Plasma membrane calcium ATPase (PMCA4): A housekeeper for RT-PCR relative quantification of polytopic membrane proteins

BACKGROUND: Although relative quantification of real-time RT-PCR data can provide valuable information, one limitation remains the selection of an appropriate reference gene. No one gene has emerged as a universal reference gene and much debate surrounds some of the more commonly used reference genes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). At this time, no gene encoding for a plasma membrane protein serves as a reference gene, and relative quantification of plasma membrane proteins is performed with genes encoding soluble proteins, which differ greatly in quantity and in targeting and trafficking from plasma membrane proteins. In this work, our aim was to identify a housekeeping gene, ideally one that codes for a plasma membrane protein, whose expression remains the same regardless of drug treatment and across a wide range of tissues to be used for relative quantification of real-time RT-PCR data for ATP binding cassette (ABC) plasma membrane transporters. RESULTS: In studies evaluating the expression levels of two commonly used reference genes coding for soluble proteins and two genes coding for membrane proteins, one plasma membrane protein, plasma membrane calcium-ATPase 4 (PMCA4), was comparable to the two reference genes already in use. In addition, PMCA4 expression shows little variation across eight drug-treated cell lines and was found to be superior to GAPDH and HPRT1, commonly used reference genes. Finally, we show PMCA4 used as a reference gene for normalizing ABC transporter expression in a drug-resistant lung carcinoma cell line. CONCLUSION: We have found that PMCA4 is a good housekeeping gene for normalization of gene expression for polytopic membrane proteins including transporters and receptors

Gist and verbatim communication concerning medication risks/benefits

To describe the information about medication risks/benefits that rheumatologists provide during patient office visits, the gist that patients with rheumatoid arthritis (RA) extract from the information provided, and the relationship between communication and medication satisfaction

Root-Associated Streptomyces Isolates Harboring melC Genes Demonstrate Enhanced Plant Colonization

Streptomycetaceae assemble into the internal, root endophytic compartment of a wide variety of plants grown in soils worldwide, suggesting their ability to survive during root microbiome assembly. A previous study found that among four nonpathogenic, root-isolated Streptomyces strains (303, 299, CL18, and 136), only 303 and 299 colonized endophytic root tissue of the majority of Arabidopsis thaliana roots when inoculated with 34 other bacterial isolates. Here we demonstrate that 303 and 299 also colonize significantly more in singly inoculated A. thaliana seedlings. The genomes of melanin-producing 303 and 299 each contain two copies of the gene encoding tyrosinase (melC2 and melD2), an enzyme necessary for melanin biosynthesis in Streptomyces. These genes were not found in the genomes of 136 or CL18. Tyrosinase activity was detected in 303 and 299 whole cell and supernatant protein extracts, suggesting functional intracellular and extracellular enzymes.. Because tyrosinase oxidizes phenolic compounds and Streptomyces colonization of A. thaliana appears to be influenced by the phenolic compound salicylic acid (SA), we measured direct sensitivity of Streptomyces isolates to the phenolic compounds catechol, ferulic acid (FA), and SA in vitro. While both 303 and 299 showed higher numbers of surviving colonies than CL18 and 136 in the presence of catechol, only 303 demonstrated a higher number of surviving colonies when isolates were challenges with FA and SA. Finally, when seedlings were singly inoculated with a collection of related plant-associated Streptomyces isolates, colonization was significantly higher in isolates possessing two tyrosinase gene copies than isolates with zero or one gene copy. Overall, we describe a connection between microbial tyrosinase activity and increased seedling colonization of nonpathogenic Streptomyces isolates in A. thaliana. We propose tyrosinase activity in Streptomyces partially protects against harmful plant-produced phenolic compounds as they transition into an endophytic lifestyle

Quality of care for older adults with chronic obstructive pulmonary disease and asthma based on comparisons to practice guidelines and smoking status

<p>Abstract</p> <p>Background</p> <p>The purpose of this study was to describe the prevalence of respiratory diseases in older adults and compare the demographic, health and smoking characteristics of those with and without these diseases. Furthermore, we evaluate the association between smoking status and patterns in health care and how concordant this care is with guidelines.</p> <p>Methods</p> <p>Using a nationally representative sample of 29,902 older adults who participated in the Medicare Current Beneficiary Survey (1992–2002), we compared guideline recommendations on the treatment and prevention of chronic obstructive pulmonary disease and asthma with survey utilization data, including the use of bronchodilators, spirometry and influenza vaccine.</p> <p>Results</p> <p>26% to 30% of older adults were diagnosed with or self-reported chronic respiratory diseases; however 69% received no pharmacological treatment and 30% of patients reporting use of pharmacological treatments did not receive short-acting bronchodilator inhalers. Current smokers appeared to receive significantly less care for respiratory diseases than non-smokers or former smokers.</p> <p>Conclusion</p> <p>Disparities between recommended and actual care for older adults with chronic lung disease require further research. The needs of older adults with co-morbidities and nicotine addiction deserve special attention in care as well as guideline development and implementation.</p

RISK ASSESSMENT OF AIR CANNONS AT SPORTING EVENTS

ABSTRAC

Effects of needs-based patient education on self-efficacy and health outcomes in people with rheumatoid arthritis: A multicentre, single blind, randomised controlled trial

