A pilot study of a crossover trial with randomized use of ankle-foot orthoses for people with Charcot-Marie-tooth disease.

OBJECTIVES: This was a pilot and feasibility study of a crossover trial with randomized use of ankle-foot orthoses by people with Charcot-Marie-tooth (CMT) disease, investigating the effects of these on gait parameters, practical aspects of use and achievement of goals. DESIGN: A randomized crossover trial. SETTING: The community and ambulatory care. PARTICIPANTS: Eight adults with CMT disease type 1 or 2. INTERVENTIONS: Ligaflex™, custom-made polypropylene and silicone ankle-foot orthoses worn in randomized order for three weeks each, with a washout week in-between; the orthoses of each participant's choice were then worn until 28 weeks. MAIN OUTCOME MEASURES: The primary outcome measure was gait velocity; other outcome measures included Goal Attainment Scaling; Likert scores, concerning aspects of orthosis use and gait analysis parameters. RESULTS: Gait velocity was greatest wearing polypropylene orthoses, median 0.96 (interquartile range (IQR) 0.75-1.18) ms(-1), compared with silicone orthoses, median 0.88 (0.71-1.12) ms(-1), and no orthosis, median 0.79 (0.56-0.84) ms(-1), P=0.006. The silicone orthoses met goals more successfully and scored more favourably for comfort, 5.0 (5.0-6.0), P=0.003 and pain, 5.5 (4.0-7.0), P=0.015. Future modifications to study methodology were identified, such as a longer period of wear and measurement of walking in different situations. CONCLUSIONS: This study confirmed the feasibility of a larger trial. It indicated differences in walking velocity and parameters concerning wear of the orthoses that could be explored further. A further crossover trial would require 27 participants in order to show a clinically meaningful difference in velocity of 0.13 ms(-1) with 90% power and alpha of 5%.

In situ oligonucleotide synthesis on carbon materials: stable substrates for microarray fabrication.

Glass has become the standard substrate for the preparation of DNA arrays. Typically, glass is modified using silane chemistries to provide an appropriate functional group for nucleic acid synthesis or oligonucleotide immobilization. We have found substantial issues with the stability of these surfaces as manifested in the unwanted release of oligomers from the surface when incubated in aqueous buffers at moderate temperatures. To address this issue, we have explored the use of carbon-based substrates. Here, we demonstrate in situ synthesis of oligonucleotide probes on carbon-based substrates using light-directed photolithographic phosphoramidite chemistry and evaluate the stabilities of the resultant DNA arrays compared to those fabricated on silanized glass slides. DNA arrays on carbon-based substrates are substantially more stable than arrays prepared on glass. This superior stability enables the use of high-density DNA arrays for applications involving high temperatures, basic conditions, or where serial hybridization and dehybridization is desired.

Rapid determination of RNA accessible sites by surface plasmon resonance detection of hybridization to DNA arrays.

RNA accessible sites are the regions in an RNA molecule that are available for hybridization with cDNA or RNA molecules. The identification of these accessible sites is a critical first step in identifying antisense-mediated gene suppression sites, as well as in a variety of other RNA-based analysis methods. Here, we present a rapid, hybridization-based, label-free method of identifying RNA accessible sites with surface plasmon resonance imaging (SPRi) on in situ synthesized oligonucleotide arrays prepared on carbon-on-metal substrates. The accessible sites of three pre-miRNAs, miRNA precursors of approximately 75 nt in length, were determined by hybridizing the RNA molecules to RNA-specific tiling arrays. An array composed of all possible 6mer oligonucleotide sequences was also utilized in this work, offering a universal platform capable of studying RNA molecules in a high throughput manner.

A qualitative examination of inappropriate hospital admissions and lengths of stay.

