Mitochondrial TNAP controls thermogenesis by hydrolysis of phosphocreatine.

Adaptive thermogenesis has attracted much attention because of its ability to increase systemic energy expenditure and to counter obesity and diabetes1-3. Recent data have indicated that thermogenic fat cells use creatine to stimulate futile substrate cycling, dissipating chemical energy as heat4,5. This model was based on the super-stoichiometric relationship between the amount of creatine added to mitochondria and the quantity of oxygen consumed. Here we provide direct evidence for the molecular basis of this futile creatine cycling activity in mice. Thermogenic fat cells have robust phosphocreatine phosphatase activity, which is attributed to tissue-nonspecific alkaline phosphatase (TNAP). TNAP hydrolyses phosphocreatine to initiate a futile cycle of creatine dephosphorylation and phosphorylation. Unlike in other cells, TNAP in thermogenic fat cells is localized to the mitochondria, where futile creatine cycling occurs. TNAP expression is powerfully induced when mice are exposed to cold conditions, and its inhibition in isolated mitochondria leads to a loss of futile creatine cycling. In addition, genetic ablation of TNAP in adipocytes reduces whole-body energy expenditure and leads to rapid-onset obesity in mice, with no change in movement or feeding behaviour. These data illustrate the critical role of TNAP as a phosphocreatine phosphatase in the futile creatine cycle.

Changes in lipid metabolism driven by steroid signalling modulate proteostasis in C. elegans.

Alzheimer's, Parkinson's and Huntington's diseases can be caused by mutations that enhance protein aggregation, but we still do not know enough about the molecular players of these pathways to develop treatments for these devastating diseases. Here, we screen for mutations that might enhance aggregation in Caenorhabditis elegans, to investigate the mechanisms that protect against dysregulated homeostasis. We report that the stomatin homologue UNC-1 activates neurohormonal signalling from the sulfotransferase SSU-1 in ASJ sensory/endocrine neurons. A putative hormone, produced in ASJ, targets the nuclear receptor NHR-1, which acts cell autonomously in the muscles to modulate polyglutamine repeat (polyQ) aggregation. A second nuclear receptor, DAF-12, functions oppositely to NHR-1 to maintain protein homeostasis. Transcriptomics analyses of unc-1 mutants revealed changes in the expression of genes involved in fat metabolism, suggesting that fat metabolism changes, controlled by neurohormonal signalling, contribute to protein homeostasis. Furthermore, the enzymes involved in the identified signalling pathway are potential targets for treating neurodegenerative diseases caused by disrupted protein homeostasis.

STUB1 Mutations as Possible Genetic Modifiers in Spinocerebellar Ataxia Type 8.

BACKGROUND: Spinocerebellar ataxia type 8 (SCA8) is a dominantly inherited expansion disorder with highly variable penetrance. ATXN8OS/ATXN8 expanded alleles have been identified in association with other types of hereditary ataxias, pointing to a possible genetic synergism. OBJECTIVES: We aimed to further investigate the molecular background of patients with SCA8 diagnosis. METHODS: Patients were selected from our cohort of 346 families. A total of 14 probands with SCA8 underwent additional investigation through exome sequencing. RESULTS: Pathogenic heterozygous STUB1 variants were found in 21.4% of SCA8 patients (3 of 14) compared to only 0.5% in the non-SCA8 group (1 of 222), indicating a statistically significant association (P < 0.05). CONCLUSIONS: The findings reported in this study might suggest a genetic synergism between STUB1 and ATXN8OS/ATXN8 expanded alleles. Further studies are needed to validate this observation and better define the clinical impact of this genetic interaction. © 2024 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Consensus on the preoperative management of patients with chronic moderate to severe shoulder pain to improve postoperative outcomes: Delphi results.

