sites of GPCRs.

Family 1a GPCRs are thought to bind small molecule ligands in a pocket comprising sequences from non-contiguous transmembrane helices. In this study, receptor-ligand binding determinants were defined by building a series of complex chimeras where multiple sequences were exchanged between related G-protein coupled receptors. Regions of P2Y(1), P2Y(2) and BLT(1) predicted to interact with nucleotide and leukotriene ligands were identified and receptors were engineered within their transmembrane helices to transpose the ligand binding site of one receptor on to another receptor. Ligand-induced activation of chimeras was compared with wild-type receptor activation in a yeast reporter gene assay. Binding of ligand to a P2Y(2)/BLT(1) chimera confirmed that the ligand binding determinants of BLT(1) are located in the upper regions of the helices and extracellular loops of this receptor and that they had been successfully transferred to a receptor that normally binds unrelated ligands.

Adverse and beneficial secondary effects of mass treatment with azithromycin to eliminate blindness due to trachoma in Nepal.

Mass administration of azithromycin to eliminate blindness due to trachoma has raised concerns regarding the emergence of antimicrobial resistance. During 2000, we compared the antimicrobial resistance of nasopharyngeal pneumococcal isolates recovered from and the prevalence of impetigo, respiratory symptoms, and diarrhea among 458 children in Nepal before and after mass administration of azithromycin. No azithromycin-resistant pneumococci were isolated except from 4.3% of children who had received azithromycin during 2 previous mass treatments (P<.001). There were decreases in the prevalence of impetigo (from 14% to 6% of subjects; adjusted odds ratio [OR], 0.41; 95% confidence interval [CI], 0.21-0.80) and diarrhea (from 32% to 11%; adjusted OR, 0.26; 95% CI, 0.14-0.43) 10 days after azithromycin treatment. The absence of macrolide-resistant isolates after 1 mass treatment with azithromycin is encouraging, although the recovery of azithromycin-resistant isolates after 2 mass treatments suggests the need for resistance monitoring when multiple rounds of antimicrobial treatment are given.

Yeast assays for G-protein-coupled receptors.

Yeast assays for G-protein-coupled receptors have many attractions due to their simplicity, low cost, and lack of endogenous receptors. Since the first report of functional coupling of the human beta 2 adrenergic receptor to the yeast pheromone-response pathway in 1990, the technology has developed to a point at which more than 30 heterologous GPCRs are now published to couple. Major breakthroughs have come from an understanding of receptor-G protein interactions, alongside advances in knowledge of the structure of heterotrimeric G proteins. Yeast screens have been used to identify ligands both from compound collections and through the autocrine expression of peptide libraries. Yeast genetics has also been applied to a functional analysis of GPCRs and peptide ligands. In this review we describe the historical development of yeast GPCR assay systems and their current applications.

Standardizing Chlamydia pneumoniae assays: recommendations from the Centers for Disease Control and Prevention (USA) and the Laboratory Centre for Disease Control (Canada).

Chlamydia pneumoniae has been associated with atherosclerosis and several other chronic diseases, but reports from different laboratories are highly variable and "gold standards" are lacking, which has led to calls for more standardized approaches to diagnostic testing. Using leading researchers in the field, we reviewed the available approaches to serological testing, culture, DNA amplification, and tissue diagnostics to make specific recommendations. With regard to serological testing, only use of microimmunofluorescence is recommended, standardized definitions for "acute infection" and "past exposure" are proposed, and the use of single immunoglobulin (Ig) G titers for determining acute infection and IgA for determining chronic infection are discouraged. Confirmation of a positive culture result requires propagation of the isolate or confirmation by use of polymerase chain reaction (PCR). Four of 18 PCR assays described in published reports met the proposed validation criteria. More consistent use of control antibodies and tissues and improvement in skill at identifying staining artifacts are necessary to avoid false-positive results of immunohistochemical staining. These standards should be applied in future investigations and periodically modified as indicated.

Reducing antibiotic use in children: a randomized trial in 12 practices.

