Capillary-Mediated Vitrification: Preservation of mRNA at Elevated Temperatures.

RNA is a fundamental tool for molecular and cellular biology research. The recent COVID-19 pandemic has proved it is also invaluable in vaccine development. However, the need for cold storage to maintain RNA integrity and the practical and economic burden associated with cold chain logistics highlight the need for new and improved preservation methods. We recently showed the use of capillary-mediated vitrification (CMV), as a tool for stabilizing temperature-sensitive enzymes. Here, we demonstrate the use of CMV as a method to preserve mRNA. The CMV process was performed by formulating a green fluorescent protein (GFP)-encoding mRNA with common excipients, applying the solution to a porous support, referred to as the scaffold, and drying the samples under vacuum for 30 min. The CMV preserved samples were stored at 55 °C for up to 100 days or 25 °C for 60 days and analyzed by electrophoresis and for transfection efficiency in a cell-based assay. The 55 °C-stressed mRNA exhibited comparable electrophoresis banding patterns and band intensity when compared to a frozen, liquid control. Additionally, the CMV stabilized mRNA maintained 97.5 ± 8.7% transfection efficiency after 77 days and 78.4 ± 3.9% after 100 days when stored 55 °C and analyzed using a cell-based assay in the CHO-K1 cell line. In contrast, a liquid control exhibited no bands on the electrophoresis gel and lost all transfection activity after being stored overnight at 55 °C. Likewise, after 60 days at 25 °C, the CMV-processed samples had full transfection activity while the activity of the liquid control was reduced to 40.1 ± 4.6%. In conclusion, CMV is a simple formulation method that significantly enhances the thermal stability of mRNA, requires minimal processing time, and could enable formulation of mRNA that can tolerate exposure to temperatures well above 25 °C during shipment and deployment in extreme environments.

Research priorities for mitochondrial disorders: Current landscape and patient and professional views.

Primary mitochondrial disorders encompass a wide range of clinical presentations and a spectrum of severity. They currently lack effective disease-modifying therapies and have a high mortality and morbidity rate. It is therefore essential to know that competitively funded research designed by academics meets the core needs of people with mitochondrial disorders and their clinicians. Priority setting partnerships are an established collaborative methodology that brings patients, carers and families, charity representatives and clinicians together to try to establish the most pressing and unanswered research priorities for a particular disease. We developed a web-based questionnaire, requesting all patients affected by primary mitochondrial disease, their carers and clinicians to pose their research questions. This yielded 709 questions from 147 participants. These were grouped into overarching themes including basic biology, causation, health services, clinical management, social impacts, prognosis, prevention, symptoms, treatment and psychological impact. Following the removal of "answered questions", the process resulted in a list of 42 discrete, answerable questions. This was further refined by web-based ranking by the community to 24 questions. These were debated at a face-to-face workshop attended by a diverse range of patients, carers, charity representatives and clinicians to create a definitive "Top 10 of unanswered research questions for primary mitochondrial disorders". These Top 10 questions related to understanding biological processes, including triggers of disease onset, mechanisms underlying progression and reasons for differential symptoms between individuals with identical genetic mutations; new treatments; biomarker discovery; psychological support and optimal management of stroke-like episodes and fatigue.

Patient Preferences in Rare Diseases: A Qualitative Study in Neuromuscular Disorders to Inform a Quantitative Preference Study.

INTRODUCTION: It has become increasingly important to include patient preference information in decision-making processes for drug development. As neuromuscular disorders represent multisystem, debilitating, and progressive rare diseases with few treatment options, this study aimed to explore unmet health care needs and patient treatment preferences for two neuromuscular disorders, myotonic dystrophy type 1 (DM1) and mitochondrial myopathies (MM) to inform early stages of drug development. METHODS: Fifteen semi-structured interviews and five focus group discussions (FGDs) were held with DM1 and MM adult patients and caregivers. Topics discussed included (1) reasons for study participation; (2) disease signs/symptoms and their impact on daily lives; (3) top desired benefits; and (4) acceptability of risks and tolerance levels for a hypothetical new treatment. Data were analyzed following a thematic 'code' approach. RESULTS: A total of 52 participants representing a wide range of disease severities participated. 'Muscle strength' and 'energy and endurance' were the disease-related unmet needs most often mentioned. Additionally, improved 'balance', 'cognition' and 'gut function' were the top desired treatment benefits, while 'damage to the liver, kidneys or eyes' was the most concerning risk. Factors influencing their tolerance to risks related to previously having experienced the risk and differentiation between permanent and temporary risks. A few differences were elicited between patients and caregivers. CONCLUSIONS: This qualitative study provided an open forum to elicit treatment-desired benefits and acceptable risks to be established by patients themselves. These findings can inform decisions for developing new treatments and the design of clinical trials for DM1 and MM.