BACKGROUND: Research has shown that a number of patients, with a variety of diagnoses, are admitted to hospital when it is not essential and can remain in hospital unnecessarily. To date, research in this area has been primarily quantitative. The purpose of this study was to explore the perceived causes of inappropriate or prolonged lengths of stay and focuses on a specific population (i.e., patients with long term neurological conditions). We also wanted to identify interventions which might avoid admission or expedite discharge as periods of hospitalisation pose particular risks for this group. METHODS: Two focus groups were conducted with a convenience sample of eight primary and secondary care clinicians working in the Derbyshire area. Data were analysed using a thematic content approach. RESULTS: The participants identified a number of key causes of inappropriate admissions and lengths of stay, including: the limited capacity of health and social care resources; poor communication between primary and secondary care clinicians and the cautiousness of clinicians who manage patients in community settings. The participants also suggested a number of strategies that may prevent inappropriate admissions or reduce length of stay (LoS), including: the introduction of new sub-acute care facilities; the introduction of auxiliary nurses to support specialist nursing staff and patient held summaries of specialist consultations. CONCLUSION: Clinicians in both the secondary and primary care sectors acknowledged that some admissions were unnecessary and some patients remain in hospital for a prolonged period. These events were attributed to problems with the current capacity or structuring of services. It was noted, for example, that there is a shortage of appropriate therapeutic services and that the distribution of beds between community and sub-acute care should be reviewed.

Appropriateness of acute admissions and last in-patient day for patients with long term neurological conditions.

BACKGROUND: To examine the appropriateness of admissions and in-patient stay for patients with long term neurological conditions (LTNCs). To identify variables predictive of appropriateness and explore management alternatives. METHODS: Adults admitted as acute patients to Derby Hospitals NHS Foundation Trust (England). Data were collected prospectively and examined by a multi-disciplinary expert panel to determine the appropriateness of admission and length of stay (LoS). Management alternatives were discussed. RESULTS: A total of 119 participants were recruited. 32 admissions were inappropriate and 83 were for an inappropriate duration. Whether a participant lived in their own home was predictive of an inappropriate admission. The number of LTNCs, number of presenting complaints and whether the participant lived alone in their own home were predictive of an inappropriate LoS. For admissions judged to be inappropriate, the panel suggested management alternatives. CONCLUSION: Patients with LTNCs are being admitted to hospital when other services, e.g. ambulatory care, are available which could meet their needs. Inefficiencies in hospital procedures, such as discharge planning and patient transfers, continue to exist. Recognition of the need to plan for discharge at admission and to ensure in-patient services are provided in a timely manner may contribute towards improved efficiency.

Carbon-on-metal films for surface plasmon resonance detection of DNA arrays.

Surface plasmon resonance (SPR) imaging affords label-free monitoring of biomolecule interactions in an array format. A surface plasmon conducting metal thin film is required for SPR measurements. Gold thin films are traditionally used in SPR experiments as they are readily functionalized with thiol-containing molecules through formation of a gold-sulfur bond. The lability of this gold-thiol linkage upon exposure to oxidizing conditions and ultraviolet light renders these surfaces incompatible with light-directed synthetic methods for fabricating DNA arrays. It is shown here that applying a thin carbon overlayer to the gold surface yields a chemically robust substrate that permits light-directed synthesis and also supports surface plasmons. DNA arrays fabricated on these carbon-metal substrates are used to analyze two classes of biomolecular interactions: DNA-DNA and DNA-protein. This new strategy allows the combinatorial study of binding interactions directly from native, unmodified biomolecules of interest and offers the possibility of discovering new ligands in complex mixtures such as cell lysates.

