There was some question over whether or not the RCTs by Onofriescu et al avanafil 100mg line. The principal investigators of each trial were contacted order avanafil 100mg on-line, but no replies were forthcoming order avanafil 50 mg line. The corresponding authors of both studies were contacted for further clarification purchase 200 mg avanafil, but no replies were received. It is therefore unclear whether or not the studies are completely separate. Characteristics of the included studies are detailed in Appendix 7. This issue may be freely reproduced for the purposes of private research and study and extracts (or indeed, the full report) may be included in professional journals 13 provided that suitable acknowledgement is made and the reproduction is not associated with any form of advertising. Applications for commercial reproduction should be addressed to: NIHR Journals Library, National Institute for Health Research, Evaluation, Trials and Studies Coordinating Centre, Alpha House, University of Southampton Science Park, Southampton SO16 7NS, UK. ASSESSMENT OF CLINICAL EFFECTIVENESS Database searches MEDLINE/EMBASE, n=3187 Science Citation Index, n=870 CENTRAL, n=46 CDSR, n=2 DARE, n=1 Conference abstracts, n=18 Total, n=4124 After de-duplication, n=2592 Excluded at screening (n=2463) Selected for full-text screening Full-text papers (n=129) Excluded at full-text assessment (n=114) • Ineligible study design, n=34 • Ineligible device, n=67 • Ineligible participants, n=3 • Ineligible outcomes, n=8 • Non-English language and unable to obtain translation, n=2 Included at full-text assessment (n=15) • RCTs, n=6 • Non-RCTs, n=9 FIGURE 2 Flow diagram outlining the study selection process. Randomised controlled trials 60 61 63 76 77, , , , All five included RCTs were available in full-text format. The BCM was the multiple-frequency device used in all five trials. One trial was conducted in Romania,60 one trial in Taiwan,76 one in Turkey77 and one in Portugal,61 and the remaining trial did not provide this information. The multiple-frequency bioimpedance device used for assessment of fluid status by all five trials was the BCM. All five trials included only adults 60 61 63 76 77, , , , aged ≥ 18 years. The main exclusion criteria reported in the trials, which assessed patients 76 77, receiving HD, were coronary stents or pacemakers; metallic devices in the body, such as joint 60 61 76 77, , , 60 76 77, , 60 61, 63 prostheses; limb amputations; and pregnancy. One trial, which assessed PD patients, excluded those who had been on one or two exchanges per day because of economic limitation and those patients with acute infection and CV events in the month prior to enrolment. The length of follow-up of the included trials ranged from 3 months61 to 2. Two trials were supported by grants from independent sources. None of these 82 83, studies enrolled paediatric populations. Two studies were conducted in the UK, two in Seoul, South 50 85, 86 87 88 30 30 85 86, , Korea, and one each in Spain, Poland, Romania and Europe. Three studies were multicentred 50 82 83 87 88, , , , and the remaining five studies were conducted in single dialysis centres. Six studies involved 30 50 85, , –88 82 83, patients receiving HD and the remaining two studies involved solely patients treated with PD. The length of follow-up in the eight non-randomised studies ranged from 16 weeks85 to 3. Three studies had no 50 83 87, , apparent links with Fresenius Medical Care and the other five studies reported either funding from 85 30 82 86 88, , , Fresenius Medical Care or some form of connection with the company. The technique used to measure blood pressure in the remaining study is unclear. TABLE 1 Summary of baseline characteristics of included studies Included studies (N = 13) Characteristic RCTs (N = 5) NRS (N = 8) Enrolled 1032 (n = 5) 993 (n = 3) Randomised 939 (n = 5) N/A Analysed 904 (n = 5) 4915 (n = 8)a Age (years): median (range) of means 60 (51. Note Dialysis vintage refers to the length of time on dialysis. This issue may be freely reproduced for the purposes of private research and study and extracts (or indeed, the full report) may be included in professional journals 15 provided that suitable acknowledgement is made and the reproduction is not associated with any form of advertising. Applications for commercial reproduction should be addressed to: NIHR Journals Library, National Institute for Health Research, Evaluation, Trials and Studies Coordinating Centre, Alpha House, University of Southampton Science Park, Southampton SO16 7NS, UK. ASSESSMENT OF CLINICAL EFFECTIVENESS Randomised controlled trials 60 61 63 76 77, , , , The five RCTs randomised a total of 939 participants: 469 to bioimpedance measurements and 470 to standard clinical assessment.