OBJECTIVE: To test whether an educational outreach intervention for families and physicians, based on the Centers for Disease Control and Prevention (CDC) principles of judicious antibiotic use, decreases antimicrobial drug prescribing for children younger than 6 years old. Setting. Twelve practices affiliated with 2 managed care organizations (MCOs) in eastern Massachusetts and northwest Washington State. Patients. All enrolled children younger than 6 years old. METHODS: Practices stratified by MCO and size were randomized to intervention or control groups. The intervention included 2 meetings of the practice with a physician peer leader, using CDC-endorsed summaries of judicious prescribing recommendations; feedback on previous prescribing rates were also provided. Parents were mailed a CDC brochure on antibiotic use, and supporting materials were displayed in waiting rooms. Automated enrollment, ambulatory visit, and pharmacy claims were used to determine rates of antibiotic courses dispensed (antibiotics/person-year) during baseline (1996-1997) and intervention (1997-1998) years. The primary analysis (for children 3 to <36 months and 36 to <72 months) assessed the impact of the intervention among children during the intervention year, controlling for covariates including patient age and baseline prescription rate. Confirmatory analyses at the practice level were also performed. RESULTS: The practices cared for 14 468 and 13 460 children in the 2 study years, respectively; 8815 children contributed data in both years. Sixty-two percent of antibiotic courses were dispensed for otitis media, 6.5% for pharyngitis, 6.3% for sinusitis, and 9.2% for colds and bronchitis. Antibiotic dispensing for children 3 to <36 months old decreased 0.41 antibiotics per person-year (18.6%) in intervention compared with 0.33 (11.5%) in control practices. Among children 36 to <72 months old, the rate decreased by 0.21 antibiotics per person-year (15%) in intervention and 0.17 (9.8%) in control practices. Multivariate analysis showed an adjusted intervention effect of 16% in the younger and 12% in the older age groups. The direction and approximate magnitude of effect were confirmed in practice-level analyses. CONCLUSIONS: A limited simultaneous educational outreach intervention for parents and providers reduced antibiotic use among children in primary care practices, even in the setting of substantial secular trends toward decreased prescribing. Future efforts to promote judicious prescribing should continue to build on growing public awareness of antibiotic overuse.

Physician control of pediatric antimicrobial use in Beijing, China, and its rural environs.

BACKGROUND: Antibiotic resistance is recognized as an increasing problem in China. It is widely believed that because antibiotics are available without a prescription, changing physician prescribing behaviors will not decrease inappropriate usage. This study identified the sources of antibiotics and the important influence that physicians have on antibiotic use by children in one region of China. METHODS: Trained medical professionals surveyed parents of children attending several kindergartens in urban Beijing and rural Gu'An, Hebei County. Parents completed a questionnaire concerning the children's recent illnesses, care-seeking patterns and antibiotic use. The team also observed hospital- and non-hospital-based pharmacy purchases of antibiotics for children, assessed the proportion accompanied by a prescription and then interviewed parents about factors influencing those purchases. RESULTS: Of 241 urban and 143 rural kindergarten parents, 76 to 82% usually obtained children's antibiotics from a hospital pharmacy (with a prescription). For 84% the first source of care was usually a physician (primarily western medicine, sometimes traditional Chinese medicine). Only 5% of antibiotics were obtained from independent vendors without prior physician consultation. Among 229 observed antibiotic purchases 72% occurred at hospital-based facilities, even after longer observation times at nonhospital pharmacies. Prescriptions accompanied all hospital-based antibiotic purchases, contrasting with 18% of nonhospital transactions (P < 0.001). Together 86% of parents self-reported that the observed purchase stemmed from a doctor's recommendation. CONCLUSIONS: Doctors directly and indirectly controlled the majority of antibiotic usage for childhood illnesses in Beijing and Gu'An (Hebei County). Physician education and implementation of treatment guidelines might substantially reduce inappropriate antimicrobial usage and help prevent antimicrobial resistance in this region.

Seasonal variation in host susceptibility and cycles of certain infectious diseases.

Seasonal cycles of infectious diseases have been variously attributed to changes in atmospheric conditions, the prevalence or virulence of the pathogen, or the behavior of the host. Some observations about seasonality are difficult to reconcile with these explanations. These include the simultaneous appearance of outbreaks across widespread geographic regions of the same latitude; the detection of pathogens in the off-season without epidemic spread; and the consistency of seasonal changes, despite wide variations in weather and human behavior. In contrast, an increase in susceptibility of the host population, perhaps linked to the annual light/dark cycle and mediated by the pattern of melatonin secretion, might account for many heretofore unexplained features of infectious disease seasonality. Ample evidence indicates that photoperiod-driven physiologic changes are typical in mammalian species, including some in humans. If such physiologic changes underlie human resistance to infectious diseases for large portions of the year and the changes can be identified and modified, the therapeutic and preventive implications may be considerable.