Calcium antagonists for Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive muscle condition starting in childhood, leading to severe disability and a shortened life span. It is due to severe deficiency of the protein dystrophin which performs both structural and signalling roles within skeletal and cardiac myocytes. Calcium accumulates in dystrophic muscle cells and plays a role in cell damage. It has been hypothesised that use of calcium antagonists might reduce this calcium load and its toxic effect on muscle cells. OBJECTIVES: To evaluate the effects of calcium antagonists on muscle function and muscle strength in people with DMD. SEARCH STRATEGY: The Cochrane Neuromuscular Disease Group Trials Register (February 2008), MEDLINE (from January 1950 to March 2008) and EMBASE (from January 1947 to March 2008) were searched. Search terms were 'calcium antagonists' or 'calcium channel blocker' or 'dantrolene' or 'verapamil' or 'nifedipine' or 'flunarizine' or 'diltiazem' or 'amlodipine' or 'nicardipine' and 'Muscular Dystrophy, Duchenne'. Bibliographies in reports of any trials were also searched. SELECTION CRITERIA: All randomised or quasi-randomised controlled trials of any calcium antagonist in people with DMD. DATA COLLECTION AND ANALYSIS: Both authors assessed all identified trials for inclusion in the study on the basis of whether they fulfilled the selection criteria. Both authors extracted data from the trials and assessed the methodological quality. Had there been more than one trial of the same intervention and outcome of sufficient methodological quality, we had planned to undertake a meta-analysis. MAIN RESULTS: Five randomised or quasi-randomised double-blind trials fulfilled the selection criteria, but were not sufficiently comparable to undertake a meta-analysis. The drugs studied were verapamil (8 participants), diltiazem (56 participants), nifedipine (105 participants) and flunarizine (27 participants). There were limitations in the description of blinding and randomisation, and definition of outcome measures. One trial, using verapamil, showed a difference between groups in muscle force measured by ergometry, but also revealed cardiac side effects. The numbers of people included in the trials were low, and so the studies may not have included enough people for sufficient power to detect small differences in muscle force or function between placebo and control groups. In addition, calcium antagonists were in an early stage of development and some of the second generation drugs that have a better side effect profile, such as amlodipine, have not been studied. AUTHORS' CONCLUSIONS: There is no evidence to show a significant beneficial effect of calcium antagonists on muscle function in DMD.

A presenilin 1 mutation (Arg278Ser) associated with early onset Alzheimer's disease and spastic paraparesis.

Early onset familial Alzheimer's disease (EOFAD) has been associated with mutations in three genes, of which presenilin 1 (PSEN1) mutations are the most frequent. Several families with an association of progressive dementia and spastic paraplegia caused by PSEN1 mutations have been described. Here we described a novel PSEN1 mutation that was associated with dementia and spastic paraplegia in a family with 5 affected individuals in three generations. The proband was a 44-year-old woman who presented with 5 years history of progressive difficulties in walking, cognition and visuospatial impairment. Her maternal grandmother, mother and two maternal aunts also had similar neurological presentation. Molecular genetic analysis showed a missense mutation predicted to substitute an arginine residue for a serine residue at position 278 in the PSEN1 polypeptide (Arg278Ser). The novel PSEN1 mutation identified in this patient adds to the diverse list of existing mutations causing EOFAD associated with spastic paraparesis.

Service provision for adults with long-term disability: a review of services for adults with chronic neuromuscular conditions in the United Kingdom.

The purpose of the study is to determine the level of service provision for adults with neuromuscular disease in the United Kingdom. The method is questionnaire survey of clinicians running specialist clinics for adults with NMD. Specialist services are mostly concentrated in areas of high population density with rural areas being less well served. Access to specialist clinical services is generally good, although funding for respiratory support is erratic. Joint paediatric/adult or adolescent clinics are relatively uncommon. Availability of therapists with an interest in muscular disease is variable. There are long waiting times for powered wheelchairs in many areas. Provision of disabled parking spaces was thought to be inadequate in most hospitals. Current service provision is inadequate to meet the complex needs of this patient population in the UK. Particular areas of concern are the transfer of care from paediatric to adult services and the availability of experienced therapists.

A questionnaire survey comparing the educational priorities of patients and medical students in the management of multiple sclerosis.