The patient depicted was treated with cisplatin 2 m onths before pre- sentation discount 100mg avanafil fast delivery. Attem pts at oral and intravenous potassium replace- m ent of up to 80 m Eq/day were unsuccessful in correcting the hypokalem ia cheap 100 mg avanafil with mastercard. O nce serum m agnesium was corrected buy avanafil 200mg without a prescription, however buy avanafil 50mg overnight delivery, serum potassium quickly norm alized. The urine chloride value is helpful in distinguishing the causes of hypokalem ia. Diuretics are a com m on cause of hypokalem ia; however, after dis- continuing diuretics, urinary potassium and chloride m ay be appropriately low. Urine diuretic screens are warranted for patients suspected of surreptious diuretic abuse. Vom iting results in chloride and sodium depletion, hyperaldosteronism , and renal potassium wasting. Posthypercapnic states are often associated with chloride depletion (from diuretics) and sodium avidity. If hypercapnia is corrected without replacing chloride, patients develop chloride-deple- tion alkalosis and hypokalem ia. The hyperaldostero- nism and increased distal sodium delivery account for the characteristic hypokalem ic m etabolic alkalosis. M oreover, im paired sodium reabsorption in the TAL results in the hypercalciuria seen in these patients, as approxim ately 25% of filtered calcium is reabsorbed in this segm ent in a process coupled to sodium reabsorption. Since potassium levels in the TAL are m uch lower than levels of sodium or chloride, lum inal potassium concentrations are rate lim iting for N a+-K+-2Cl- co-transporter activity. Defects in ATP-sensitive potassium channels would be predicted to alter potassium recy- cling and dim inish N a+-K+-2Cl- cotrans- porter activity. Since approxim ately 30% of fil- m ore avid sodium and calcium reabsorption tered sodium is reabsorbed by this segm ent of the nephron, defective sodium reabsorption by the proxim al nephrons. FIGURE 3-14 CHARACTERISTICS OF HYPOKALEM IA W ITH Distinguishing characteristics of HYPERTENSION AND M ETABOLIC ALKALOSIS hypokalem ia associated with hypertension and m etabolic alkalosis. The am iloride- sensitive sodium channel on the apical m em brane of the distal tubule consists of hom ologous , , and subunits. Each subunit is com posed of two transm em brane-spanning dom ains, an extracel- lular loop, and intracellular am ino and carboxyl term inals. Truncation m utations of either the or subunit carboxyl term i- nal result in greatly increased sodium conductance, which creates a favorable electrochem ical gradient for potassium secretion. FIGURE 3-16 M echanism of hypokalem ia in the syndrom e of apparent m ineralo- corticoid excess (AM E). Cortisol and aldosterone have equal affini- ty for the intracellular m ineralocorticoid receptor (M R); however, in aldosterone-sensitive tissues such as the kidney, the enzym e 11 -hydroxysteroid dehydrogenase (11 -H SD) converts cortisol to cortisone. Since cortisone has a low affinity for the M R, the enzym e 11 -H SD serves to protect the kidney from the effects of glucocorticoids. In hereditary or acquired AM E, 11 -H SD is defective or is inactiveted (by licorice or carbenoxalone). Cortisol, which is present at concentrations approxim ately 1000-fold that of aldosterone, becom es a m ineralocorticoid. The hyperm ineralo- corticoid state results in increased transcription of subunits of the sodium channel and the N a+-K+-ATPase pum p. The favorable elec- trochem ical gradient then favors potassium secretion [7,15]. These enzymes have identical intron-extron structures and are closely linked on chromosome 8. The chimeric gene is now under the contol of ACTH, and aldosterone secretion is enhanced, thus causing hypokalemia and hypertension. By inhibiting pituitary release of ACTH, glucocorticoid administration leads to a fall in aldosterone levels and correction of the clinical and biochemical abnormalities of GRA. The presence of Aldo S activity in the FIGURE 3-17 zona fasciculata gives rise to characteristic ele- Genetics of glucocorticoid-remediable aldosteronism (GRA): schematic representation of vations in 18-oxidation products of cortisol unequal crossover in GRA. The genes for aldosterone synthase (Aldo S) and 11 -hydroxylase (18-hydroxycortisol and 18-oxocortisol), (11 -OHase) are normally expressed in separate zones of the adrenal cortex. Hypokalemia: Clinical M anifestations CLINICAL M ANIFESTATIONS OF HYPOKALEM IA Cardiovascular Renal/electrolyte Abnormal electrocardiogram