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.

The effect of an algorithm-based sedation guideline on the duration of mechanical ventilation in an Australian intensive care unit

To examine the effect of an algorithm-based sedation guideline developed in a North American intensive care unit (ICU) on the duration of mechanical ventilation of patients in an Australian ICU. The intervention was tested in a pre-intervention, post-intervention comparative investigation in a 14-bed adult intensive care unit. Adult mechanically ventilated patients were selected consecutively (n =322) The pre-intervention and post-intervention groups were similar except for a higher number of patients with a neurological diagnosis in the pre-intervention group. An algorithm-based sedation guideline including a sedation scale was introduced using a multifaceted implementation strategy. The median duration of ventilation was 5.6 days in the post-intervention group, compared with 4.8 days for the pre-intervention group (P = 0.99). The length of stay was 8.2 days in the post-intervention group versus 7.1 days in the pre-intervention group (P = 0.04). There were no statistically significant differences for the other secondary outcomes, including the score on the Experience of Treatment in ICU 7 item questionnaire, number of tracheostomies and number of self-extubations. Records of compliance to recording the sedation score during both phases revealed that patients were slightly more deeply sedated when the guideline was used. The use of the algorithm-based sedation guideline did not reduce duration of mechanical ventilation in the setting of this study.

Changes in joint stability with muscle contraction measured from transmission of mechanical vibration

A non-invasive in vivo technique was developed to evaluate changes in wrist joint stability properties induced by increased co-activation of the forearm muscles in a gripping task. Mechanical vibration at 45, 50 and 55 Hz was applied to the radial head in ten healthy volunteers. Vibrations of the styloid process of the radius and the distal end of the metacarpal bone of the index finger were measured with triaxial accelerometers. Joint stability properties were quantified by the transfer function gain between accelerations on either side of the wrist-joint. Gain was calculated with the muscles at rest and at five force levels ranging from 5% to 25% of maximum grip force (%MF). During contraction the gain was significantly greater than in control trial (0%MF) for all contractions levels at 45 and 50 Hz and a trend for 15%MF and higher at 55 Hz. Group means of contraction force and gain were significantly correlated at 45 (R-2 = 0.98) and 50 Hz (R-2 = 0.72), but not at 55 Hz (R-2 = 0.10). In conclusion, vibration transmission gain may provide a method to evaluate changes in joint stability properties. (c) 2005 Published by Elsevier Ltd.

Screening of obstructive and central apnoea/hypopnoea in children using variability: A preliminary study

Aim: Polysomnography (PSG) is the current standard protocol for sleep disordered breathing (SDB) investigation in children. Presently, there are limited reliable screening tests for both central (CE) and obstructive (OE) respiratory events. This study compared three indices, derived from pulse oximetry and electrocardiogram ( ECG), with the PSG gold standard. These indices were heart rate (HR) variability, arterial blood oxygen de-saturation (SaO(2)) and pulse transit time (PTT). Methods: 15 children (12 male) from routine PSG studies were recruited (aged 3 - 14 years). The characteristics of the three indices were based on known criteria for respiratory events (RPE). Their estimation singly and in combination was evaluated with simultaneous scored PSG recordings. Results: 215 RPE and 215 tidal breathing events were analysed. For OE, the obtained sensitivity was HR (0.703), SaO(2) (0.047), PTT (0.750), considering all three indices (0) and either of the indices (0.828) while specificity was (0.891), (0.938), (0.922), (0.953) and (0.859) respectively. For CE, the sensitivity was HR (0.715), SaO(2) (0.278), PTT (0.662), considering all indices (0.040) and either of the indices (0.868) while specificity was (0.815), (0.954), (0.901), (0.960) and (0.762) accordingly. Conclusions: Preliminary findings herein suggest that the later combination of these non-invasive indices to be a promising screening method of SDB in children.

Rapid screening of 4000 individuals for germ-line variations in the BRAF gene

Background: The BRAF gene is frequently somatically altered in malignant melanoma. A majority of variations are at the valine 600 residue leading to a V600E substitution that constitutively activates the kinase. We screened 4000 patient and control DNAs for germ-line variations at the valine 600 residue. Methods: We developed a novel assay by adapting single-base variation assays and software for MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry to screen for all 5 reported variants at codon 600 of the BRAF gene. We screened a case-control collection comprising samples from 1082 melanoma patients and 154 of their unaffected relatives from 1278 families and from 2744 individuals from 659 unselected twin families with no history of melanoma. A panel of 66 melanoma cell lines was used for variation-positive controls. Results: All melanoma cell lines that we had found previously to carry a codon 600 variation were verified in this study. Three of the 4 possible variants (V600E n = 47, V600K n = 2, V600R n = 1) were detected, but no case of V600D was available. No germ-line variants were found in the samples from the 3980 melanoma patients or from the control individuals. Conclusions: This new assay is a high-throughput, automated alternative to standard sequencing and can be used as a rapid initial screen for somatic variants associated with melanoma. Germ-line variants at valine 600 are unlikely to exist and do not contribute to the reported role of the BRAF gene in melanoma predisposition. (c) 2006 American Association for Clinical Chemistry.