OBJECTIVE: To compare the educational priorities patients and students raise concerning the management of multiple sclerosis (MS). DESIGN/SETTING: A single-centre comparative questionnaire survey conducted in a foundation trust hospital which provides teaching for one UK medical school. PARTICIPANTS: A total of 255 people with multiple sclerosis (pwMS) and 125 final year medical students attending a mandatory module were invited to participate. MAIN OUTCOME MEASUREMENTS: Questionnaires were developed and piloted for this study and analysed on the basis of the International Classification of Functioning, Disability and Health terminology. RESULTS: Questionnaires were returned by 125 (50%) pwMS (age range 36-86 years; median 58) and 96 (77%) medical students (age range 22-37 years; median 23). The most commonly reported priority listed by people with MS and students concerned 'environmental contextual factors' (95.5% and 99%, respectively). PwMS focused primarily on the 'social and attitudinal aspects' of the environment (53.6%), while students expressed greater interest in the use of medications (91.7%) and investigations (14.6%) (p < 0.001). People with greater psychological or physical impact of the condition were more likely to prioritise 'health condition' topics. CONCLUSIONS: PwMS and medical students identify different topics when asked to list aspects of management of MS which they deem to be important for medical student teaching. These differences in educational priorities should be taken into consideration when teaching students about MS. The findings may also apply to other long-term neurological conditions and warrant further investigation.

Controlling oligonucleotide surface density in light-directed DNA array fabrication.

Over the past two decades high-density DNA arrays have developed into a central technology for nucleic acid analyses. Important application areas include whole-genome gene expression studies, high throughput analyses of single nucleotide polymorphisms, and, most recently, the determination of binding site specificities for transcription factors and other critical elements involved in gene regulation. A key parameter in the performance of DNA arrays is the density of the surface-bound oligonucleotides, which strongly affects both thermodynamic and kinetic aspects of DNA hybridization. In this report, we describe an approach for the control of oligonucleotide density in photolithographically fabricated DNA arrays, based upon a controlled UV light deprotection procedure. Modulation of the UV exposure permits a desired degree of deprotection of surface synthesis sites; a subsequent capping reaction to inactivate the exposed sites leaves only a desired fraction of active sites remaining for synthesis, corresponding to a lower oligonucleotide density. It is shown that the procedure is reasonably general, in that it is readily transferable to alternative substrate materials with similar results.

A tetrafluorophenyl activated ester self-assembled monolayer for the immobilization of amine-modified oligonucleotides.

A tetrafluorophenyl (TFP) ester-terminated self-assembled monolayer (SAM) for the fabrication of DNA arrays on gold surfaces is described. Activated ester SAMs are desirable for biomolecule array fabrication because they readily react with amine-containing molecules to form a stable amide linkage. N-Hydroxysuccinimide (NHS) ester SAMs are commonly used for this purpose but are subject to a competing hydrolysis side reaction, limiting their effectiveness under basic conditions. TFP was evaluated here as an alternative activated ester leaving group with a potentially greater stability under basic conditions. It is shown that TFP SAMs are much more stable to basic pH than their NHS analogs and are also more hydrophobic, which is an advantage in the fabrication of high-density spotted arrays. DNA arrays prepared on TFP SAMs at pH 10 have a 5-fold greater surface density of DNA molecules, reduced fluorescence background, and smaller spot radii than those prepared on NHS SAM analogs.

Ionizable isotopic labeling reagent for relative quantification of amine metabolites by mass spectrometry.

A powerful approach to relative quantification by mass spectrometry is to employ labeling reagents that target specific functional groups in molecules of interest. A quantitative comparison of two or more samples may be readily accomplished by using a chemically identical but isotopically distinct labeling reagent for each sample. The samples may then be combined, subjected to purification steps, and mass analyzed. Comparison of the signal intensities obtained from the isotopically labeled variants of the target analyte(s) provides quantitative information on their relative concentrations in the sample. In this report, we describe the synthesis and use of heavy and light isotopic forms of methyl acetimidate for the relative quantification of amine-containing species. The principal advantages of methyl acetimidate as a labeling reagent are that the reaction product is positively charged and hydrophobicity is increased, both of which enhance electrospray ionization efficiency and increase detection sensitivity. The quantitative nature of the analysis was demonstrated in model metabolomics experiments in which heavy and light labeled Arabidopsis extracts were combined in different ratios. Finally, the labeling strategy was employed to determine differences in the amounts of amine-containing metabolites for Arabidopsis seeds germinated under two different conditions.