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Background: Allogeneic bone marrow transplant (alloBMT) is curative for hematologic malignancies through the graft-versus-tumor (GVT) effect but has been ineffective for solid tumors like osteosarcoma (OS). OS expresses CD155 which interacts strongly with inhibitory receptors TIGIT and CD96 but also binds to activating receptor DNAM-1 on natural killer (NK) cells. CD155 has never been targeted after alloBMT. Combining adoptively transferred allogeneic NK (alloNK) cells with CD155 blockade after alloBMT may enhance a GVT effect against OS., Methods: Murine NK cells were activated and expanded ex vivo with soluble IL-15/IL-15Rα. AlloNK and syngeneic NK (synNK) cell phenotype, cytotoxicity, cytokine production, and degranulation against CD155-expressing murine OS cell line K7M2 were assessed in vitro. Mice bearing pulmonary OS metastases underwent alloBMT and alloNK cell infusion with anti-CD155 either before or after tumor induction, with select groups receiving anti-DNAM-1 pretreated alloNK cells. Tumor growth, GVHD and survival were monitored, and differential gene expression of lung tissue was assessed by RNA microarray., Results: AlloNK cells exhibited superior cytotoxicity against CD155-expressing OS compared to synNK cells, and this activity was enhanced by CD155 blockade. CD155 blockade increased alloNK cell degranulation and interferon gamma production through DNAM-1. In vivo, CD155 blockade with alloNK infusion increased survival when treating OS that relapsed after alloBMT. No benefit was seen for treating established OS before alloBMT. Treatment with combination CD155 and anti-DNAM-1 pretreated alloNK ameliorated survival and tumor control benefits seen with CD155 blockade alone. RNA microarray showed mice treated with alloNK and CD155 blockade had increased expression of cytotoxicity genes and the NKG2D ligand H60a, whereas mice treated with anti-DNAM-1 pretreated alloNK cells resulted in upregulation of NK cell inhibitory receptor genes. Whereas blocking DNAM-1 on alloNK abrogated cytotoxicity, blocking NKG2D had no effect, implying DNAM-1:CD155 engagement drives alloNK activation against OS., Conclusions: These results demonstrate the safety and efficacy of infusing alloNK cells with CD155 blockade to mount a GVT effect against OS and show benefits are in part through DNAM-1. Defining the hierarchy of receptors that govern alloNK responses is critical to translating alloNK cell infusions and immune checkpoint inhibition for solid tumors treated with alloBMT., What Is Already Known on This Topic: Allogeneic bone marrow transplant (alloBMT) has yet to show efficacy in treating solid tumors, such as osteosarcoma (OS). CD155 is expressed on OS and interacts with natural killer (NK) cell receptors, such as activating receptor DNAM-1 and inhibitory receptors TIGIT and CD96 and has a dominant inhibitory effect on NK cell activity. Targeting CD155 interactions on allogeneic NK cells could enhance anti-OS responses, but this has not been tested after alloBMT., What This Study Adds: CD155 blockade enhances allogeneic natural killer cell-mediated cytotoxicity against OS and improved event-free survival after alloBMT in an in vivo mouse model of metastatic pulmonary OS. Addition of DNAM-1 blockade abrogated CD155 blockade-enhanced allogeneic NK cell antitumor responses., How This Study Might Affect Research Practice or Policy: These results demonstrate efficacy of allogeneic NK cells combined with CD155 blockade to mount an antitumor response against CD155-expressing OS. Translation of combination adoptive NK cell and CD155 axis modulation offers a platform for alloBMT treatment approaches for pediatric patients with relapsed and refractory solid tumors.

Electrocatalytic CO 2 reduction processes are generally coupled with the oxidation of water. Process economics can greatly improve by replacing the water oxidation with a more valuable oxidation reaction, a process called paired electrolysis. Here we report the feasibility of pairing CO 2 reduction with the oxidation of glycerol on Ni 3 S 2 /NF anodes to produce formate at both anode and cathode. Initially we optimized the oxidation of glycerol to maximize the Faraday efficiency to formate by using design of experiments. In flow cell electrolysis, excellent selectivity (up to 90 % Faraday efficiency) was achieved at high current density (150 mA/cm 2 of geometric surface area). Then we successfully paired the reduction of CO 2 with the oxidation of glycerol. A prerequisite for industrial application is to obtain reaction mixtures with a high concentration of formate to enable efficient downstream separation. We show that the anodic process is limited in formate concentration, as Faraday efficiency to formate greatly decreases when operating at 2.5 M formate (∼10 w%) in the reaction mixture due to over-oxidation of formate. We identify this as a major bottleneck for the industrial feasibility of this paired electrolysis process., (© 2023 The Authors. ChemPlusChem published by Wiley-VCH GmbH.)

