A yellow fluorescent protein-based assay for high-throughput screening of glycine and GABA(A) receptor chloride channels

There is a significant clinical need to identify novel ligands with high selectivity and potency for GABA(A), GABA(C) and glycine receptor Cl- channels. Two recently developed, yellow fluorescent protein variants (YFP-I152L and YFP-V163S) are highly sensitive to quench by small anions and are thus suited to reporting anionic influx into cells. The aim of this study was to establish the optimal conditions for using these constructs for high-throughput screening of GABA(A), GABA(C) and glycine receptors transiently expressed in HEK293 cells. We found that a 70% fluorescence reduction was achieved by quenching YFP-I152L with a 10 s influx of I- ions, driven by an extemal I- concentration of at least 50 mM. The fluorescence quench was rapid, with a mean time constant of 3 s. These responses were similar for all anion receptor types studied. We also show the assay is sufficiently sensitive to measure agonist and antagonist concentration-responses using either imaging- or photomultiplier-based detection systems. The robustness, sensitivity and low cost of this assay render it suited for high-throughput screening of transiently expressed anionic ligand-gated channels. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Afterglows, redshifts, and properties of swift gammaray bursts

We present optical, near-IR, and radio follow-up of 16 Swift bursts, including our discovery of nine afterglows and a redshift determination for three. These observations, supplemented by data from the literature, provide an afterglow recovery rate of 52% in the optical/near-IR, much higher than in previous missions (BeppoSAX, HETE-2, INTEGRAL, and IPN). The optical/near-IR afterglows of Swift events are on average 1.8 mag fainter at t = 12 hr than those of previous missions. The X-ray afterglows are similarly fainter than those of pre-Swift bursts. In the radio the limiting factor is the VLA threshold, and the detection rate for Swift bursts is similar to that for past missions. The redshift distribution of pre-Swift bursts peaked at z similar to 1, whereas the six Swift bursts with measured redshifts are distributed evenly between 0.7 and 3.2. From these results we conclude that ( 1) the pre-Swift distributions were biased in favor of bright events and low-redshift events, ( 2) the higher sensitivity and accurate positions of Swift result in a better representation of the true burst redshift and brightness distributions ( which are higher and dimmer, respectively), and (3) similar to 10% of the bursts are optically dark, as a result of a high redshift and/or dust extinction. We remark that the apparent lack of low-redshift, low-luminosity Swift bursts and the lower event rate than prelaunch estimates ( 90 vs. 150 per year) are the result of a threshold that is similar to that of BATSE. In view of these inferences, afterglow observers may find it advisable to make significant changes in follow-up strategies of Swift events. The faintness of the afterglows means that large telescopes should be employed as soon as the burst is localized. Sensitive observations in RIz and near-IR bands will be needed to discriminate between a typical z similar to 2 burst with modest extinction and a high-redshift event. Radio observations will be profitable for a small fraction (similar to 10%) of events. Finally, we suggest that a search for bright host galaxies in untriggered BAT localizations may increase the chance of finding nearby low-luminosity GRBs.

Cardiovascular disease and PPARdelta: Targeting the risk factors

Metabolism, in part, is regulated by the peroxisome proliferator-activated receptors (PPARs). The PPARs act as nutritional lipid sensors and three mammalian PPAR subtypes designated PPARalpha (NR1C1), PPARgamma (NR1C3) and PPARdelta (NR1C2) have been identified. This subgroup of nuclear hormone receptors binds DNA and controls gene expression at the nexus of pathways that regulate lipid and glucose homeostasis, energy storage and expenditure in an organ-specific manner. Recent evidence has demonstrated activation of PPARdelta in the major mass peripheral tissue (ie, adipose and skeletal muscle). It enhances glucose tolerance, insulin-stimulated glucose disposal, lipid catabolism, energy expenditure, cholesterol efflux and oxygen consumption. These effects positively influence the blood-lipid profile. Furthermore, PPARdelta activation produces a predominant type I/slow twitch/oxidative muscle fiber phenotype that leads to increased endurance, insulin sensitivity and resistance to obesity. PPARdelta has rapidly emerged as a potential target in the battle against dyslipidemia, insulin insensitivity, type II diabetes and obesity, with therapeutic efficacy in the treatment of cardiovascular disease risk factors. GW-501516 is currently undergoing phase II safety and efficacy trials in human volunteers for the treatment of dyslipidemia. The outcome of these clinical trials are eagerly awaited against a background of conflicting reports about cancer risks in genetically predisposed animal models. This review focuses on the potential pharmacological utility of selective PPARdelta agonists in the context of risk factors associated with metabolic and cardiovascular disease.