Treating cardiovascular disease with antimicrobial agents: a survey of knowledge, attitudes, and practices among physicians in the United States.

To assess physicians' knowledge, attitudes, and prescribing behaviors with regard to the association between Chlamydia pneumoniae and cardiovascular disease, we surveyed 750 physicians in Alaska, 1172 in West Virginia, and 569 infectious disease (ID) specialists in a nationwide network during February-May 1999. Eighty-five percent knew of the association between C. pneumoniae and atherosclerosis, but this awareness was more common among ID specialists and cardiologists than among generalists (96% vs. 77%; P<.001). Knowledge scores were significantly higher among ID specialists and cardiologists (P<.001) and among physicians who saw relatively more patients who had myocardial infarction and/or were at risk of atherosclerotic disease. Four percent of physicians had treated or recommended treating cardiovascular diseases with antimicrobial agents; this percentage was significantly higher among cardiologists, physicians who empirically treat patients with peptic ulcers with antimicrobial agents, and physicians with a relatively high knowledge score.

Comparison of two commercial microimmunofluorescence kits and an enzyme immunoassay kit for detection of serum immunoglobulin G antibodies to Chlamydia pneumoniae.

We compared the MRL and the Labsystems Chlamydia pneumoniae microimmunofluorescence (MIF) immunoglobulin G (IgG) kits and the Labsystems enzyme immunoassay (EIA) kit in a blinded study of 83 serum samples in which we evaluated titers, cross-reactivity to other species, and reproducibility. There was no statistically significant difference between the MRL and the Labsystems MIF kits in the endpoint titers of IgG antibody to C. pneumoniae. The correlation between the results obtained with these two MIF kits was excellent (r = 0.95; P = 0.001). The cross-reactivity of the C. pneumoniae-positive sera with C. trachomatis- and C. psittaci-positive sera was assessed for each MIF kit. For C. pneumoniae-positive sera with titers of > or =32, the Labsystems MIF kit exhibited more cross-reactivity to C. psittaci than the MRL kit did. The values obtained with the Labsystems EIA kit represented single dilutions of serum specimens expressed as enzymeimmuno units on a continuous scale. The results obtained with the Labsystems EIA kit correlated moderately well with those obtained with each MIF kit when they were compared for their abilities to detect IgG antibodies to C. pneumoniae (for the MRL MIF kit, r = 0.79 [P = 0.001]; for the Labsystems MIF kit, r = 0.78 [P = 0.001]). The results obtained with the commercial MRL and Labsystems MIF kits and the Labsystems EIA kit tested were reproducible; and the kits were standardized, had quality control reagents, and are suitable for detection of C. pneumoniae antibodies in serum and for use in interlaboratory studies. Validation of the use of these kits for clinical diagnosis still needs further evaluation.

Evaluation of Binax NOW, an assay for the detection of pneumococcal antigen in urine samples, performed among pediatric patients.

In our evaluation of a new assay for the detection of pneumococcal antigen in urine (Binax NOW; Binax), the test result was no more likely to be positive among 88 children with radiographically confirmed pneumonia than among 198 control subjects; however, it was significantly more likely to be positive among children who were nasopharyngeal carriers of pneumococci. This test is not likely to be useful for distinguishing children with pneumococcal pneumonia from those who are merely colonized.

Self-compatible B mutants in coprinus with altered pheromone-receptor specificities.

A successful mating in the mushroom Coprinus cinereus brings together a compatible complement of pheromones and G-protein-coupled receptors encoded by multiallelic genes at the B mating-type locus. Rare B gene mutations lead to constitutive activation of B-regulated development without the need for mating. Here we characterize a mutation that arose in the B6 locus and show that it generates a mutant receptor with a single amino acid substitution (R96H) at the intracellular end of transmembrane domain III. Using a heterologous yeast assay and synthetic pheromones we show that the mutation does not make the receptor constitutively active but permits it to respond inappropriately to a normally incompatible pheromone encoded within the same B6 locus. Parallel experiments carried out in Coprinus showed that a F67W substitution in this same pheromone enabled it to activate the normally incompatible wild-type receptor. Together, our experiments show that a single amino acid replacement in either pheromone or receptor can deregulate the specificity of ligand-receptor recognition and confer a self-compatible B phenotype. In addition, we use the yeast assay to demonstrate that different receptors and pheromones found at a single B locus belong to discrete subfamilies within which receptor activation cannot normally occur.