Objective: To establish the prevalence of unmet need for spasticity management in care home residents in two counties of the United Kingdom., Design: Cross-sectional observational study with a six-month follow-up arm for participants with identified unmet needs., Setting: 22 care homes in Derbyshire and Nottinghamshire., Subjects: 60 care home residents with upper motor neuron syndrome-related spasticity., Interventions: No intervention. When unmet needs around spasticity management were identified, the participant's general practitioner was advised of these in writing., Main Measures: Resistance to Passive Movement Scale to assess spasticity; recording of (a) the presence of factors which may aggravate spasticity, (b) potential complications of spasticity, (c) spasticity-related needs and (d) current interventions to manage spasticity. Two assessors judged the presence or absence of needs for spasticity management and whether these needs were met by current care., Results: Out of 60 participants, 14 had no spasticity-related needs; 46 had spasticity-related needs; 11 had needs which were being met by current care and 35 participants had spasticity-related needs at baseline which were not being met by their current care. These were most frequently related to the risk of contracture development or problems with skin hygiene or integrity in the upper limb. In total, 6 participants had one or more pressure sores and 35 participants had one or more established joint contractures. A total of 31 participants were available for follow-up. Informing general practitioners of unmet needs resulted in no change to spasticity management in 23/31 cases., Conclusion: Care home residents in this study had high levels of unmet need for spasticity management.

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.

This study used a mixed-methods approach with questionnaires and semistructured interviews to explore the views and experiences of people with neuromuscular conditions who use powered mobile arm supports (PMASs). All 170 users of PMASs who had neuromuscular conditions and lived within 200 mi of the study center were contacted by post. Of the 22 who expressed an interest, 13, aged 13 to 69 yr, took part. Participants had been using the PMAS from 6 mo to 8 yr, and the majority had Duchenne muscular dystrophy. Interviews took place in participants' homes or by telephone, and participants completed the ABILHAND and the Upper-Limb Functional Index and provided details regarding their diagnosis and experience with the PMAS. Themes that emerged were the positive influence of PMASs on psychological factors and social participation; increased range of and independence in several activities, including eating, drinking, and exercise; variations in funding and time taken for provision; participants' initial reactions; timing of initial use; reasons for nonuse; and suggestions for future development. We concluded that, in this group, PMASs improved confidence, dignity, and the ability to engage in social situations, as well as increased independence in several activities. The majority of negative aspects of use would be amenable to change.

Background: Multiple observational studies have suggested that breast cancer gene (BRCA)-associated ovarian cancers have improved survival compared with BRCA-negative ovarian cancers. However, most of those studies combined BRCA1 and BRCA2 patients or evaluated only BRCA1 patients. The objective of the current study was to examine whether BRCA1-associated and BRCA2-associated ovarian cancers were associated with different outcomes., Methods: This was a single-institution, retrospective analysis of patients who had a new diagnosis of histologically confirmed stage III or IV serous ovarian, fallopian tube, or primary peritoneal cancer between January 1, 1996 and February 1, 2011 and who underwent BRCA mutation testing on 1 of 2 institutional review board-approved follow-up studies. Patients who had been tested for BRCA mutations beyond 24 months of diagnosis were excluded from analysis to minimize selection bias from including patients who were referred for genetic testing because of long survival., Results: Data from 190 patients (143 BRCA-negative patients, 30 BRCA1-positive patients, and 17 BRCA2-positive patients) were analyzed. During the study period, 73 deaths were observed (60 BRCA-negative patients, 10 BRCA1-positive patients, 3 BRCA2-positive patients). The median follow-up for the remaining 117 survivors was 2.5 years. At 3 years, 69.4%, 90.7%, and 100% of BRCA-negative patients, BRCA1-positive patients, and BRCA2-positive patients were alive, respectively. On univariate analysis, age, BRCA2 mutations, debulking status, and type of first-line therapy (intravenous or intraperitoneal) were significant predictors of overall survival. On multivariate analysis, BRCA2 mutations (hazard ratio, 0.20; 95% confidence interval, 0.06-0.65; P = .007), but not BRCA1 mutations (hazard ratio, 0.70; 95% confidence interval, 0.36-1.38; P = .31), predicted for improved overall survival compared with BRCA-negative patients. When carriers of BRCA2 mutations were directly compared with carriers of BRCA1 mutations, BRCA2 mutations appeared to confer improved overall survival (hazard ratio, 0.29; 95% confidence interval, 0.08-1.05; P = .060), although this finding did not reach significance., Conclusions: The current data suggests that BRCA2 mutations confer an overall survival advantage compared with either being BRCA-negative or having a BRCA1 mutation in high-grade serous ovarian cancer. This finding may have important implications for clinical trial design., (Copyright © 2011 American Cancer Society.)

