Anaesthesia and Intensive Care

The anaesthetist has seen his role steadily expand and it is hard to think of one medical specialty in which anaesthetists are not involved at least to some extent. We present a range of papers which demonstrates the breadth of anaesthesia: from its traditional realm in the operating theatre to critical care and resuscitation onto chronic pain and obstetric analgesia.peer-reviewe

The use of two novel techniques in the coagulopathic trauma patient in intensive care

Severe Trauma is a multisystem disorder with a high mortality in an often young population. It offers a big challenge to the intensivist, and any help that is available might prove decisive in saving the patient’s life. This case report highlights the use of two novel technologies in critically ill trauma patients, namely the use of activated recombinant factor VII (Novoseven®) to correct coagulopathy, and the use of a central venous oxygen saturation monitor (PresepTM with VigileoTM) to optimise perfusion.peer-reviewe

A cross-sectional survey of a dermatology outpatient service in Malta

A survey of the outpatient service provided by a consultant dermatologist at the national dermatology department in Malta was carried out. The aims of this study were to identify the main conditions being treated and to analyze management and referral practices. Possible implications for future training of primary care physicians were also investigated. The survey was carried out for one week every season over a 12-month period, giving a total study period of four weeks. Data was collected on a total of 662 patients (401 new patients and 261 follow-ups). The average waiting time for a routine clinic appointment for new cases was 4 weeks, but 18% of patients were seen within 48 hours of referral and 7% were seen within one week. Age-specific attendance rates were highest for females over 50 years and males over 60. Overall, the commonest conditions seen were chronic leg ulcers, psoriasis, skin infections and seborrhoeic keratoses. Skin biopsy was the most frequent investigation performed and topical treatment was the commonest form of therapy. Private general practitioners and government doctors based in health centres accounted for 51% and 29% of all referrals respectively. A diagnosis was offered in 65% of referral notes. Of these, 44% had a diagnosis matching that given by the dermatologist at the patient's first visit. Treatment was attempted prior to referral in 64% of patients with acne but in only 15% of patients with viral warts.peer-reviewe

Trends in sun exposure awareness and protection practices in Malta : 1999-2004

Rising skin cancer incidence rates have led to sun awareness campaigns in Malta since the early 1990s. The aim of this study is to assess the impact of these campaigns by analysing trends in sun exposure-related knowledge and behaviour amongst the Maltese people. A total of 559 Maltese pedestrians aged 16-50 years were interviewed in 1999. The same questionnaire was used to interview 304 pedestrians in 2004. More people admitted spending leisure time outdoors during peak sunshine hours in 2004 than in 1999 (85.9% vs 62.4%, p<0.001). There was a drop in people regularly wearing a hat during outdoor leisure activities from 32.4% to 18.4% (p<0.001), and from 37.5% to 9.3% (p<0.001) during outdoor work activities. Rates of regular sunscreen use remained constant at about 50% using it for outdoor leisure activities, but dropped from 25.0% to 9.3% for outdoor work (p=0.02). In 2004, 96.1% of participants having children aged less than 12 years stated that they regularly used sunscreen on their children (87.0% in 1999, p=0.01), while 66.2% said that their children regularly wore a hat (78.4% in 1999, p=0.05). More people were regarding a suntan as unhealthy in 2004 than in 1999 (62.8% versus 37.0%, p<0.001). The mass media remained the most important source of health information.peer-reviewe

Evidence for specific subunit distribution and interactions in the quaternary structure of α-crystallin

The quaternary structure of alpha-crystallin is dynamic, a property which has thwarted crystallographic efforts towards structural characterization. In this study, we have used collision-induced dissociation mass spectrometry to examine the architecture of the polydisperse assemblies of alpha-crystallin. For total alpha-crystallin isolated directly from fetal calf lens using size-based chromatography, the alpha B-crystallin subunit was found to be preferentially dissociated from the oligomers, despite being significantly less abundant overall than the alpha A-crystallin subunits. Furthermore, upon mixing molar equivalents of purified alpha A- and alpha B-crystallin, the levels of their dissociation were found to decrease and increase, respectively, with time. Interestingly though, dissociation of subunits from the alpha A- and alpha B-crystallin homo-oligomers was comparable, indicating that strength of the alpha A:alpha A, and alpha B:alpha B subunit interactions are similar. Taken together, these data suggest that the differences in the number of subunit contacts in the mixed assemblies give rise to the disproportionate dissociation of alpha B-crystallin subunits. Limited proteolysis mass spectrometry was also used to examine changes in protease accessibility during subunit exchange. The C-terminus of alpha A-crystallin was more susceptible to proteolytic attack in homo-oligomers than that of aB-crystallin. As subunit exchange proceeded, proteolysis of the alpha A-crystallin C-terminus increased, indicating that in the hetero-oligomeric form this tertiary motif is more exposed to solvent. These data were used to propose a refined arrangement for the interactions of the alpha-crystallin domains and C-terminal extensions of subunits within the alpha-crystallin assembly. In particular, we propose that the palindromic IPI motif of alpha B-crystallin gives rise to two orientations of the C-terminus