BACKGROUND: Appropriate preoperative management of patients with chronic moderate to severe shoulder pain who are candidates for surgery owing to rotator cuff disease or glenohumeral osteoarthritis may improve surgery and patient outcomes, but published evidence in this regard is scarce. Therefore, the availability of recommendations on preoperative interventions based on expert consensus may serve as guidance. METHODS: A Delphi study was conducted to develop a preoperative management algorithm based on a national expert consensus. A Delphi questionnaire was developed by a scientific committee following a systematic review of the relevant literature published during the past 10 years using PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) criteria. It consisted of 48 statements divided into 5 blocks (block I, assessment and diagnosis of preoperative pain; block II, preoperative function and psychosocial aspects; block III, therapeutic objectives; block IV, treatment; and block V, follow-up and referral), and 28 experienced shoulder surgeons from across the country were invited to answer. RESULTS: All participants responded to the Delphi questionnaire in the first round, and 25 responded in the second round (89.3% of those invited). Overall, 46 of 49 final statements reached a consensus, on the basis of which a final preoperative management algorithm was defined by the scientific committee. First, surgeons should assess shoulder pain intensity and characteristics, shoulder functionality, and psychosocial aspects using specific validated questionnaires. Preoperative therapeutic objectives should include shoulder pain control, depression and/or nocturnal sleep improvement, opioid consumption adjustment, and substance abuse cessation. Postoperative objectives regarding the degree of shoulder pain reduction or improvement in functionality and/or quality of life should be established in agreement with the patient. Treatment of preoperative chronic moderate to severe shoulder pain should comprise nonpharmacologic as well as pharmacologic interventions. Follow-up of the shoulder pain levels, treatment adherence, and mental health status of these patients may be carried out by the surgical team (surgeon and anesthesiologist) together with the primary care team. Patients with very intense shoulder pain levels may be referred to a pain unit following specific protocols. CONCLUSION: A preoperative management algorithm for patients with chronic moderate to severe shoulder pain who are candidates for surgery owing to rotator cuff disease or glenohumeral osteoarthritis was defined based on a national expert consensus. Main points include comprehensive patient management starting with an objective assessment of shoulder pain and function, as well as quality of life; establishment of preoperative and postoperative therapeutic targets; prescription of individualized therapeutic interventions; and multidisciplinary patient follow-up. Implementation of these recommendations into clinical practice may result in better preoperative shoulder pain management and more successful surgical outcomes and patient satisfaction.

Differential efficacy with epidural blood and fibrin patches for the treatment of post-dural puncture headache.

BACKGROUND: Accidental dural puncture (ADP) is the most frequent major complication when performing an epidural procedure in obstetrics. Consequently, loss of pressure in the cerebrospinal fluid (CSF) leads to the development of post-dural puncture headache (PDPH), which occurs in 16%-86% of cases. To date, the efficacy of epidural fibrin patches (EFP) has not been evaluated in a controlled clinical trial, nor in comparative studies with epidural blood patches (EBP). METHODS: The objective of the present study was to compare the efficacy of EFP with respect to EBP for the treatment of refractory accidental PDPH. This prospective, randomized, open-label, parallel, comparative study included 70 puerperal women who received an EBP or EFP (35 in each group) after failure of the conventional analgesic treatment for accidental PDPH in a hospital. RESULTS: A higher percentage of women with EFP than EBP achieved complete PDPH relief after 2 (97.1% vs. 54.3%) and 12 h (100.0% vs. 65.7%) of the patch injection. The percentage of patients who needed rescue analgesia was significantly lower with EFP after 2 (2.9% vs. 48.6%) and 12 h (0.0% vs. 37.1%). After 24 h, PDPH was resolved in all women who received EFP. The recurrence of PDPH was reported in one woman from the EBP group (2.9%), who subsequently required a second patch. The mean length of hospital stay was significantly lower with EFP (3.9 days) than EBP (5.9 days). Regarding satisfaction, the mean value (Likert scale) was significantly higher with EFP (4.7 vs. 3.0). CONCLUSIONS: EFP provided better outcomes than EBP for the treatment of obstetric PDPH in terms of efficacy, safety, and patient satisfaction.

Annexin A5 stabilizes matrix vesicle-biomimetic lipid membranes: unravelling a new role of annexins in calcification.

Matrix vesicles are a special class of extracellular vesicles thought to actively contribute to both physiologic and pathologic mineralization. Proteomic studies have shown that matrix vesicles possess high amounts of annexin A5, suggesting that the protein might have multiple roles at the sites of calcification. Currently, Annexin A5 is thought to promote the nucleation of apatitic minerals close to the inner leaflet of the matrix vesicles' membrane enriched in phosphatidylserine and Ca2+. Herein, we aimed at unravelling a possible additional role of annexin A5 by investigating the ability of annexin A5 to adsorb on matrix-vesicle biomimetic liposomes and Langmuir monolayers made of dipalmitoylphosphatidylserine (DPPS) and dipalmitoylphosphatidylcholine (DPPC) in the absence and in the presence of Ca2+. Differential scanning calorimetry and dynamic light scattering measurements showed that Ca2+ at concentrations in the 0.5-2.0 mM range induced the aggregation of liposomes probably due to the formation of DPPS-enriched domains. However, annexin A5 avoided the aggregation of liposomes at Ca2+ concentrations lower than 1.0 mM. Surface pressure versus surface area isotherms showed that the adsorption of annexin A5 on the monolayers made of a mixture of DPPC and DPPS led to a reduction in the area of excess compared to the theoretical values, which confirmed that the protein favored attractive interactions among the membrane lipids. The stabilization of the lipid membranes by annexin A5 was also validated by recording the changes with time of the surface pressure. Finally, fluorescence microscopy images of lipid monolayers revealed the formation of spherical lipid-condensed domains that became unshaped and larger in the presence of annexin A5. Our data support the model that annexin A5 in matrix vesicles is recruited at the membrane sites enriched in phosphatidylserine and Ca2+ not only to contribute to the intraluminal mineral formation but also to stabilize the vesicles' membrane and prevent its premature rupture.