Functional alterations Predisposition for digitalis toxicity Decreased glomerular filtration rate Atrial ventricular arrhythmias Decreased renal blood flow Hypertension Renal concentrating defect Neuromuscular Increased renal ammonia production Smooth muscle Chloride wasting Constipation/ileus Metabolic alkalosis Bladder dysfunction Hypercalciuria Skeletal muscle Phosphaturia W eakness/cramps Structural alterations Tetany Dilation and vacuolization of Paralysis proximal tubules Myalgias/rhabdomyolysis Medullary cyst formation Interstitial nephritis Endocrine/metabolic Decreased insulin secretion Carbohydrate intolerance Increased renin FIGURE 3-19 Decreased aldosterone Electrocardiographic changes associated with hypokalemia.
The directly visualizing the subcellular localization of GPCRs secondary antibody is typically coupled to a fluorochrome and for performing biochemical studies of specific receptor (such as fluorescein) 100 mg avanafil free shipping, a recognizable particle (such as colloi- trafficking mechanisms purchase avanafil 50 mg without a prescription. GPCRs can be detected in situ in cell or tissue preparations using immunochemical techniques and receptor-specific an- Biochemical Methods to AssaySpecific tibodies purchase avanafil 100 mg visa. Antibodies that recognize the native receptor pro- Receptor Trafficking Processes tein can be used to examine the localization of endogenously expressed receptors buy avanafil 200 mg on-line, whereas epitope-tagging methods (see Whereas microscopic imaging can readily provide a great above) can be used to detect mutated versions of the receptor deal of qualitative information about GPCR localization protein or as a means to detect recombinant receptors for and trafficking, it can be quite challenging to quantitiate A B FIGURE 22. Visualization of HA epitope-tagged dopamine D1 receptors in transfected cells, using a fluorochrome-labeled secondary antibody and fluorescence microscopy. The ability of this receptortoundergoregulatedinternalizationis indicatedbythedopamine-inducedredistribution of immunoreactive receptors from the plasma membrane (visualized as linear staining at the cell periphery) to endocytic vesicles (visualized as punctate structures located throughout the cytoplasm). In addition to being extremely a specific subcellular localization or to measure accurately useful for examining posttranslational modifications of the rate or extent of specific trafficking processes. The im- GPCRs, in some cases it is possible to use these techniques portance of these processes has motivated the development to isolate receptor-containing complexes that presumably of biochemical methods for examining GPCR trafficking. The In addition to their utility for receptor localization, antibod- basic idea is to immunopurify a specific GPCR from cell ies specifically recognizing GPCRs facilitate biochemical or tissue extracts (or from a partially purified subcellular studies of GPCR trafficking using techniques adapted from fraction prepared from a cell or tissue lysate) using an anti- other areas of cell and molecular biology. For example, one body recognizing the native receptor or an engineered epi- method that has been extremely useful for quantitative stud- tope tag, and then to analyze proteins specifically associated ies of GPCR endocytosis is cell-surface biotinylation cou- with this complex using a different antibody. In general, pled with immunoprecipitation of receptors. Proteins pres- this is accomplished by immunoprecipitation of the receptor ent in the plasma membrane of cells can be specifically followed by analysis of associated proteins in the complex labeled by incubating intact cells in the presence of biotin by immunoblotting with the appropriate additional anti- coupled to an activated ester, which is membrane-imper- body. In some cases, the protein complexes are sufficiently meant and therefore forms a covalent bond only with ex- stable that they remain associated through the initial immu- posed amine moieties present in plasma membrane proteins. In other cases this is not true, In general, biotinylation in this manner does not adversely and the complexes dissociate before the receptor can be affect GPCR function, allowing biotinylation to be used as purified from the extract. In this case, various chemical a chemical tag for surface receptors. Using variations of this basic biochemistry, it is possi- teraction with heterotrimeric G proteins (52) and with - ble to measure a wide variety of membrane trafficking pro- arrestins (53), and to examine the regulation of these protein cesses. For example, internalization of GPCRs