Susceptibility of Mutant SOD1 to Form a Destabilized Monomer Predicts Cellular Aggregation and Toxicity but Not In vitro Aggregation Propensity

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the rapid and progressive degeneration of upper and lower motor neurons in the spinal cord, brain stem and motor cortex. The first gene linked to ALS was the gene encoding the free radical scavenging enzyme superoxide dismutase-1 (SOD1) that currently has over 180, mostly missense, ALS-associated mutations identified. SOD1-associated fALS patients show remarkably broad mean survival times (~17 years death post-diagnosis) that are mutation dependent. A hallmark of SOD1-associated ALS is the deposition of SOD1 into large insoluble aggregates in motor neurons. This is thought to be a consequence of mutation induced structural destabilization and/or oxidative damage leading to the misfolding and aggregation of SOD1 into a neurotoxic species. Here we aim to understand the relationship between SOD1 variant toxicity, structural stability, and aggregation propensity using a combination of cell culture and purified protein assays. Cell based assays indicated that aggregation of SOD1 variants correlate closely to cellular toxicity. However, the relationship between cellular toxicity and disease severity was less clear. We next utilized mass spectrometry to interrogate the structural consequences of metal loss and disulfide reduction on fALS-associated SOD1 variant structure. All variants showed evidence of unfolded, intermediate, and compact conformations, with SOD1G37R, SOD1G93A and SOD1V148G having the greatest abundance of intermediate and unfolded SOD1. SOD1G37R was an informative outlier as it had a high propensity to unfold and form oligomeric aggregates, but it did not aggregate to the same extent as SOD1G93A and SOD1V148G in in vitro aggregation assays. Furthermore, seeding the aggregation of DTT/EDTA-treated SOD1G37R with preformed SOD1G93A fibrils elicited minimal aggregation response, suggesting that the arginine substitution at position-37 blocks the templating of SOD1 onto preformed fibrils. We propose that this difference may be explained by multiple strains of SOD1 aggregate and this may also help explain the slow disease progression observed in patients with SOD1G37R

Susceptibility of mutant SOD1 to form a destabilized monomer predicts cellular aggregation and toxicity but not in vitro aggregation propensity

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the rapid and progressive degeneration of upper and lower motor neurons in the spinal cord, brain stem and motor cortex. The first gene linked to ALS was the gene encoding the free radical scavenging enzyme superoxide dismutase-1 (SOD1) that currently has over 180, mostly missense, ALS-associated mutations identified. SOD1-associated fALS patients show remarkably broad mean survival times (~17 years death post-diagnosis) that are mutation dependent. A hallmark of SOD1-associated ALS is the deposition of SOD1 into large insoluble aggregates in motor neurons. This is thought to be a consequence of mutation induced structural destabilization and/or oxidative damage leading to the misfolding and aggregation of SOD1 into a neurotoxic species. Here we aim to understand the relationship between SOD1 variant toxicity, structural stability, and aggregation propensity using a combination of cell culture and purified protein assays. Cell based assays indicated that aggregation of SOD1 variants correlate closely to cellular toxicity. However, the relationship between cellular toxicity and disease severity was less clear. We next utilized mass spectrometry to interrogate the structural consequences of metal loss and disulfide reduction on fALS-associated SOD1 variant structure. All variants showed evidence of unfolded, intermediate, and compact conformations, with SOD1G37R, SOD1G93A and SOD1V148G having the greatest abundance of intermediate and unfolded SOD1. SOD1G37R was an informative outlier as it had a high propensity to unfold and form oligomeric aggregates, but it did not aggregate to the same extent as SOD1G93A and SOD1V148G in in vitro aggregation assays. Furthermore, seeding the aggregation of DTT/EDTA-treated SOD1G37R with preformed SOD1G93A fibrils elicited minimal aggregation response, suggesting that the arginine substitution at position-37 blocks the templating of SOD1 onto preformed fibrils. We propose that this difference may be explained by multiple strains of SOD1 aggregate and this may also help explain the slow disease progression observed in patients with SOD1G37R

