Validation of epidermal AMBRA1 and loricrin (AMBLor) as a prognostic biomarker for nonulcerated American Joint Committee on Cancer stage I/II cutaneous melanoma.

BACKGROUND: Combined expression of the autophagy-regulatory protein AMBRA1 (activating molecule in Beclin1-regulated autophagy) and the terminal differentiation marker loricrin in the peritumoral epidermis of stage I melanomas can identify tumour subsets at low risk of -metastasis. OBJECTIVES: To validate the combined expression of peritumoral AMBRA1 and loricrin (AMBLor) as a prognostic biomarker able to identify both stage I and II melanomas at low risk of tumour recurrence. METHODS: Automated immunohistochemistry was used to analyse peritumoral AMBRA1 and loricrin expression in geographically distinct discovery (n = 540) and validation (n = 300) cohorts of nonulcerated American Joint Committee on Cancer (AJCC) stage I and II melanomas. AMBLor status was correlated with clinical outcomes in the discovery and validation cohorts separately and combined. RESULTS: Analysis of AMBLor in the discovery cohort revealed a recurrence-free survival (RFS) rate of 95.5% in the AMBLor low-risk group vs. 81.7% in the AMBLor at-risk group (multivariate log-rank, P < 0.001) and a negative predictive value (NPV) of 96.0%. In the validation cohort, AMBLor analysis revealed a RFS rate of 97.6% in the AMBLor low-risk group vs. 78.3% in the at-risk group (multivariate log-rank, P < 0.001) and a NPV of 97.6%. In a multivariate model considering AMBLor, Breslow thickness, age and sex, analysis of the combined discovery and validation cohorts showed that the estimated effect of AMBLor was statistically significant, with a hazard ratio of 3.469 (95% confidence interval 1.403-8.580, P = 0.007) and an overall NPV of 96.5%. CONCLUSIONS: These data provide further evidence validating AMBLor as a prognostic biomarker to identify nonulcerated AJCC stage I and II melanoma tumours at low risk of disease recurrence.

Health professional and patient views of a novel prognostic test for melanoma: A theoretically informed qualitative study.

OBJECTIVES: Cutaneous melanoma rates are steadily increasing. Up to 20% of patients diagnosed with AJCC Stage I/II melanomas will develop metastatic disease. To date there are no consistently reliable means to accurately identify truly high versus low-risk patient subpopulations. There is hence an urgent need for more accurate prediction of prognosis to determine appropriate clinical management. Validation of a novel prognostic test based on the immunohistochemical expression of two protein biomarkers in the epidermal microenvironment of primary melanomas was undertaken; loss of these biomarkers had previously been shown to be associated with a higher risk of recurrence or metastasis. A parallel qualitative study exploring secondary care health professional and patient views of the test was undertaken and this paper reports the perceived barriers and enablers to its implementation into the melanoma care pathway. METHODS: Qualitative methods were employed drawing upon the Theoretical Domains Framework (TDF) in the exploration and analysis. An inductive-deductive analysis was performed, with all data coded using a thematic then TDF framework. FINDINGS: 20 dermatologists, plastic surgeons, cancer nurse specialists, oncologists and histopathologists participated. Nine TDF domains were relevant to all health professional groups and the 'Skills' and 'Beliefs about Capabilities' domains were relevant only to histopathologists. 'Optimism' and 'Beliefs about consequences' were strong enablers particularly for clinicians. 'Environmental context and resources' (impact on pathology services) and 'Knowledge' (the need for robust evidence about the test reliability) were the main perceived barriers. 19 patients and one carer were interviewed. For the patients eight domains were relevant. ('Knowledge', 'Emotions', 'Beliefs about consequences', 'Social Role and identity', 'Behavioural regulation', 'Memory, attention and decision processes', 'Reinforcement' and 'Skills'). The consequences of the implementation of the test were reassurance about future risk, changes to the follow-up pathway on which there were mixed views, and the need to ensure they maintained self-surveillance (Beliefs about consequences). The test was acceptable to all patient interviewees but the resultant changes to management would need to be supported by mechanisms for fast-track back into the clinic, further information on self-surveillance and clear management plans at the time the result is conveyed (Behavioural regulation). CONCLUSIONS: Health professionals and patients perceived positive consequences-for patients and for health services-of adopting the test. However, its implementation would require exploration of the resource implications for pathology services, psychological support for patients with a high-risk test result and mechanisms to reassure and support patients should the test lead to reduced frequency or duration of follow-up. Exploring implementation at an early stage with health professionals presented challenges related to the provision of specific details of the test and its validation.