NPP1 and TNAP hydrolyze ATP synergistically during biomineralization.

Matrix vesicles (MVs) are a special class of extracellular vesicles released by mineralizing cells during bone and tooth mineralization that initiate the precipitation of apatitic minerals by regulating the extracellular ratio between inorganic phosphate (Pi), a calcification promoter, and pyrophosphate (PPi), a calcification inhibitor. The Pi/PPi ratio is thought to be controlled by two ecto-phosphatases present on the outer leaflet of the MVs' membrane: ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) that produces PPi as well as Pi from ATP and tissue-nonspecific alkaline phosphatase (TNAP) that hydrolyzes both ATP and PPi to generate Pi. However, if and how these enzymes act in concert in MVs are still unclear. Herein, we investigated the role of NPP1 and TNAP in ATP hydrolysis during MV-mediated biomineralization using proteoliposomes as a biomimetic model for MVs. Proteoliposomes composed by 1,2-dipalmitoylphosphatidylcholine (DPPC) and harboring NPP1 alone, TNAP alone, or both together at different molar ratios (1:1, 10:1, and 1:10) were fabricated. After 48 h of incubation with ATP, TNAP-containing proteoliposomes consumed more ATP than NPP1-containing vesicles (270 and 210 nmol, respectively). Both types of vesicles comparatively formed ADP (205 and 201 nmol, respectively), while NPP1-containing vesicles hydrolyzed AMP less efficiently than TNAP-containing proteoliposomes (10 and 25 nmol, respectively). In vitro mineralization assays showed that in the presence of ATP, TNAP-harboring proteoliposomes mineralized through a sigmoidal single-step process, while NPP1-harboring vesicles displayed a two-step mineralization process. ATR-FTIR analyses showed that the minerals produced by TNAP-harboring proteoliposomes were structurally more similar to hydroxyapatite than those produced by NPP1-harboring vesicles. Our results with proteoliposomes indicate that the pyrophosphohydrolase function of NPP1 and the phosphohydrolase activity of TNAP act synergistically to produce a Pi/PPi ratio conducive to mineralization and the synergism is maximal when the two enzymes are present at equimolar concentrations. The significance of these findings for hypophosphatasia is discussed.

Increased PHOSPHO1 and alkaline phosphatase expression during the anabolic bone response to intermittent parathyroid hormone delivery.

The administration of intermittent parathyroid hormone (iPTH) is anabolic to the skeleton. Recent studies with cultured osteoblasts have revealed that the expression of PHOSPHO1, a bone-specific phosphatase essential for the initiation of mineralisation, is regulated by PTH. Therefore, this study sought to determine whether the bone anabolic response to iPTH involves modulation of expression of Phospho1 and of other enzymes critical for bone matrix mineralisation. To mimic iPTH treatment, primary murine osteoblasts were challenged with 50 nM PTH for 6 h in every 48 h period for 8 days (4 cycles), 14 days (7 cycles) and 20 days (10 cycles) in total. The expression of both Phospho1 and Smpd3 was almost completely inhibited after 4 cycles, whereas 10 cycles were required to stimulate a similar response in Alpl expression. To explore the in vivo role of PHOSPHO1 in PTH-mediated osteogenesis, the effects of 14- and 28-day iPTH (80 µg/kg/day) administration was assessed in male wild-type (WT) and Phospho1-/- mice. The expression of Phospho1, Alpl, Smpd3, Enpp1, Runx2 and Trps1 expression was enhanced in the femora of WT mice following iPTH administration but remained unchanged in the femora of Phospho1-/- mice. After 28 days of iPTH administration, the anabolic response in the femora of WT was greater than that noted in Phospho1-/- mice. Specifically, cortical and trabecular bone volume/total volume, as well as cortical thickness, were increased in femora of iPTH-treated WT but not in iPTH-treated Phospho1-/- mice. Trabecular bone osteoblast number was also increased in iPTH-treated WT mice but not in iPTH-treated Phospho1-/-  mice. The increased levels of Phospho1, Alpl, Enpp1 and Smpd3 in WT mice in response to iPTH administration is consistent with their contribution to the potent anabolic properties of iPTH in bone. Furthermore, as the anabolic response to iPTH was attenuated in mice deficient in PHOSPHO1, this suggests that the osteoanabolic effects of iPTH are at least partly mediated via bone mineralisation processes.

Disentangling the origins of confidence in speeded perceptual judgments through multimodal imaging.

