Functional and Structural Characterisation of Mutant Tropoelastin Constructs

The major component of elastic fibres is elastin, formed from its soluble precursor, tropoelastin. The tropoelastin nanostructure is characterised by an N-terminal coil region, hinge region, bridge region and C-terminal foot region. Tropoelastin assembly into elastic fibres consists of distinct stages of tropoelastin synthesis, coacervation and cross-linking into mature fibres. However, the contributions of specific structural regions in tropoelastin to elastic fibre assembly are insufficiently understood. This thesis explores the significance of specific residues/regions in human tropoelastin. Mutant constructs were designed in which the bridge region R515, the hinge region E345/E414, or the N-terminal D72 have been inactivated by alanine substitution. Another mutant that contains domain 22, which is typically spliced out in native human tropoelastin, was also produced. Functional impairment of the R515A isoform has been reported in assays modelling each stage of assembly. A system was optimised to define the elastogenic potential of R515A. When added to human cells, R515A tropoelastin assembled less efficiently into elastic fibres with atypical morphology, which is partially attributed to an altered bridge and C-terminal structure. The E345A, E414A, E345A+E414A, D72A and EX22 constructs were extensively characterised via their ability to coacervate, cross-link, interact with cells, and form elastic fibres. All mutant constructs displayed varying degrees of impaired self-assembly. The shapes of the mutant species were further analysed to correlate their functional attributes to structural effects of the mutation/s. All mutants showed conformational changes consistent with biochemical properties and the expected mutation site/s. These results identify for the first time the role of these regions in maintaining the wild-type structure of human tropoelastin, and the importance of this native structure to normal tropoelastin assembly and function

Genetic methods for Rapid Detection of Medically Important Nosocomial Bactera

The role of the microbiology laboratory is (1) to provide infection control information, so that highly transmissible isolates may be identified and appropriate control measures instigated as rapidly as possible and (2) to provide adequate information to the clinician enabling correct antibiotic choices to be made, particularly in the critically ill. Microbiological data is by definition slow as it is culture dependent: this study focused on the development of genetic, culture-independent methods for detection of resistance in nosocomial pathogens that could be introduced into the routine microbiology department and would fit into the routine workflow with a consequent reduction in time to result. Initially a duplex real-time polymerase chain reaction was developed for the rapid identification and detection of S. aureus and methicillin-resistance. This was optimised for immediate as-needs testing of positive blood cultures signalling with “Gram positive cocci, possibly staphylococcus” evident on Gram stain, on a random access real-time PCR platform. This technology, allowing early identification of S. aureus and its susceptibility to methicillin, by simple automated methodology, may soon become the standard for all microbiology laboratories servicing the critically ill. The second part of the study involved the development of a selective broth and multiplex PCR for detection of three important nosocomial isolates at this institution, methicillin-resistant S. aureus (MRSA), carbapenem-resistant Enterobacteriaceae, and multi-resistant Acinetobacter baumannii (MRAB). A multiplex PCR using four primer sets was designed to detect low colonisation levels of these isolates after overnight incubation in selective broth, significantly reducing the time to result and associated costs. This potentially useful epidemiological screening tool is practical, reproducible and sensitive with the potential of moving to an automated test (using real-time PCR, for example) in the future. The availability of early negative results judged by simple visual scanning (or by densitometry), means that the result is less operator-dependent, potentially reducing error rate. The last part of the study dealt with an important resistance phenotype, aminoglycoside resistance. There had been no recent comprehensive local surveys performed to determine the frequency of aminoglycoside resistance amongst the Enterobacteriaceae, or to identify the genetic determinants and their transmissibility. The isolates collected for the study were all resistant to at least one of gentamicin, tobramycin or amikacin. Identification of integron cassette arrays and use of specific internal primers identified at least one genetic determinant for gentamicin and tobramycin resistance in 22 of 23 isolates. Three isolates had two aminoglycoside resistance genes, and three isolates had three aminoglycoside resistance genes identified (Table 6.1). Transferable gentamicin-resistant plasmids were predominant amongst Klebsiella spp., but less so amongst Enterobacter spp. and E. coli. Gentamicin-resistant Klebsiella spp. were often ESBL positive, the genetic determinants of which were typically co-transferred on a conjugative plasmid. The importance of screening at a local level was demonstrated by the unexpected predominance of aac(6')-IIc amongst Enterobacter spp. and the detection of a new gene (aac(6')-LT). This part of the study has provided an understanding of the primary aminoglycoside resistance genes present in the local setting and their association with other resistances. This knowledge will allow development of assays for patient screening (clinical isolates and colonising flora), to better understand the epidemiology of aminoglycoside resistance and to allow better choice of antibiotic therapy related to presence or absence of these genes