Design, Conduct, and Use of Patient Preference Studies in the Medical Product Life Cycle: A Multi-Method Study.

Objectives: To investigate stakeholder perspectives on how patient preference studies (PPS) should be designed and conducted to allow for inclusion of patient preferences in decision-making along the medical product life cycle (MPLC), and how patient preferences can be used in such decision-making. Methods: Two literature reviews and semi-structured interviews (n = 143) with healthcare stakeholders in Europe and the US were conducted; results of these informed the design of focus group guides. Eight focus groups were conducted with European patients, industry representatives and regulators, and with US regulators and European/Canadian health technology assessment (HTA) representatives. Focus groups were analyzed thematically using NVivo. Results: Stakeholder perspectives on how PPS should be designed and conducted were as follows: 1) study design should be informed by the research questions and patient population; 2) preferred treatment attributes and levels, as well as trade-offs among attributes and levels should be investigated; 3) the patient sample and method should match the MPLC phase; 4) different stakeholders should collaborate; and 5) results from PPS should be shared with relevant stakeholders. The value of patient preferences in decision-making was found to increase with the level of patient preference sensitivity of decisions on medical products. Stakeholders mentioned that patient preferences are hardly used in current decision-making. Potential applications for patient preferences across industry, regulatory and HTA processes were identified. Four applications seemed most promising for systematic integration of patient preferences: 1) benefit-risk assessment by industry and regulators at the marketing-authorization phase; 2) assessment of major contribution to patient care by European regulators; 3) cost-effectiveness analysis; and 4) multi criteria decision analysis in HTA. Conclusions: The value of patient preferences for decision-making depends on the level of collaboration across stakeholders; the match between the research question, MPLC phase, sample, and preference method used in PPS; and the sensitivity of the decision regarding a medical product to patient preferences. Promising applications for patient preferences should be further explored with stakeholders to optimize their inclusion in decision-making.

Inositol trisphosphate receptor-mediated Ca2+ signalling stimulates mitochondrial function and gene expression in core myopathy patients.

Core myopathies are a group of childhood muscle disorders caused by mutations of the ryanodine receptor (RyR1), the Ca2+ release channel of the sarcoplasmic reticulum. These mutations have previously been associated with elevated inositol trisphosphate receptor (IP3R) levels in skeletal muscle myotubes derived from patients. However, the functional relevance and the relationship of IP3R mediated Ca2+ signalling with the pathophysiology of the disease is unclear. It has also been suggested that mitochondrial dysfunction underlies the development of central and diffuse multi-mini-cores, devoid of mitochondrial activity, which is a key pathological consequence of RyR1 mutations. Here we used muscle biopsies of central core and multi-minicore disease patients with RyR1 mutations, as well as cellular and in vivo mouse models of the disease to characterize global cellular and mitochondrial Ca2+ signalling, mitochondrial function and gene expression associated with the disease. We show that RyR1 mutations that lead to the depletion of the channel are associated with increased IP3-mediated nuclear and mitochondrial Ca2+ signals and increased mitochondrial activity. Moreover, western blot and microarray analysis indicated enhanced mitochondrial biogenesis at the transcriptional and protein levels and was reflected in increased mitochondrial DNA content. The phenotype was recapitulated by RYR1 silencing in mouse cellular myotube models. Altogether, these data indicate that remodelling of skeletal muscle Ca2+ signalling following loss of functional RyR1 mediates bioenergetic adaptation.

Pathological consequences of MICU1 mutations on mitochondrial calcium signalling and bioenergetics.