Economic evaluation of robot-assisted training versus an enhanced upper limb therapy programme or usual care for patients with moderate or severe upper limb functional limitation due to stroke: results from the RATULS randomised controlled trial.

OBJECTIVE: To determine whether robot-assisted training is cost-effective compared with an enhanced upper limb therapy (EULT) programme or usual care. DESIGN: Economic evaluation within a randomised controlled trial. SETTING: Four National Health Service (NHS) centres in the UK: Queen's Hospital, Barking, Havering and Redbridge University Hospitals NHS Trust; Northwick Park Hospital, London Northwest Healthcare NHS Trust; Queen Elizabeth University Hospital, NHS Greater Glasgow and Clyde; and North Tyneside General Hospital, Northumbria Healthcare NHS Foundation Trust. PARTICIPANTS: 770 participants aged 18 years or older with moderate or severe upper limb functional limitation from first-ever stroke. INTERVENTIONS: Participants randomised to one of three programmes provided over a 12-week period: robot-assisted training plus usual care; the EULT programme plus usual care or usual care. MAIN ECONOMIC OUTCOME MEASURES: Mean healthcare resource use; costs to the NHS and personal social services in 2018 pounds; utility scores based on EQ-5D-5L responses and quality-adjusted life years (QALYs). Cost-effectiveness reported as incremental cost per QALY and cost-effectiveness acceptability curves. RESULTS: At 6 months, on average usual care was the least costly option (£3785) followed by EULT (£4451) with robot-assisted training being the most costly (£5387). The mean difference in total costs between the usual care and robot-assisted training groups (£1601) was statistically significant (p<0.001). Mean QALYs were highest for the EULT group (0.23) but no evidence of a difference (p=0.995) was observed between the robot-assisted training (0.21) and usual care groups (0.21). The incremental cost per QALY at 6 months for participants randomised to EULT compared with usual care was £74 100. Cost-effectiveness acceptability curves showed that robot-assisted training was unlikely to be cost-effective and that EULT had a 19% chance of being cost-effective at the £20 000 willingness to pay (WTP) threshold. Usual care was most likely to be cost-effective at all the WTP values considered in the analysis. CONCLUSIONS: The cost-effectiveness analysis suggested that neither robot-assisted training nor EULT, as delivered in this trial, were likely to be cost-effective at any of the cost per QALY thresholds considered. TRIAL REGISTRATION NUMBER: ISRCTN69371850.

Analysing the Action Research Arm Test (ARAT): a cautionary tale from the RATULS trial.

Many studies of stroke rehabilitation use the Action Research Arm Test (ARAT) as an outcome, which measures upper limb function by scoring the ability to complete functional tasks. This report describes an issue encountered when analysing the ARAT subscales in a trial of upper limb therapies after stroke. The subscales of the ARAT at three months followed a 'U-shaped' distribution, and therefore, comparing means or medians was not appropriate. A simple alternative approach was chosen that dichotomised the subscales. When analysing the ARAT, the shape of the distributions must be checked in order to choose the most appropriate descriptive and inferential statistical techniques. In particular, if the data follows a 'U-shaped' distribution, a simple dichotomising or a more sophisticated approach is needed. These should also be considered for heavily skewed distributions, often arising from substantial floor or ceiling effects. Inappropriate analyses can lead to misleading conclusions.

Evaluation of the enhanced upper limb therapy programme within the Robot-Assisted Training for the Upper Limb after Stroke trial: descriptive analysis of intervention fidelity, goal selection and goal achievement.