Style and interpretation in the nineteenth‐century German violin school with particular reference to the three sonatas for pianoforte and violin by Johannes Brahms

From the mid nineteenth to early twentieth centuries the performance of Brahms’s music was intricately bound with the performance style of artists within his circle. In violin playing Joseph Joachim (1831‐1907) was the foremost exponent of the German violin school. The stylistic characteristics of this school, which included selective use of a pre‐modern style of vibrato, prominent application of portamento, predominantly legato approach to bow strokes and the frequent and noticeable modification of tempo and rhythm, were considered indispensable expressive devices by Joachim, Brahms and others associated with this circle. While the use of such devices in the nineteenth century has been well documented in published research over the past 15 years or so, there is currently much contention about the extent to which such devices were employed. Importantly, in addition to written documentation and solo recordings, this thesis examines recordings of chamber ensembles—whose members had a connection to the German violin school and/or Brahms—that as yet have been little consulted as primary source evidence. Spectrogram analyses of many of these recordings provide definitive evidence of vibrato that was narrow in width, fast, and applied selectively. Other new evidence in my thesis strongly supports the hypothesis that portamento, tempo modification and rhythmic alteration were used to a much greater extent than today, and this significantly enhanced the rhetorical features in Brahms’s music. A detailed Performance Edition with Critical Notes about Brahms’s three Sonatas for Pianoforte and Violin Opp. 78, 100 and 108, applies the evidence elucidated throughout the thesis

The patient with severe traumatic brain injury: clinical decision-making: the first 60 min and beyond

Purpose of reviewThere is an urgent need to discuss the uncertainties and paradoxes in clinical decision-making after severe traumatic brain injury (s-TBI). This could improve transparency, reduce variability of practice and enhance shared decision-making with proxies.Recent findingsClinical decision-making on initiation, continuation and discontinuation of medical treatment may encompass substantial consequences as well as lead to presumed patient benefits. Such decisions, unfortunately, often lack transparency and may be controversial in nature. The very process of decision- making is frequently characterized by both a lack of objective criteria and the absence of validated prognostic models that could predict relevant outcome measures, such as long-term quality and satisfaction with life. In practice, while treatment-limiting decisions are often made in patients during the acute phase immediately after s-TBI, other such severely injured TBI patients have been managed with continued aggressive medical care, and surgical or other procedural interventions have been undertaken in the context of pursuing a more favorable patient outcome. Given this spectrum of care offered to identical patient cohorts, there is clearly a need to identify and decrease existing selectivity, and better ascertain the objective criteria helpful towards more consistent decision-making and thereby reduce the impact of subjective valuations of predicted patient outcome.SummaryRecent efforts by multiple medical groups have contributed to reduce uncertainty and to improve care and outcome along the entire chain of care. Although an unlimited endeavor for sustaining life seems unrealistic, treatment-limiting decisions should not deprive patients of a chance on achieving an outcome they would have considered acceptable.Scientific Assessment and Innovation in Neurosurgical Treatment Strategie

External validation of a model to predict the survival of patients presenting with a spinal epidural metastasis

The surgical treatment of spinal metastases is evolving. The major problem is the selection of patients who may benefit from surgical treatment. One of the criteria is an expected survival of at least 3 months. A prediction model has been previously developed. The present study has been performed in order to validate externally the model and to demonstrate that this model can be generalized to other institutions and other countries than the Netherlands. Data of 356 patients from five centers in Germany, Spain, Sweden, and the Netherlands who were treated for metastatic epidural spinal cord compression were collected. Hazard ratios in the test population corresponded with those of the developmental population. However, the observed and the expected survival were different. Analysis revealed that the baseline hazard function was significantly different. This tempted us to combine the data and develop a new prediction model. Estimating iteratively, a baseline hazard was composed. An adapted prediction model is presented. External validation of a prediction model revealed a difference in expected survival, although the relative contribution of the specific hazard ratios was the same as in the developmental population. This study emphasized the need to check the baseline hazard function in external validation. A new model has been developed using an estimated baseline hazar

Inter-method reliability of the modified Rankin Scale in patients with subarachnoid hemorrhage