Loss of function mutations of the protein MICU1, a regulator of mitochondrial Ca2+ uptake, cause a neuronal and muscular disorder characterised by impaired cognition, muscle weakness and an extrapyramidal motor disorder. We have shown previously that MICU1 mutations cause increased resting mitochondrial Ca2+ concentration ([Ca2+]m). We now explore the functional consequences of MICU1 mutations in patient derived fibroblasts in order to clarify the underlying pathophysiology of this disorder. We propose that deregulation of mitochondrial Ca2+ uptake through loss of MICU1 raises resting [Ca2+]m, initiating a futile Ca2+ cycle, whereby continuous mitochondrial Ca2+ influx is balanced by Ca2+ efflux through the sodium calcium exchanger (NLCXm). Thus, inhibition of NCLXm by CGP-37157 caused rapid mitochondrial Ca2+ accumulation in patient but not control cells. We suggest that increased NCLX activity will increase sodium/proton exchange, potentially undermining oxidative phosphorylation, although this is balanced by dephosphorylation and activation of pyruvate dehydrogenase (PDH) in response to the increased [Ca2+]m. Consistent with this model, while ATP content in patient derived or control fibroblasts was not different, ATP increased significantly in response to CGP-37157 in the patient but not the control cells. In addition, EMRE expression levels were altered in MICU1 patient cells compared to the controls. The MICU1 mutations were associated with mitochondrial fragmentation which we show is related to altered DRP1 phosphorylation. Thus, MICU1 serves as a signal-noise discriminator in mitochondrial calcium signalling, limiting the energetic costs of mitochondrial Ca2+ signalling which may undermine oxidative phosphorylation, especially in tissues with highly dynamic energetic demands. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Calcium signaling as a mediator of cell energy demand and a trigger to cell death.

Calcium signaling is pivotal to a host of physiological pathways. A rise in calcium concentration almost invariably signals an increased cellular energy demand. Consistent with this, calcium signals mediate a number of pathways that together serve to balance energy supply and demand. In pathological states, calcium signals can precipitate mitochondrial injury and cell death, especially when coupled to energy depletion and oxidative or nitrosative stress. This review explores the mechanisms that couple cell signaling pathways to metabolic regulation or to cell death. The significance of these pathways is exemplified by pathological case studies, such as those showing loss of mitochondrial calcium uptake 1 in patients and ischemia/reperfusion injury.

Loss-of-function mutations in MICU1 cause a brain and muscle disorder linked to primary alterations in mitochondrial calcium signaling.

Mitochondrial Ca(2+) uptake has key roles in cell life and death. Physiological Ca(2+) signaling regulates aerobic metabolism, whereas pathological Ca(2+) overload triggers cell death. Mitochondrial Ca(2+) uptake is mediated by the Ca(2+) uniporter complex in the inner mitochondrial membrane, which comprises MCU, a Ca(2+)-selective ion channel, and its regulator, MICU1. Here we report mutations of MICU1 in individuals with a disease phenotype characterized by proximal myopathy, learning difficulties and a progressive extrapyramidal movement disorder. In fibroblasts from subjects with MICU1 mutations, agonist-induced mitochondrial Ca(2+) uptake at low cytosolic Ca(2+) concentrations was increased, and cytosolic Ca(2+) signals were reduced. Although resting mitochondrial membrane potential was unchanged in MICU1-deficient cells, the mitochondrial network was severely fragmented. Whereas the pathophysiology of muscular dystrophy and the core myopathies involves abnormal mitochondrial Ca(2+) handling, the phenotype associated with MICU1 deficiency is caused by a primary defect in mitochondrial Ca(2+) signaling, demonstrating the crucial role of mitochondrial Ca(2+) uptake in humans.

Comparison of clinical body composition methods in people taking weight-inducing atypical antipsychotic medications.

The purpose of this study was to compare the accuracy of clinical methods to estimate body fat (%BF) in people who take weight-inducing atypical antipsychotic medications. Forty-seven people (35 males, 12 females) with previously diagnosed psychotic illness who had been taking atypical antipsychotic medications for more than 6 months took part in this study. Percentage body fat was estimated using bioelectrical impedance analysis (BIA) and anthropometry from previously published prediction equations and compared with that measured using the deuterium dilution technique which served as the criterion measure. Bland-Altman analyses were used to assess the agreement between measures. In the males, %BF determined using BIA with the Lukaski equation was the only clinical method with mean differences that were not significant from criterion values. While in the females, %BF determined from BMI was the only method that was significantly different from the criterion values. All of the methods of estimating %BF except Watson equations provided consistent estimates across the weight range. Therefore, this study suggests that in a group of people who predominantly had schizophrenia and were taking atypical antipsychotic medications, BIA using the equation of Lukaski was the best indicator of %BF, although on an individual basis the accuracy was poor. BMI underestimated %BF to a greater significant extent than BIA. The use of BIA rather than BMI may provide a better indicator of adiposity in people who take weight inducing antipsychotic medications.

Bioelectric impedance is a better indicator of obesity in men with schizophrenia than body mass index.