Background: Tracheal aspirates (TAs) from critically ill neonates accumulate bacterial endotoxin and demonstrate mobilization of endotoxin-binding proteins, but the potential bioactivity of endotoxin in TAs is unknown. We characterized innate immune activation in TAs of mechanically ventilated neonates., Methods: Innate immune activation in TAs of mechanically ventilated neonates was characterized using a targeted 84-gene quantitative real-time (qRT) PCR array. Protein expression of cytokines was confirmed by multiplex assay. Expression and localization of the endotoxin-inducible antimicrobial protein Calgranulin C (S100A12) was assessed by flow cytometry. Endotoxin levels were measured in TA supernatants using the Limulus amoebocyte lysate assay., Results: Analyses by qRT-PCR demonstrated expression of pattern recognition receptors, Toll-like receptor-nuclear factor κB and inflammasome pathways, cytokines/chemokines and their receptors, and anti-infective proteins in TA cells. Endotoxin positivity increased with postnatal age. As compared with endotoxin-negative TAs, endotoxin-positive TAs demonstrated significantly greater tumor necrosis factor (TNF), interleukin (IL)-6, IL-10, and serpin peptidase inhibitor, clade E, member 1 (SERPINE1) mRNA, and IL-10, TNF, and IL-1β protein. Expression of S100A12 protein was localized to TA neutrophils., Conclusion: Correlation of endotoxin with TA inflammatory responses suggests endotoxin bioactivity and the possibility that endotoxin antagonists could mitigate pulmonary inflammation and its sequelae in this vulnerable population.

Objective: BRCA-associated and sporadic ovarian cancers have different pathologic and clinical features. Our goal was to determine if BRCA mutation status is an independent predictor of residual tumor volume following primary surgical cytoreduction., Methods: We conducted a retrospective analysis of patients with FIGO stage IIIC-IV high-grade serous ovarian cancer classified for the presence or absence of germline BRCA mutations. The primary outcome was tumor-debulking status categorized as complete gross resection (0mm), optimal but visible disease (1-10 mm), or suboptimal debulking (>10 mm) following primary surgical cytoreduction. Overall survival by residual tumor size and BRCA status was also assessed as a secondary endpoint., Results: Data from 367 patients (69 BRCA mutated, 298 BRCA wild-type) were analyzed. Rate of optimal tumor debulking (0-10 mm) in BRCA wild-type and BRCA-mutated patients were 70.1% and 84.1%, respectively (P=0.02). On univariate analysis, increasing age (10-year OR, 1.33; 95% CI, 1.07-1.65; P=0.01) and wild-type BRCA status (OR, 0.47; 95% CI, 0.23-0.94, P=0.03) were both significantly associated with suboptimal surgical outcome. On multivariate analysis, BRCA mutation status was no longer associated with residual tumor volume (OR, 0.63; 95% CI, 0.31-1.29; P=0.21) while age remained a borderline significant predictor (10-year OR, 1.25; 95% CI, 1.01-1.56; P=0.05). Both smaller residual tumor volume and mutant BRCA status were significantly associated with improved overall survival., Conclusion: BRCA mutation status is not associated with the rate of optimal tumor debulking at primary surgery after accounting for differences in patient age. Improved survival of BRCA carriers is unlikely the result of better surgical outcomes but instead intrinsic tumor biology., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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%.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Previously, it has been reported that energy consumed as alcohol prior to lunch does not result in subsequent reductions in voluntary food intake, and in some situations alcohol can increase subsequent appetite. The present study extends these findings by examining the effects of beliefs about alcohol content. Eighteen unrestrained men ate lunch 20 min after a preload of either water, an alcoholic beer or a non-alcoholic beer matched for energy content. Food intake was significantly less following the non-alcoholic beer than after alcohol or water, but when preload energy was included subjects had a higher overall energy intake on the day they consumed alcohol compared with both water and no-alcohol conditions. There were no significant differences in hunger or fullness ratings following the three drinks before or after the test meal, but the specific relationship between rated hunger and intake within the test meal was altered by the drink manipulation. The rate at which hunger decreased, and fullness increased, was slower after alcohol than after water or no-alcohol. The drinks did not alter the pleasantness of the test meal or increase hunger at the start of eating. When contrasted with previous work, these data confirm that alcohol consumed before lunch fails to reduce subsequent food intake, but also suggests that changes in rated appetite are influenced by beliefs about alcohol content.