The influence of the N-terminal region proximal to the core domain on the assembly and chaperone activity of αB-crystallin

αB-Crystallin (HSPB5) is a small heat-shock protein that is composed of dimers that then assemble into a polydisperse ensemble of oligomers. Oligomerisation is mediated by heterologous interactions between the C-terminal tail of one dimer and the core α-crystallin domain of another and stabilised by interactions made by the N-terminal region. Comparatively little is known about the latter contribution, but previous studies have suggested that residues in the region 54-60 form contacts that stabilise the assembly. We have generated mutations in this region (P58A, S59A, S59K, R56S/S59R and an inversion of residues 54-60) to examine their impact on oligomerisation and chaperone activity in vitro. By using native mass spectrometry, we found that all the αB-crystallin mutants were assembly competent, populating similar oligomeric distributions to wild-type, ranging from 16-mers to 30-mers. However, circular dichroism spectroscopy, intrinsic tryptophan and bis-ANS fluorescence studies demonstrated that the secondary structure differs to wild type, the 54-60 inversion mutation having the greatest impact. All the mutants exhibited a dramatic decrease in exposed hydrophobicity. We also found that the mutants in general were equally active as the wild-type protein in inhibiting the amorphous aggregation of insulin and seeded amyloid fibrillation of α-synuclein in vitro, except for the 54-60 inversion mutant, which was significantly less effective at inhibiting insulin aggregation. Our data indicate that alterations in the part of the N-terminal region proximal to the core domain do not drastically affect the oligomerisation of αB-crystallin, reinforcing the robustness of αB-crystallin in functioning as a molecular chaperone

Understanding the α-crystallin cell membrane conjunction

PURPOSE. It is well established that levels of soluble α-crystallin in the lens cytoplasm fall steadily with age, accompanied by a corresponding increase in the amount of membrane-bound α-crystallin. Less well understood, is the mechanism driving this age-dependent membrane association. The aim of this study was to investigate the role of the membrane and its associated proteins and peptides in the binding of α-crystallin. METHODS. Fibre cell membranes from human and bovine lenses were separated from soluble proteins by centrifugation. Membranes were stripped of associated proteins with successive aqueous, urea and alkaline solutions. Protein constituents of the respective membrane isolates were examined by SDS-PAGE and Western immunoblotting. Recombinant αA- and αB-crystallins were fluorescently-labeled with Alexa350® dye and incubated with the membrane isolates and the binding capacity of membrane for α-crystallin was determined. RESULTS. The binding capacity of human membranes was consistently higher than that of bovine membranes. Urea- and alkali-treated membranes from the nucleus had similar binding capacities for αA-crystallin, which were significantly higher than both cortical membrane extracts. αB-Crystallin also had a higher affinity for nuclear membrane. However, urea-treated nuclear membrane had three times the binding capacity for αB-crystallin as compared to the alkali-treated nuclear membrane. Modulation of the membrane-crystallin interaction was achieved by the inclusion of an N-terminal peptide of αB-crystallin in the assays, which significantly increased the binding. Remarkably, following extraction with alkali, full length αA- and αB-crystallins were found to remain associated with both bovine and human lens membranes. CONCLUSIONS. Fiber cell membrane isolated from the lens has an inherent capacity to bind α-crystallin. For αB-crystallin, this binding was found to be proportional to the level of extrinsic membrane proteins in cells isolated from the lens nucleus, indicating these proteins may play a role in the recruitment of αB-crystallin. No such relationship was evident for αA-crystallin in the nucleus, or for cortical membrane binding. Intrinsic lens peptides, which increase in abundance with age, may also function to modulate the interaction between soluble α-crystallin and the membrane. In addition, the tight association between α-crystallin and the lens membrane suggests that the protein may be an intrinsic component of the membrane structure