has been interactions by ligand-induced activation of the receptor. Recent studies provide strong support for this idea and, specifically, provide evidence for homo- and heterodimeri- Methods for Examining Specific Protein zation of individual GPCRs in vivo. This principle is per- Interactions Involved in GPCR Function haps best established for receptor tyrosine kinases, where it and Regulation is well established that oligomerization of receptors is re- A salient lesson emerging from recent cell biological studies quired for appropriate ligand-dependent signal transduction is that GPCR signal transduction can be viewed, in essence, (54). A relatively early hint that GPCRs may also undergo as a dynamically regulated network of protein–protein in- oligomerization came from studies of the 2-adrenergic re- teractions that occur in specific subcellular locations. There- ceptor using epitope-tagging techniques, where it was ob- fore, an important goal of current and future research is to served that receptors tagged with one epitope could specifi- define these critical protein interactions and elucidate their cally coimmunoprecipitate receptors tagged with a distinct temporal and spatial regulation in intact cells and tissues. More recently, evidence for oligomerization of many Coimmunoprecipitation Techniques to GPCRs has been reported. A particularly compelling exam- Examine Defined Protein Interactions ple of this is the recent observation that distinct subtypes with GPCRs in Intact Cells of GABA-B receptor hetero-oligomerize in cells, and that As discussed above, it is possible to rapidly purify GPCRs oligomerization is essential for the formation of recombi- from cell or tissue extracts using receptor-specific antibodies nant receptors possessing the functional properties charac- 22: G-Protein–Coupled Receptors 285 teristic of native GABA-B receptors observed in vivo interactions. A cDNA library prepared from a tissue of inter- (57,58). Both the bait and prey expressed opioid receptors (59). In a recently published study transcription of the reporter gene. However, if the fused (60), glutathione S-transferase (GST)-fusion proteins en- bait and prey polypeptides form a sufficiently stable pro- coding the C-terminal tail of the D5 receptor were shown tein–protein interaction, they bring their corresponding to interact with the GABA-A receptor present in rat hippo- DNA binding and transcriptional activation domains into campal extracts. Additionally, using an antibody recogniz- close proximity, thus reconstituting transcriptional activa- ing the dopamine D5 receptor, it was possible to coimmu- tion of the reporter gene. Transformed yeast cells containing noprecipitate the GABA-A receptor from cell extracts. In addition to known proteins that mediate and regulate Protein interactions suggested to occur by the yeast two- GPCR signaling (heterotrimeric G proteins, GRKs, ar- hybrid system can be examined using various in vitro bio- restins), which were originally identified by functional as- chemical techniques, such as affinity chromatography facili- says using biochemical purification, cDNA cloning meth- tated by GST-fusion proteins. In addition to serving as an ods have facilitated the identification of additional protein independent assay for previously defined candidate interact- interactions with GPCRs that were completely unantici- ing proteins, this method can be used to identify novel pated (61). These novel protein interactions, while their protein interactions with GPCRs de novo (63).
The PCAM could serve as a systematic way of recording needs and actions to be shared across the primary care team (GPs effective 50mg avanafil, nurses order 50mg avanafil, links or other social care roles) generic avanafil 50 mg mastercard. Patient and public involvement One of the key benefits of including PPI in clinical trials and on trial design is that they are likely to make studies more feasible order avanafil 50 mg on line, at least in terms of patient recruitment. Research has shown that trials with higher levels of PPI are four times more likely to recruit to target. These can help to identify necessary adjustments to improve recruitment and retention. The PPI partners in this study did indeed help to shape the recruitment strategy for patients, which was to opt in to either a focus group study or involved opting in to completing questionnaires and a possible interview. The outcomes needed to include measures of physical health, mental health and social needs. We also required information on actions undertaken by nurses (advice, referrals, signposting) and on whether or not patients had taken up