OBJECTIVE: To report the fidelity of the enhanced upper limb therapy programme within the Robot-Assisted Training for the Upper Limb after stroke (RATULS) randomized controlled trial, the types of goals selected and the proportion of goals achieved. DESIGN: Descriptive analysis of data on fidelity, goal selection and achievement from an intervention group within a randomized controlled trial. SETTING: Out-patient stroke rehabilitation within four UK NHS centres. SUBJECTS: 259 participants with moderate-severe upper limb activity limitation (Action Research Arm Test 0-39) between one week and five years post first stroke. INTERVENTION: The enhanced upper limb therapy programme aimed to provide 36 one-hour sessions, including 45 minutes of face-to-face therapy focusing on personal goals, over 12 weeks. RESULTS: 7877/9324 (84%) sessions were attended; a median of 34 [IQR 29-36] per participant. A median of 127 [IQR 70-190] repetitions were achieved per participant per session attended. Based upon the Canadian Occupational Performance Measure, goal categories were: self-care 1449/2664 (54%); productivity 374/2664 (14%); leisure 180/2664 (7%) and 'other' 661/2664 (25%). For the 2051/2664 goals for which data were available, 1287 (51%) were achieved, ranging between 27% by participants more than 12 months post stroke with baseline Action Research Arm Test scores 0-7, and 88% by those less than three months after stroke with scores 8-19. CONCLUSIONS: Intervention fidelity was high. Goals relating to self-care were most commonly selected. The proportion of goals achieved varied, depending on time post stroke and baseline arm activity limitation.

Robot-assisted training compared with an enhanced upper limb therapy programme and with usual care for upper limb functional limitation after stroke: the RATULS three-group RCT.

BACKGROUND: Loss of arm function is common after stroke. Robot-assisted training may improve arm outcomes. OBJECTIVE: The objectives were to determine the clinical effectiveness and cost-effectiveness of robot-assisted training, compared with an enhanced upper limb therapy programme and with usual care. DESIGN: This was a pragmatic, observer-blind, multicentre randomised controlled trial with embedded health economic and process evaluations. SETTING: The trial was set in four NHS trial centres. PARTICIPANTS: Patients with moderate or severe upper limb functional limitation, between 1 week and 5 years following first stroke, were recruited. INTERVENTIONS: Robot-assisted training using the Massachusetts Institute of Technology-Manus robotic gym system (InMotion commercial version, Interactive Motion Technologies, Inc., Watertown, MA, USA), an enhanced upper limb therapy programme comprising repetitive functional task practice, and usual care. MAIN OUTCOME MEASURES: The primary outcome was upper limb functional recovery 'success' (assessed using the Action Research Arm Test) at 3 months. Secondary outcomes at 3 and 6 months were the Action Research Arm Test results, upper limb impairment (measured using the Fugl-Meyer Assessment), activities of daily living (measured using the Barthel Activities of Daily Living Index), quality of life (measured using the Stroke Impact Scale), resource use costs and quality-adjusted life-years. RESULTS: A total of 770 participants were randomised (robot-assisted training, n = 257; enhanced upper limb therapy, n = 259; usual care, n = 254). Upper limb functional recovery 'success' was achieved in the robot-assisted training [103/232 (44%)], enhanced upper limb therapy [118/234 (50%)] and usual care groups [85/203 (42%)]. These differences were not statistically significant; the adjusted odds ratios were as follows: robot-assisted training versus usual care, 1.2 (98.33% confidence interval 0.7 to 2.0); enhanced upper limb therapy versus usual care, 1.5 (98.33% confidence interval 0.9 to 2.5); and robot-assisted training versus enhanced upper limb therapy, 0.8 (98.33% confidence interval 0.5 to 1.3). The robot-assisted training group had less upper limb impairment (as measured by the Fugl-Meyer Assessment motor subscale) than the usual care group at 3 and 6 months. The enhanced upper limb therapy group had less upper limb impairment (as measured by the Fugl-Meyer Assessment motor subscale), better mobility (as measured by the Stroke Impact Scale mobility domain) and better performance in activities of daily living (as measured by the Stroke Impact Scale activities of daily living domain) than the usual care group, at 3 months. The robot-assisted training group performed less well in activities of daily living (as measured by the Stroke Impact Scale activities of daily living domain) than the enhanced upper limb therapy group at 3 months. No other differences were clinically important and statistically significant. Participants found the robot-assisted training and the enhanced upper limb therapy group programmes acceptable. Neither intervention, as provided in this trial, was cost-effective at current National Institute for Health and Care Excellence willingness-to-pay thresholds for a quality-adjusted life-year. CONCLUSIONS: Robot-assisted training did not improve upper limb function compared with usual care. Although robot-assisted training improved upper limb impairment, this did not translate into improvements in other outcomes. Enhanced upper limb therapy resulted in potentially important improvements on upper limb impairment, in performance of activities of daily living, and in mobility. Neither intervention was cost-effective. FUTURE WORK: Further research is needed to find ways to translate the improvements in upper limb impairment seen with robot-assisted training into improvements in upper limb function and activities of daily living. Innovations to make rehabilitation programmes more cost-effective are required. LIMITATIONS: Pragmatic inclusion criteria led to the recruitment of some participants with little prospect of recovery. The attrition rate was higher in the usual care group than in the robot-assisted training or enhanced upper limb therapy groups, and differential attrition is a potential source of bias. Obtaining accurate information about the usual care that participants were receiving was a challenge. TRIAL REGISTRATION: Current Controlled Trials ISRCTN69371850. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 24, No. 54. See the NIHR Journals Library website for further project information.