The acute effects of mild traumatic brain injury on finger tapping with and without word repetition

This study aimed to investigate the acute effects of mild Traumatic Brain Injury (mTBI) on the performance of a finger tapping and word repetition dual task in order to determine working memory impairment in mTBI Sixty-four (50 male, 14 female) right-handed cases of mTBI and 26 (18 male and 8 female) right-handed cases of orthopaedic injuries were tested within 24 hours of injury. Patients with mTBI completed fewer correct taps in 10 seconds than patients with orthopaedic injuries, and female mTBI cases repeated fewer words. The size of the dual task decrement did not vary between groups. When added to a test battery including the Rapid Screen of Concussion (RSC; Comerford, Geffen, May, Medland T Geffen, 2002) and the Digit Symbol Substitution Test,finger tapping speed accounted for 1% of between groups variance and did not improve classification rates of male participants. While the addition of tapping rate did not improve the sensitivity and specificity of the RSC and DSST to mTBI in males, univariate analysis of motor performance in females indicated. that dual task performance might be diagnostic. An increase in female sample Size is warranted. These results confirm the view that there is a generalized slowing of processing ability following mTBI.

Accounting for management costs in sensitivity analyses of matrix population models

Traditional sensitivity and elasticity analyses of matrix population models have been used to p inform management decisions, but they ignore the economic costs of manipulating vital rates. For exam le, the growth rate of a population is often most sensitive to changes in adult survival rate, but this does not mean that increasing that rate is the best option for managing the population because it may be much more expensive than other options. To explore how managers should optimize their manipulation of vital rates, we incorporated the cost of changing those rates into matrix population models. We derived analytic expressions for locations in parameter space where managers should shift between management of fecundity and survival, for the balance between fecundity and survival management at those boundaries, and for the allocation of management resources to sustain that optimal balance. For simple matrices, the optimal budget allocation can often be expressed as simple functions of vital rates and the relative costs of changing them. We applied our method to management of the Helmeted Honeyeater (Lichenostomus melanops cassidix; an endangered Australian bird) and the koala (Phascolarctos cinereus) as examples. Our method showed that cost-efficient management of the Helmeted Honeyeater should focus on increasing fecundity via nest protection, whereas optimal koala management should focus on manipulating both fecundity and survival simultaneously, These findings are contrary to the cost-negligent recommendations of elasticity analysis, which would suggest focusing on managing survival in both cases. A further investigation of Helmeted Honeyeater management options, based on an individual-based model incorporating density dependence, spatial structure, and environmental stochasticity, confirmed that fecundity management was the most cost-effective strategy. Our results demonstrate that decisions that ignore economic factors will reduce management efficiency.

A sensitive HPLC method for measuring bacterial proteolysis and proteinase activity in UHT milk

A sensitive quantitative reversed-phase HPLC method is described for measuring bacterial proteolysis and proteinase activity in UHT milk. The analysis is performed on a TCA filtrate of the milk. The optimum concentration of TCA was found to be 4%; at lower concentrations, non-precipitated protein blocked the HPLC while higher concentrations yielded lower amounts of peptides. The method showed greater sensitivity and reproducibility than a fluorescamine-based method. Quantification of the HPLC method was achieved by use of an external dipeptide standard or a standard proteinase. (c) 2006 Elsevier Ltd. All rights reserved.

A new instrument for targeting falls prevention interventions was accurate and clinically applicable in a hospital setting