Body mass index (BMI) is commonly used as an indicator of obesity, although in both clinical and research settings the use of bioelectric impedance analysis (BIA) is commonplace. The purpose of this study was to examine the relationship between BMI, BIA and percentage body fat to determine whether either is a superior indicator of obesity in men with schizophrenia. The reference method of deuterium dilution was used to measure total body water and, subsequently, percentage body fat in 31 men with schizophrenia. Comparisons with the classification of body fat using BMI and BIA were made. The correlation between percentage body fat and BMI was 0.64 whereas the correlation between percentage body fat and BIA was 0.90. The sensitivity and specificity in distinguishing between obese and overweight participants was 0.55 and 0.80 for BMI and 0.86 and 0.75 for BIA. BIA proved to be a better indicator of obesity than BMI. BMI misclassified a large proportion of men with schizophrenia as overweight when they had excess adiposity of sufficient magnitude to be considered as obese. Because of the widespread use of BMI as an indicator of obesity among people with schizophrenia, the level of obesity among men with schizophrenia may be in excess of that previously indicated.

Energy expenditure and physical activity in clozapine use: implications for weight management.

OBJECTIVE: The management of atypical antipsychotic-induced weight gain is a significant challenge for people with mental illness. Fundamental research into energy metabolism in people taking atypical antipsychotic medication has been neglected. The current study of men with schizophrenia taking clozapine aimed to measure total energy expenditure (TEE) and energy expended on physical activity--activity energy expenditure (AEE) and to consider the clinical implications of the findings. METHOD: The well-established reference method of doubly labelled water (DLW) was used to measure TEE and AEE in men with schizophrenia who had been taking clozapine for more than 6 months. Resting energy expenditure was determined using indirect calorimetry. RESULTS: The TEE was 2511+/-606 kcal day-1 which was significantly different to World Health Organization recommendations (more than 20% lower). The Physical activity level (PAL) was 1.39+/-0.27 confirming the sedentary nature of people with schizophrenia who take clozapine. CONCLUSIONS: The findings support the need for weight management strategies for people with schizophrenia who take clozapine to focus on the enhancement of energy expenditure by increasing physical activity and reducing inactivity or sedentary behaviours, rather than relying primarily on strategies to reduce energy intake.

Resting energy expenditure is lower than predicted in people taking atypical antipsychotic medication.

Resting energy expenditure (REE) is lower than predicted in persons taking atypical antipsychotic medication, and weight management is a significant clinical challenge for some of them. However, to date there have been no published guidelines to assist clinicians in choosing appropriate prediction equations to estimate energy expenditure in persons taking atypical antipsychotic medications. The objectives of this study were to measure REE in a group of men taking the atypical antipsychotic clozapine and to determine whether REE can be accurately predicted for this population using previously published regression equations. REE was measured using indirect calorimetry via a ventilated hood on eight men who had completed at least 6 months of treatment with clozapine. Comparisons between measured REE and predicted REE using five different equations were undertaken. The commonly-used Harris-Benedict and Schofield equations systematically overestimated REE. Predictions of REE from other equations were too variable for clinical use. When estimating energy requirements as part of a weight-management program in men who have been taking clozapine for 6 months, predictions of REE from the equations of Harris-Benedict and Schofield should be reduced by 280 kcal/day.

Atypical antipsychotic weight gain: a major clinical challenge.

OBJECTIVE: The major aim of this paper is to review findings from weight management intervention studies to consider clozapine and/or olanzapine induced weight gain. A parallel aim is to summarize the challenges facing future research and provide an overview of best practice in the management of weight in mental health patients. METHOD: A systematic literature search was conducted using Medline, Cinahl and PsychINFO data bases and reference lists from relevant published articles. Five studies which reported weight control practices in patients taking atypical antipsychotic medications were located and reviewed. RESULTS: The studies reviewed provide some important descriptive clinical insights; however, common shortcomings include small subject numbers and methodological drawbacks such as lack of a control group. CONCLUSIONS: There is some evidence that weight gain associated with atypical antipsychotic medication can be ameliorated by lifestyle changes such as improved nutritional practices and increased physical activity. Lifestyle interventions for individuals with psychotic disorders may need to be adapted to be most effective; for example, using strategies to counter increased appetite and to enhance physical activity. Clinicians need to be vigilant and persistent in monitoring and intervening if weight gain occurs. A standardized screening tool and clinical pathway would help clinicians to target appropriate interventions for each person prescribed atypical antipsychotic medication.