Many people who have arm weakness following a stroke feel that insufficient attention is paid by rehabilitation services to recovery of their arm. Unfortunately, it is currently unclear how best to provide rehabilitation to optimise recovery, but robot-assisted training and therapy programmes that focus on practising functional tasks are promising and require further evaluation. The Robot-Assisted Training for the Upper Limb after Stroke (RATULS) trial evaluated three approaches to rehabilitation for people with moderate or severe difficulty using their arm. These approaches were robot-assisted training using the Massachusetts Institute of Technology-Manus robotic gym system (InMotion commercial version, Interactive Motion Technologies, Inc., Watertown, MA, USA), an enhanced upper limb therapy programme based on repetitive practice of functional tasks and usual care. Robot-assisted training and the enhanced upper limb therapy programme were provided in an outpatient setting for 45 minutes per session, three times per week, for 12 weeks, in addition to usual care. The Massachusetts Institute of Technology-Manus robotic gym system was selected as it was felt to be the best available technology. The participant sits at a table, places their affected arm onto the Massachusetts Institute of Technology-Manus arm support and attempts to move their arm to play a game on the computer screen. Movements are assisted by the Massachusetts Institute of Technology-Manus if the patient cannot perform the movements themselves. The results of the RATULS trial show that robot-assisted training did not result in additional improvement in stroke survivors' arm use when compared with the enhanced upper limb therapy programme or usual care. Stroke survivors who received enhanced upper limb therapy experienced meaningful improvements in undertaking activities of daily living, when compared with those participants who received either robot-assisted training or usual care. Participants who received enhanced upper limb therapy also experienced benefits in their mobility, compared with usual care participants. Participants and therapists found both therapies acceptable, and described various benefits. A health economic analysis found that neither robot-assisted training nor the enhanced upper limb therapy programme was a cost-effective treatment for the NHS.

PBC-10: a short quality of life measure for clinical screening in primary biliary cholangitis.

BACKGROUND: Current guidelines in primary biliary cholangitis ( PBC) recommend routine screening for symptoms. However, at present there are no validated practical tools suitable for screening use in practice. AIM: To develop a short quality of life questionnaire for PBC METHODS: The short PBC HRQL questionnaire was derived and validated by analysing the PBC-40 questionnaires from the UK-PBC Research Cohort. Construct validity was assessed using the European Quality of Life Five Dimensions (EQ5D) questionnaire. Test-retest analysis was done by asking a subgroup of patients to complete the questionnaire twice within 2-4 weeks. RESULTS: A total of 2219 patients completed PBC-40 questionnaire in 2013. Stepwise regression identified 10 questions that contributed to more than 95% of the PBC-40 score variance and covered the main domains of PBC. The short HRQL questionnaire, PBC-10, had good internal consistency (Cronbach's α 0.905) and item-total correlations. PBC-10 demonstrated no ceiling effects but a floor effect was noted. Further validation on 2502 patients who completed the PBC questionnaire in 2017 confirmed the psychometric properties of PBC-10. Further analysis on 186 patients showed that PBC-10 demonstrated good internal consistency (Cronbach's α = 0.936), had good reproducibility (intra-class correlation coefficient = 0.945), good correlation with the EQ5D (r = .736), and was responsive to change. A change of 4 points in the PBC-10 score would be considered clinically important. CONCLUSION: PBC-10 is a short and valid questionnaire for assessing the HRQL in patients with PBC in clinical practice.

Robot assisted training for the upper limb after stroke (RATULS): a multicentre randomised controlled trial.