Objectives The Educational Needs Assessment Tool (ENAT) is a self-completed questionnaire, which allows patients with arthritis to prioritise their educational needs. The aim of this study was to evaluate the effects of needs-based patient education on self-efficacy, health outcomes and patient knowledge in people with rheumatoid arthritis (RA). Methods Patients with RA were enrolled into this multicentre, single-blind, parallel-group, pragmatic randomised controlled trial. Patients were randomised to either the intervention group (IG) where patients completed ENAT, responses of which were used by the clinical nurse specialist to guide patient education; or control group (CG) in which they received patient education without the use of ENAT. Patients were seen at weeks 0, 16 and 32. The primary outcome was selfefficacy (Arthritis Self Efficacy Scale (ASES)-Pain and ASES-Other symptoms). Secondary outcomes were health status (short form of Arthritis Impact Measurement Scale 2, AIMS2-SF) and patient knowledge questionnaire-RA. We investigated between-group differences using analysis of covariance, adjusting for baseline variables. Results A total of 132 patients were recruited (IG=70 and CG=62). Their mean (SD) age was 54 (12.3) years, 56 (13.3) years and disease duration 5.2 (4.9) years, 6.7 (8.9) years for IG and CG, respectively. There were significant between-group differences, in favour of IG at week 32 in the primary outcomes, ASES-Pain, mean difference (95% CI) -4.36 (1.17 to 7.55), t=-2.72, p=0.008 and ASES-Other symptoms, mean difference (95% CI) -5.84 (2.07 to 9.62), t=-3.07, p=0.003. In secondary outcomes, the between-group differences favoured IG in AIMS2-SF Symptoms and AIMS2-SF Affect. There were no between-group differences in other secondary outcomes. Conclusions The results suggest that needs-based education helps improve patients' self-efficacy and some aspects of health status

A destabilized bacterial luciferase for dynamic gene expression studies

Fusions of genetic regulatory elements with reporter genes have long been used as tools for monitoring gene expression and have become a major component in synthetic gene circuit implementation. A major limitation of many of these systems is the relatively long half-life of the reporter protein(s), which prevents monitoring both the initiation and the termination of transcription in real-time. Furthermore, when used as components in synthetic gene circuits, the long time constants associated with reporter protein decay may significantly degrade circuit performance. In this study, short half-life variants of LuxA and LuxB from Photorhabdus luminescens were constructed in Escherichia coli by inclusion of an 11-amino acid carboxy-terminal tag that is recognized by endogenous tail-specific proteases. Results indicated that the addition of the C-terminal tag affected the functional half-life of the holoenzyme when the tag was added to luxA or to both luxA and luxB, but modification of luxB alone did not have a significant effect. In addition, it was also found that alteration of the terminal three amino acid residues of the carboxy-terminal tag fused to LuxA generated variants with half-lives of intermediate length in a manner similar to that reported for GFP. This report is the first instance of the C-terminal tagging approach for the regulation of protein half-life to be applied to an enzyme or monomer of a multi-subunit enzyme complex and will extend the utility of the bacterial luciferase reporter genes for the monitoring of dynamic changes in gene expression

The involvement of cancer patients in the four stages of decision-making preceding continuous sedation until death: A qualitative study

BACKGROUND: Involving patients in decision-making is considered to be particularly appropriate towards the end of life. Professional guidelines emphasize that the decision to initiate continuous sedation should be made in accordance with the wishes of the dying person and be preceded by their consent. AIM: To describe the decision-making process preceding continuous sedation until death with particular attention to the involvement of the person who is dying. DESIGN: Qualitative case studies using interviews. SETTING/PARTICIPANTS: Interviews with 26 physicians, 30 nurses and 24 relatives caring for 24 patients with cancer who received continuous sedation until death in Belgium, the United Kingdom and the Netherlands. RESULTS: We distinguished four stages of decision-making: initiation, information exchange, deliberation and the decision to start continuous sedation until death. There was wide variation in the role the patient had in the decision-making process. At one end of the spectrum (mostly in the United Kingdom), the physician discussed the possible use of sedation with the patient, but took the decision themselves. At the other end (mostly in Belgium and the Netherlands), the patient initiated the conversation and the physician's role was largely limited to evaluating if and when the medical criteria were met. CONCLUSION: Decision-making about continuous sedation until death goes through four stages and the involvement of the patient in the decision-making varies. Acknowledging the potential sensitivity of raising the issue of end-of-life sedation, we recommend building into clinical practice regular opportunities to discuss the goals and preferences of the person who is dying for their future medical treatment and care

CD4+ T Cell Effects on CD8+ T Cell Location Defined Using Bioluminescence

T lymphocytes of the CD8+ class are critical in delivering cytotoxic function and in controlling viral and intracellular infections. These cells are “helped” by T lymphocytes of the CD4+ class, which facilitate their activation, clonal expansion, full differentiation and the persistence of memory. In this study we investigated the impact of CD4+ T cells on the location of CD8+ T cells, using antibody-mediated CD4+ T cell depletion and imaging the antigen-driven redistribution of bioluminescent CD8+ T cells in living mice. We documented that CD4+ T cells influence the biodistribution of CD8+ T cells, favoring their localization to abdominal lymph nodes. Flow cytometric analysis revealed that this was associated with an increase in the expression of specific integrins. The presence of CD4+ T cells at the time of initial CD8+ T cell activation also influences their biodistribution in the memory phase. Based on these results, we propose the model that one of the functions of CD4+ T cell “help” is to program the homing potential of CD8+ T cells