Background and Objective: To describe the diagnostic accuracy and practical application of the Peter James Centre Falls Risk Assessment Tool (PJC-FRAT), a multidisciplinary falls risk screening and intervention deployment instrument. Methods: In phase 1, the accuracy of the PJC-FRAT was prospectively compared to a gold standard (the STRATIFY) on a cohort of subacute hospital patients (n = 122). In phase 2, the PJC-FRAT was temporally reassessed using a subsequent cohort (n = 316), with results compared to those of phase 1. Primary outcomes were falls (events), fallers (patients who fell), and hospital completion rates of the PJC-FRAT. Results: In phase 1, PJC-FRAT accuracy of identifying falters showed sensitivity of 73% (bootstrap 95% confidence interval CI = 55, 90) and specificity of 75% (95% CI = 66, 83), compared with the STRATIFY (cutoff >= 2/5) sensitivity of 77% (95% CI = 59, 92) and specificity of 51% (95% CI = 41, 61). This difference was not significant. In phase 2, accuracy of nursing staff using the PJC-FRAT was lower. PJC-FRAT completion rates varied among disciplines over both phases: nurses and physiotherapists, >= 90%; occupational therapists, >= 82%; and medical officers, >= 57%. Conclusion: The PJC-FRAT was practical and relatively accurate as a predictor of falls and a deployment instrument for falls prevention interventions, although continued staff education may be necessary to maintain its accuracy. (c) 2006 Elsevier Inc. All rights reserved.

Can myocardial contrast echo provide incremental benefit to exercise echo? Implications of the intensity and duration of hyperaemia

Background. Stress myocardial contrast echo (MCE) is technically challenging with exercise (Ex) because of cardiacmovementandshort duration ofhyperemia.Vasodilators solve these limitations, but are less potent for inducing abnormal wall motion (WM). We sought whether a combined dipyridamole (DI; 0.56 mg/kg i.v. 4 min) and Ex stress protocol would enable MCE to provide incremental benefit toWManalysis for detection of CAD. Methods. Standard echo images were followed by real time MCE at rest and following stress in 85 pts, 70 undergoing quantitative coronary angiography and 15 low risk pts.WMAfrom standard and LVopacification images, and then myocardial perfusion were assessed sequentially in a blinded fashion. A subgroup of 13 pts also underwent Ex alone, to assess the contribution of DI to quantitative myocardial flow reserve (MFR). Results. Significant (>50%) stenoses were present in 43 pts, involving 69 territories. Addition of MCE improved SE sensitivity for detection of CAD (91% versus 74%, P = 0.02) and better appreciation of disease extent (87% versus 65%territories, P=0.003), with a non-significant reduction in specificity. In 55 territories subtended by a significant stenosis, but with no resting WM abnormality, ability to identify ischemia was also significantly increased by MCE (82% versus 60%, P = 0.002). MFR was less with Ex alone than with DIEx stress (2.4 ± 1.6 versus 4.0 ± 1.9, P = 0.05), suggesting prolongation of hyperaemia with DI may be essential to the results. Conclusions. Dipyridamole-exercise MCE adds significant incremental benefit to standard SE, with improved diagnostic sensitivity and more accurate estimation of extent of CAD.

Evaluation and comparison of mammalian subcellular localization prediction methods

Background: Determination of the subcellular location of a protein is essential to understanding its biochemical function. This information can provide insight into the function of hypothetical or novel proteins. These data are difficult to obtain experimentally but have become especially important since many whole genome sequencing projects have been finished and many resulting protein sequences are still lacking detailed functional information. In order to address this paucity of data, many computational prediction methods have been developed. However, these methods have varying levels of accuracy and perform differently based on the sequences that are presented to the underlying algorithm. It is therefore useful to compare these methods and monitor their performance. Results: In order to perform a comprehensive survey of prediction methods, we selected only methods that accepted large batches of protein sequences, were publicly available, and were able to predict localization to at least nine of the major subcellular locations (nucleus, cytosol, mitochondrion, extracellular region, plasma membrane, Golgi apparatus, endoplasmic reticulum (ER), peroxisome, and lysosome). The selected methods were CELLO, MultiLoc, Proteome Analyst, pTarget and WoLF PSORT. These methods were evaluated using 3763 mouse proteins from SwissProt that represent the source of the training sets used in development of the individual methods. In addition, an independent evaluation set of 2145 mouse proteins from LOCATE with a bias towards the subcellular localization underrepresented in SwissProt was used. The sensitivity and specificity were calculated for each method and compared to a theoretical value based on what might be observed by random chance. Conclusion: No individual method had a sufficient level of sensitivity across both evaluation sets that would enable reliable application to hypothetical proteins. All methods showed lower performance on the LOCATE dataset and variable performance on individual subcellular localizations was observed. Proteins localized to the secretory pathway were the most difficult to predict, while nuclear and extracellular proteins were predicted with the highest sensitivity.