BACKGROUND: Loss of arm function is a common problem after stroke. Robot-assisted training might improve arm function and activities of daily living. We compared the clinical effectiveness of robot-assisted training using the MIT-Manus robotic gym with an enhanced upper limb therapy (EULT) programme based on repetitive functional task practice and with usual care. METHODS: RATULS was a pragmatic, multicentre, randomised controlled trial done at four UK centres. Stroke patients aged at least 18 years with moderate or severe upper limb functional limitation, between 1 week and 5 years after their first stroke, were randomly assigned (1:1:1) to receive robot-assisted training, EULT, or usual care. Robot-assisted training and EULT were provided for 45 min, three times per week for 12 weeks. Randomisation was internet-based using permuted block sequences. Treatment allocation was masked from outcome assessors but not from participants or therapists. The primary outcome was upper limb function success (defined using the Action Research Arm Test [ARAT]) at 3 months. Analyses were done on an intention-to-treat basis. This study is registered with the ISRCTN registry, number ISRCTN69371850. FINDINGS: Between April 14, 2014, and April 30, 2018, 770 participants were enrolled and randomly assigned to either robot-assisted training (n=257), EULT (n=259), or usual care (n=254). The primary outcome of ARAT success was achieved by 103 (44%) of 232 patients in the robot-assisted training group, 118 (50%) of 234 in the EULT group, and 85 (42%) of 203 in the usual care group. Compared with usual care, robot-assisted training (adjusted odds ratio [aOR] 1·17 [98·3% CI 0·70-1·96]) and EULT (aOR 1·51 [0·90-2·51]) did not improve upper limb function; the effects of robot-assisted training did not differ from EULT (aOR 0·78 [0·48-1·27]). More participants in the robot-assisted training group (39 [15%] of 257) and EULT group (33 [13%] of 259) had serious adverse events than in the usual care group (20 [8%] of 254), but none were attributable to the intervention. INTERPRETATION: Robot-assisted training and EULT did not improve upper limb function after stroke compared with usual care for patients with moderate or severe upper limb functional limitation. These results do not support the use of robot-assisted training as provided in this trial in routine clinical practice. FUNDING: National Institute for Health Research Health Technology Assessment Programme.

Robot Assisted Training for the Upper Limb after Stroke (RATULS): study protocol for a randomised controlled trial.

BACKGROUND: Loss of arm function is a common and distressing consequence of stroke. We describe the protocol for a pragmatic, multicentre randomised controlled trial to determine whether robot-assisted training improves upper limb function following stroke. METHODS/DESIGN: Study design: a pragmatic, three-arm, multicentre randomised controlled trial, economic analysis and process evaluation. SETTING: NHS stroke services. PARTICIPANTS: adults with acute or chronic first-ever stroke (1 week to 5 years post stroke) causing moderate to severe upper limb functional limitation. Randomisation groups: 1. Robot-assisted training using the InMotion robotic gym system for 45 min, three times/week for 12 weeks 2. Enhanced upper limb therapy for 45 min, three times/week for 12 weeks 3. Usual NHS care in accordance with local clinical practice Randomisation: individual participant randomisation stratified by centre, time since stroke, and severity of upper limb impairment. PRIMARY OUTCOME: upper limb function measured by the Action Research Arm Test (ARAT) at 3 months post randomisation. SECONDARY OUTCOMES: upper limb impairment (Fugl-Meyer Test), activities of daily living (Barthel ADL Index), quality of life (Stroke Impact Scale, EQ-5D-5L), resource use, cost per quality-adjusted life year and adverse events, at 3 and 6 months. Blinding: outcomes are undertaken by blinded assessors. Economic analysis: micro-costing and economic evaluation of interventions compared to usual NHS care. A within-trial analysis, with an economic model will be used to extrapolate longer-term costs and outcomes. Process evaluation: semi-structured interviews with participants and professionals to seek their views and experiences of the rehabilitation that they have received or provided, and factors affecting the implementation of the trial. SAMPLE SIZE: allowing for 10% attrition, 720 participants provide 80% power to detect a 15% difference in successful outcome between each of the treatment pairs. Successful outcome definition: baseline ARAT 0-7 must improve by 3 or more points; baseline ARAT 8-13 improve by 4 or more points; baseline ARAT 14-19 improve by 5 or more points; baseline ARAT 20-39 improve by 6 or more points. DISCUSSION: The results from this trial will determine whether robot-assisted training improves upper limb function post stroke. TRIAL REGISTRATION: ISRCTN, identifier: ISRCTN69371850 . Registered 4 October 2013.