Duchenne muscular dystrophy (DMD) causes a progressive impairment of muscle function leading to hypercapnic respiratory failure. Most studies of respiratory function in DMD have been cross-sectional rather than longitudinal, and these data have not been related to survival. We retrospectively studied 58 patients with DMD with at least 2 yr of follow-up spirometry and known vital status. Spirometry was abnormal at entry: median FEV(1) 1.60 L (range 0.4 to 2.6 L), FVC 1.65 L (range 0.45 to 2.75 L), FVC 64% predicted (range 29 to 97%). Individual rates of change of vital capacity varied, with a median annual change of -0.18 L (range 0.04 to -0.74 L), -8.0% predicted FVC (range 2 to -39%). During the study 37 patients died; the median age of death, calculated by Kaplan-Meier analysis, was 21.5 yr (range 15 to 28.5 yr). The age when vital capacity fell below 1 L was a strong marker of subsequent mortality (5-yr survival 8%). The maximal vital capacity recorded and its rate of decline (however expressed) predicted survival time. Repeated spirometric measurement provides a simple and relatively powerful means of assessing disease progression in these patients and should be considered when planning treatment trials.

REM-related oxygen desaturation occurs in advanced Duchenne muscular dystrophy (DMD) and might be an independent predictor of disease progression. We have followed 18 patients for 10 yr after an initial respiratory sleep study or until death or onset of nasal ventilation. We measured baseline spirometry, blood gas tensions, maximal respiratory pressures, and body mass index. In 11 cases, VC was recorded serially. Median survival was 50 (range, 13 to 89) mo from initial study and unrelated to age at time of study, BMI, or mouth pressures but correlated with PaCO2 (r = -0.72, p < 0.005, n = 17), minimal nocturnal SaO2 (r = 0.62, p < 0.007, n = 18) and VC (r = 0. 65, p < 0.005, n = 17). Cox regression analysis showed a VC of less than 1 L at the time of study to be the best single predictor of subsequent survival. The only measure associated with age of death was the age at which the VC fell below 1 L (r = 0.79, p < 0.004). These data suggest measurement of PaCO2 or serial assessment of VC should be studied further as valid methods of assessing prognosis in DMD.

Somnolence in myotonic dystrophy (DM) has not been measured using a reliable daytime somnolence scale. The aim of this study was to compare somnolence in DM patients with healthy controls and Charcot-Marie-Tooth disease (CMT) patients using such a scale and to compare this with potential contributory factors. We investigated 35 subjects with adult-onset DM, 16 healthy controls and 13 CMT controls. The Epworth Sleepiness Scale (ESS) was the principal measurement of daytime somnolence. Nocturnal sleep was assessed using a sleep diary. Other assessments measured daytime respiratory function, cognitive function, motor impairment, disability, swallowing capacity and depression. DM and CMT patients had greater daytime sleepiness than unaffected controls. In the DM group significant correlations were found between somnolence and measures of disability, sleep quality and some measures of depression. It was concluded that there is an abnormal level of daytime somnolence in DM, which is partially associated with disability.

We describe a family with a proximal myopathy, subclinical EMG myotonia, cataracts and deafness. Transmission through two generations and down the male line confirms autosomal dominant inheritance. There was no abnormal expansion of the CTG triplet repeat in the last exon of the dystrophia myotonica protein kinase (DMPK) gene associated with myotonic dystrophy. Heteroduplex analysis of all but the promoter region of the DMPK gene has excluded point mutations in this gene as an underlying cause for this myotonic disorder. The family was not sufficiently informative to exclude linkage to the sodium channel gene SCN4A or the chloride channel gene CLC1. This family clearly fulfils the recently established diagnostic criteria for PROMM (proximal myotonic myopathy) and in addition shows consistent severe deafness as a hitherto undescribed feature of PROMM. We discuss the diagnostic criteria of PROMM in relation to this family and other recent papers, all of which would now fulfil the aforementioned diagnostic criteria for PROMM.