Background and objectives The modified Rankin Scale (mRS) is one of the most frequently used outcome measures in trials in patients with an aneurysmal subarachnoid hemorrhage (aSAH). The assessment method of the mRS is often not clearly described in trials, while the method used might influence the mRS score. The aim of this study is to evaluate the inter-method reliability of different assessment methods of the mRS.Methods This is a prospective, randomized, multicenter study with follow-up at 6 weeks and 6 months. Patients aged >= 18 years with aSAH were randomized to either a structured interview or a self-assessment of the mRS. Patients were seen by a physician who assigned an mRS score, followed by either the structured interview or the self-assessment. Inter-method reliability was assessed with the quadratic weighted kappa score and percentage of agreement. Assessment of feasibility of the self-assessment was done by a feasibility questionnaire.Results The quadratic weighted kappa was 0.60 between the assessment of the physician and structured interview and 0.56 between assessment of the physician and self-assessment. Percentage agreement was, respectively, 50.8 and 19.6%. The assessment of the mRS through a structured interview and by self-assessment resulted in systematically higher mRS scores than the mRS scored by the physician. Self-assessment of the mRS was proven feasible.Discussion The mRS scores obtained with different assessment methods differ significantly. The agreement between the scores is low, although the reliability between the assessment methods is good. This should be considered when using the mRS in clinical trials.Scientific Assessment and Innovation in Neurosurgical Treatment Strategie

Quality indicators for patients with traumatic brain injury in European intensive care units

Background: The aim of this study is to validate a previously published consensus-based quality indicator set for the management of patients with traumatic brain injury (TBI) at intensive care units (ICUs) in Europe and to study its potential for quality measur

Changing care pathways and between-center practice variations in intensive care for traumatic brain injury across Europe

Purpose: To describe ICU stay, selected management aspects, and outcome of Intensive Care Unit (ICU) patients with traumatic brain injury (TBI) in Europe, and to quantify variation across centers. Methods: This is a prospective observational multicenter study conducted across 18 countries in Europe and Israel. Admission characteristics, clinical data, and outcome were described at patient- and center levels. Between-center variation in the total ICU population was quantified with the median odds ratio (MOR), with correction for case-mix and random variation between centers. Results: A total of 2138 patients were admitted to the ICU, with median age of 49 years; 36% of which were mild TBI (Glasgow Coma Scale; GCS 13–15). Within, 72 h 636 (30%) were discharged and 128 (6%) died. Early deaths and long-stay patients (> 72 h) had more severe injuries based on the GCS and neuroimaging characteristics, compared with short-stay patients. Long-stay patients received more monitoring and were treated at higher intensity, and experienced worse 6-month outcome compared to short-stay patients. Between-center variations were prominent in the proportion of short-stay patients (MOR = 2.3, p < 0.001), use of intracranial pressure (ICP) monitoring (MOR = 2.5, p < 0.001) and aggressive treatme

Machine learning algorithms performed no better than regression models for prognostication in traumatic brain injury

Objective: We aimed to explore the added value of common machine learning (ML) algorithms for prediction of outcome for moderate and severe traumatic brain injury. Study Design and Setting: We performed logistic regression (LR), lasso regression, and ridge regression with key baseline predictors in the IMPACT-II database (15 studies, n = 11,022). ML algorithms included support vector machines, random forests, gradient boosting machines, and artificial neural networks and were trained using the same predictors. To assess generalizability of predictions, we performed internal, internal-external, and external validation on the recent CENTER-TBI study (patients with Glasgow Coma Scale <13, n = 1,554). Both calibration (calibration slope/intercept) and discrimination (area under the curve) was quantified. Results: In the IMPACT-II database, 3,332/11,022 (30%) died and 5,233(48%) had unfavorable outcome (Glasgow Outcome Scale less than 4). In the CENTER-TBI study, 348/1,554(29%) died and 651(54%) had unfavorable outcome. Discrimination and calibration varied widely between the studies and less so between the studied algorithms. The mean area under the curve was 0.82 for mortality and 0.77 for unfavorable outcomes in the CENTER-TBI study. Conclusion: ML algorithms may not outperform traditional regression approaches in a low-dimensional setting for outcome prediction after moderate or severe traumatic brain injury. Similar to regression-based prediction models, ML algorithms should be rigorously validated to ensure applicability to new populations

Frequency of fatigue and its changes in the first 6 months after traumatic brain injury: results from the CENTER-TBI study

Background: Fatigue is one of the most commonly reported subjective symptoms following traumatic brain injury (TBI). The aims were to assess frequency of fatigue over the first 6 months after TBI, and examine whether fatigue changes could be predicted by demographic characteristics, injury severity and comorbidities. Methods: Patients with acute TBI admitted to 65 trauma centers were enrolled in the study Collaborative European NeuroTrauma Effectiveness Research in TBI (CENTER-TBI). Subj