Biodentine and mineral trioxide aggregate induce similar cellular responses in a fibroblast cell line.

INTRODUCTION: The aim of this study was to assess the cell viability and messenger RNA expression of interleukin (IL)-1α and IL-6 in 3T3 fibroblast cells when in direct contact with Biodentine (Septodont, Saint Maur de Fossés, France) and mineral trioxide aggregate (MTA). METHODS: Biodentine and MTA were coated onto coverslips and allowed to set. An uncoated coverslip and one coated with GC Fuji IX (GC Corporation, Tokyo, Japan) were used as controls. Coverslips were cultured with 3T3 fibroblast cells. Cell viability was assessed quantitatively using AlamarBlue dye (Serotec, Oxford, UK) after 3, 6, 24, and 72 hours. Morphologic cell changes of 3T3 cells in contact with BD and MTA were observed by scanning electron microscopy, and cytokine expression was assessed at the messenger RNA level by semiquantitative reverse-transcription polymerase chain reaction after 3 and 24 hours of direct contact with the materials. RESULTS: Cells in contact with Biodentine and MTA showed similar viability to untreated control cells at all time points, with the exception of 6 hours when viability was decreased with both treatments. Examination by scanning electron microscopy revealed cells adhering to most of the Biodentine surface after 24 hours. However, for MTA samples, significantly fewer cells were observed. The messenger RNA expression of IL-1α and IL-6 by cells in contact with Biodentine was similar to cells in contact with MTA. CONCLUSIONS: Biodentine and MTA showed similar cytotoxicity and induced a similar pattern of cytokine expression.

Altered expression of ZnT10 in Alzheimer's disease brain.

There is an increasing body of evidence suggesting that metal homeostasis is dysregulated in the pathology of Alzheimer's disease (AD). Although expression levels of several transporters belonging the SLC30 family, which comprises predominantly zinc transporters, have been studied in the AD brain, SLC30A10 (ZnT10) has not been studied in this context. To determine if dysregulated expression of ZnT10, which may transport both Zn and Mn, could be a factor that contributes to AD, we investigated if there were differences in ZnT10 mRNA levels in specimens of frontal cortex from AD patients and controls and also if brain tissue from the APP/PS1 transgenic (Tg) mouse model showed abnormal levels of ZnT10 mRNA expression. Our results show that ZnT10 is significantly (P<0.01) decreased in the frontal cortex in AD. Furthermore, we observed a significant decrease in ZnT10 mRNA levels in the APP/PS1-Tg mice compared with wild-type controls (P<0.01). Our results suggest that this dysregulation in ZnT10 could further contribute to disease progression.

Effects of Sirt1 on DNA methylation and expression of genes affected by dietary restriction.

Changes in DNA methylation across the life course may contribute to the ageing process. We hypothesised that some effects of dietary restriction to extend lifespan and/or mitigate against features of ageing result from changes in DNA methylation, so we determined if genes that respond to dietary restriction also show age-related changes in DNA methylation. In support of our hypothesis, the intersection of lists of genes compiled from published sources that (1) were differentially expressed in response to dietary restriction and (2) showed altered methylation with increased age was greater than expected. We also hypothesised that some effects of Sirt1, which may play a pivotal role in beneficial effects of dietary restriction, are mediated through DNA methylation. We thus measured effects of Sirt1 overexpression and knockdown in a human cell line on DNA methylation and expression of a panel of eight genes that respond to dietary restriction and show altered methylation with age. Six genes were affected at the level of DNA methylation, and for six expressions were affected. In further support of our hypothesis, we observed by DNA microarray analysis that genes showing differential expression in response to Sirt1 knockdown were over-represented in the complied list of genes that respond to dietary restriction. The findings reveal that Sirt1 has effects on DNA methylation across the genome and affects, in particular, the expression of genes that respond to dietary restriction. Sirt1-mediated effects on DNA methylation and, consequently, gene expression may thus be one of the mechanisms underlying the response to dietary restriction.

Identification of the human zinc transcriptional regulatory element (ZTRE): a palindromic protein-binding DNA sequence responsible for zinc-induced transcriptional repression.