Cardiac disease is a well-known complication of myotonic dystrophy, understanding of which has been increased by recent advances in both molecular techniques and cardiological investigations. Conduction disturbances and tachyarrhythmias occur commonly in myotonic dystrophy. These have been shown to have a broad correlation in severity with both neuromuscular disease and the extent of the molecular defect in some, but not all, studies. Clinical evidence of generalised cardiomyopathy is unusual. The rate of progression differs widely between individuals; sudden death may be caused by ventricular arrhythmias or complete heart block, and this can be at an early stage of disease. A familial tendency towards cardiac complications has been shown in some studies. The histopathology is of fibrosis, primarily in the conducting system and sino-atrial node, myocyte hypertrophy and fatty infiltration. Electron microscopy shows prominent I-bands and myofibrillar degeneration. Myotonin protein kinase, the primary product of the myotonic dystrophy gene, may be located at the intercalated discs and have a different isoform in cardiac tissue. The role of other genes or the normal myotonic dystrophy allele in myotonic heart disease has yet to be determined. Suggestions for clinical management include a careful cardiac history and a 12-lead ECG at least every year, with a low threshold for use of 24 h Holter monitoring. Extra care should be taken before, during and after general anaesthetics, which carry a high frequency of cardiorespiratory complications. Finally, myotonic dystrophy should be considered in previously undiagnosed patients presenting to a cardiologist or general physician with suspected arrhythmia or conduction block.

The three-dimensional crystal structure of pi class glutathione S-transferase YfYf from mouse liver complexed with the inhibitor S-(p-nitrobenzyl)glutathione has been determined at 1.8 A resolution by X-ray diffraction. In addition two complexes with glutathione sulphonic acid and S-hexylglutathione have been determined at resolutions of 1.9 and 2.2 A, respectively. The high resolution of the S-(p-nitrobenzyl)glutathione complex allows a detailed analysis of the active site including the hydrophobic (H-) subsite. The nitrobenzyl moiety occupies a hydrophobic pocket with its aromatic ring sandwiched between Phe8 and the hydroxyl group of Tyr108. An insertion of two residues Gly41 and Leu42, with respect to the pig enzyme, splits helix alpha B into an alpha-helix and a 3(10) helix. Water bridges between carbonyl oxygen atoms of the alpha-helix at its C terminus and the amide NH groups of the 3(10) helix at its N terminus provide structural continuity between these two secondary elements. Tyr7 appears to be the only residue close to the sulphur atom of glutathione, while three conserved water molecules lie in the surrounding area in all complexes. The enzyme mechanism is discussed on the basis of the structural analysis.

Mouse liver glutathione S-transferase YfYf (Pi class) reacts with [14C]ethacrynic acid to form a covalent adduct with a stoichiometry of 1 mol per mol of subunit. Proteolytic digestion of the enzyme-[14C]ethacrynic acid adduct with V8 protease produced an 11 kDa fragment containing radioactivity. Sequencing revealed this to be an N-terminal peptide (minus the first 15 residues, terminating at Glu-112) which contains only one cysteine residue (Cys-47). This is tentatively identified as the site of ethacrynic attachment. Kinetic studies reveal that glutathione S-conjugates protect against inactivation by ethacrynic acid, but the level of protection is not consistent with their potency as product inhibitors. A model is proposed in which glutathione S-conjugates and ethacrynic acid compete for the free enzyme, and a second molecule of ethacrynic acid reacts covalently with the enzyme-ethacrynic acid complex. The native protein contains one thiol reactive with 5,5'-dithiobis-(2-nitrobenzoic acid) at neutral pH. The resultant mixed disulphide, like the ethacrynic acid adduct, is inactive, but treatment with cyanide (which incorporates on a mol for mol basis) restores activity to 35% of that of the native enzyme.

Mouse glutathione S-transferase GST YfYf (an orthologue of GST P or 7-7 in the rat and of GST pi in the human) was found to have a subunit Mr of 24,500 and cross-reacted with anti-(rat GST YfYf). N-Terminal analysis showed a close similarity to the rat, human and bovine orthologues. On isoelectric focusing the native enzyme had a pI of 8.3 and a pI of 7.3 in the presence of urea. Initial-rate studies with 1-chloro-2,4-dinitrobenzene (CDNB) and GSH as substrates and inhibition studies with the product of the enzyme-catalysed conjugation of CDNB and GSH, S-(2,4-dinitrophenyl)glutathione, indicated a rapid-equilibrium random mechanism for the enzyme. The diuretic drug ethacrynic acid was found to be simultaneously a competitive inhibitor and an uncompetitive activator of the enzyme (with CDNB as the substrate whose concentration was varied). By using a computer simulation program (EKPLOT) a model was developed that would explain the experimental data. It is proposed that ethacrynic acid can compete with CDNB at the active site but simultaneously bind to an allosteric site on the enzyme, causing an elevation in the Vmax. for the conjugation of CDNB and GSH. The implications of such an activation mechanism for an enzyme potentially conjugating a range of xenobiotic compounds are discussed.