this advice, referral or signposting to services. The complexity of the study design, and its attempt to gather multiple outcomes at both the nurse level and the patient level, was not lost on our PPI members, as we worked together to gather the required knowledge in the most efficient manner. This was probably helped by the degree of knowledge of research that our PPI members had and their enthusiasm for the study. They contributed to the many discussions throughout the study on how to address this and were reassured that the team had tried all possible avenues within the time scale available to achieve practice recruitment and retention. In reflecting on our PPI as a team, we thought it best to allow our PPI members to write their own contributions to this. We asked them to reflect on their experience of working with us and whether or not we could have done anything differently to enable their participation in the study. Their responses are included in the following two subsections. My experience and comments on the Patient Centred Assessment Method process; by patient representative 1 As a patient representative I appreciate the requirement and desirability for academia to be sometimes balanced by a lay point of view and if not present the journey from concept to publication may not be as comprehensive as it could be. I sit as a patient representative (I prefer the term representative patient) on several committees and research groups so feel qualified to state that my experience with this feasibility study was one of the best in terms of support, inclusiveness, consideration and birthday cakes. The panel made an effort to explain any terms or acronyms I or my fellow PR [patient representative] were unfamiliar with and always listened to our viewpoints and took the time to solicit our thoughts. As to the actual content of the study I share the disappointment on the paucity of the total numbers of patients and practices involved but strongly believe that this holistic approach will show many benefits. My particular thanks must go to Professor Maxwell, Dr Carina Hibberd and Ms Nadine Dougall. My experience and comments on the Patient Centred Assessment Method process; by patient representative 2 As a sufferer from a sluggish (not to say absent) NHS protocol in dealing with anxiety issues caused by the diagnosis of a cardiac problem, I joined the Living Better Project Steering Committee which was an RCGP-led study aiming to improve the care of people with LTCs in primary care, hoping for procedural improvement. Unfortunately the results of the research resulting from the Living Better project did not translate into the hoped for improved protocol to establish a route to identify co-lateral problems which frequently resulted alongside a chronic disease diagnosis. But, in PCAM, I saw an opportunity to introduce a method to improve this situation within the existing structure. Encouraged to be involved from the start in discussing the initial documentation and to join the Steering Committee by the key researchers it has been a pleasure to be involved with and to follow the development of this ambitious project. Always encouraged to participate fully in committee discussions and to contribute ideas throughout, and to feel free to criticise, my lay colleague and I were kept involved in all of the developing problems by the project leaders as well as in the successes. Our involvement in the discussion of the final report has also been thorough. The infectious enthusiasm of the researchers and their stoicism when things were difficult have been singular. There is no doubt in my mind that, in PCAM, there is the germ of an idea which will become part of NHS protocol in the years to come. This issue may be freely reproduced for the purposes of private research and study and extracts (or indeed, the full report) may be included in professional journals 75 provided that suitable acknowledgement is made and the reproduction is not associated with any form of advertising. Applications for commercial reproduction should be addressed to: NIHR Journals Library, National Institute for Health Research, Evaluation, Trials and Studies Coordinating Centre, Alpha House, University of Southampton Science Park, Southampton SO16 7NS, UK. DISCUSSION Strengths and limitations The strength of this study is that it was designed as a feasibility study that has fully tested practice, nurse and patient recruitment and retention. The combination of the five studies, which all contribute to the overall aims of the study, means that, often, multiple sources of information could be used to contribute to overall study findings.