Many genes with crucial roles in zinc homeostasis in mammals respond to fluctuating zinc supply through unknown mechanisms, and uncovering these mechanisms is essential to understanding the process at cellular and systemic levels. We detected zinc-dependent binding of a zinc-induced protein to a specific sequence, the zinc transcriptional regulatory element (ZTRE), in the SLC30A5 (zinc transporter ZnT5) promoter and showed that substitution of the ZTRE abrogated the repression of a reporter gene in response to zinc. We identified the ZTRE in other genes, including (through an unbiased search) the CBWD genes and (through targeted analysis) in multiple members of the SLC30 family, including SLC30A10, which is repressed by zinc. The function of the CBWD genes is currently unknown, but roles for homologs in metal homeostasis are being uncovered in bacteria. We demonstrated that CBWD genes are repressed by zinc and that substitution of the ZTRE in SLC30A10 and CBWD promoter-reporter constructs abrogates this response. Other metals did not affect expression of the transcriptional regulator, binding to the ZTRE or promoter-driven reporter gene expression. These findings provide the basis for elucidating how regulation of a network of genes through this novel mechanism contributes to zinc homeostasis and how the cell orchestrates this response.

Efflux function, tissue-specific expression and intracellular trafficking of the Zn transporter ZnT10 indicate roles in adult Zn homeostasis.

Zn is essential to the structure and function of numerous proteins and enzymes so requires tight homeostatic control at both the systemic and cellular level. Two families of Zn transporters - ZIP (SLC39) and ZnT (SLC30) - contribute to Zn homeostasis. There are at least 10 members of the human ZnT family, and the expression profile and regulation of each varies depending on tissue type. Little is known about the role and expression pattern of ZnT10; however in silico data predict restricted expression to foetal tissue. We show a differential expression profile for ZnT10 in adult human tissue by RT-qPCR and detect highest levels of expression in small intestine, liver and brain tissues. We present data revealing the functional activity of ZnT10 to be in the efflux direction. Using a plasmid construct to express ZnT10 with an N-terminal FLAG-epitope tag, we reveal subcellular localisation in a neuroblastoma cell line (SH-SY5Y) to be at the Golgi apparatus under standard conditions of culture, with trafficking to the plasma membrane observed at higher extracellular Zn concentrations. We demonstrate down-regulation by Zn of ZnT10 mRNA levels in cultured intestinal and neuroblastoma cell lines and demonstrate reduced transcription from the ZnT10 promoter at an elevated extracellular Zn concentration. These features of ZnT10 localisation, regulation and function, together with the discovery that ZnT10 is expressed a high levels in brain tissue, indicate that ZnT10 has a role in regulating Zn homeostasis in the brain so may have relevance to the development of neurodegenerative disease.

Hippocampal neuronal atrophy and cognitive function in delayed poststroke and aging-related dementias.

BACKGROUND AND PURPOSE: We have previously shown delayed poststroke dementia in elderly (≥75 years old) stroke survivors is associated with medial temporal lobe atrophy; however, the basis of the structural and functional changes is unknown. METHODS: Using 3-dimensional stereological methods, we quantified hippocampal pyramidal neuronal volumes and densities in a total of 95 postmortem samples from demented and nondemented poststroke survivors within our prospective Cognitive Function after Stroke study and subjects pathologically diagnosed with vascular dementia, Alzheimer disease, and mixed Alzheimer disease and vascular dementia syndrome. RESULTS: Hippocampal CA1 but not CA2 subfield neuron density was affected in poststroke, Alzheimer disease, vascular dementia, and mixed dementia groups relative to control subjects (P<0.05). Neuronal volume was reduced in the poststroke dementia relative to poststroke nondemented group in both CA1 and CA2, although there were no apparent differences in neuronal density. Poststroke nondemented neuronal volumes were similar to control subjects but greater than in all dementias (P<0.05). Neuronal volumes positively correlated with global cognitive function and memory function in both CA1 and CA2 in poststroke subjects (P<0.01). Degrees of neuronal atrophy and loss were similar in the poststroke dementia and vascular dementia groups. However, in the entorhinal cortex layer V, neuronal volumes were only impaired in the mixed and Alzheimer disease groups (P<0.05). CONCLUSIONS: Our results suggest hippocampal neuronal atrophy is an important substrate for dementia in both cerebrovascular and neurodegenerative disease.