The human capacity to compute the likelihood that a decision is correct-known as metacognition-has proven difficult to study in isolation as it usually cooccurs with decision making. Here, we isolated postdecisional from decisional contributions to metacognition by analyzing neural correlates of confidence with multimodal imaging. Healthy volunteers reported their confidence in the accuracy of decisions they made or decisions they observed. We found better metacognitive performance for committed vs. observed decisions, indicating that committing to a decision may improve confidence. Relying on concurrent electroencephalography and hemodynamic recordings, we found a common correlate of confidence following committed and observed decisions in the inferior frontal gyrus and a dissociation in the anterior prefrontal cortex and anterior insula. We discuss these results in light of decisional and postdecisional accounts of confidence and propose a computational model of confidence in which metacognitive performance naturally improves when evidence accumulation is constrained upon committing a decision.

Efficacy and Safety of Celiac Plexus Neurolysis in the Treatment of Chronic Pain Secondary to Oncological Pathology of the Upper Hemiabdomen: A Systematic Review and Meta-Analysis.

Objectives: The management of chronic pain among patients with abdominal cancer is complex; against that, the neurolysis of the celiac plexus (CPN) is the best technique at the moment to determine the efficacy and safety in the treatment of chronic pain secondary to oncological pathology of the upper abdomen. Material and Methods: This was a systematic review of controlled clinical trials between 2000 and 2021, in the sources MEDLINE/PubMed, Cochrane, Scopus, Web of Science, and Google Scholar. Three independent evaluators analysed the results of the bibliographical research. The quality of the studies was assessed with the Jadad scale and the mean difference (95% confidence interval) and heterogeneity of the studies (I2) were calculated with Review Manager 5.3. Results: Seven hundred and forty-four publications were identified, including 13 studies in the qualitative synthesis and three studies in the quantitative synthesis. No difference was found in the decrease in pain intensity between 1 and 12 weeks after the intervention, comparing the experimental group with the control (P > 0.05). The adverse effects related to neurolysis were not serious and transitory, mentioning the most frequent adverse effects and reporting a percentage between 21% and 67% (with 17% for echoendoscopic neurolysis and 49% for percutaneous neurolysis). Conclusion: Celiac plexus neurolysis for the treatment of severe chronic pain secondary to oncological pathology in the upper hemiabdomen produces similar pain relief as conventional pharmacological analgesic treatment. It is a safe analgesic technique since the complications are mild and transitory.

TNAP upregulation is a critical factor in Tauopathies and its blockade ameliorates neurotoxicity and increases life-expectancy.

Tauopathies are a family of neurodegenerative diseases characterized by the presence of abnormally hyperphosphorylated Tau protein. Several studies have proposed that increased extracellular Tau (eTau) leads to the spread of cerebral tauopathy. However, the molecular mechanisms underlying eTau-induced neurotoxicity remain unclear. Previous in vitro studies reported that the ecto-enzyme tissue-nonspecific alkaline phosphatase (TNAP) dephosphorylate eTau at different sites increasing its neurotoxicity. Here, we confirm TNAP protein upregulation in the brains of Alzheimer's patients and found a similar TNAP increase in Pick's disease patients and P301S mice, a well-characterized mouse model of tauopathies. Interestingly, the conditional overexpression of TNAP causes intracellular Tau hyperphosphorylation and aggregation in cells neighbouring those overexpressing the ectoenzyme. Conversely, the genetic disruption of TNAP reduced the dephosphorylation of eTau and decreased neuronal hyperactivity, brain atrophy, and hippocampal neuronal death in P301S mice. TNAP haploinsufficiency in P301S mice prevents the decreased anxiety-like behaviour, motor deficiency, and increased memory capacity and life expectancy. Similar results were observed by the in vivo pharmacological blunting of TNAP activity. This study provides the first in vivo evidence demonstrating that raised TNAP activity is critical for Tau-induced neurotoxicity and suggest that TNAP blockade may be a novel and efficient therapy to treat tauopathies.

CIBERER: Spanish national network for research on rare diseases: A highly productive collaborative initiative.

CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research.

Brain-Computer Interfaces for Treatment of Focal Dystonia.

Task-specificity in isolated focal dystonias is a powerful feature that may successfully be targeted with therapeutic brain-computer interfaces. While performing a symptomatic task, the patient actively modulates momentary brain activity (disorder signature) to match activity during an asymptomatic task (target signature), which is expected to translate into symptom reduction.

ß-defensin 1 Gene Polymorphisms are Associated with Kidney Disease in Northwestern Mexicans with Type 2 Diabetes.

Diabetic kidney disease (DKD) is one of the more limiting complications to the quality of life of diabetes mellitus patients. Studies including cultured cells, animal models, and case-control studies highlight the role of human ß-defensin-1 (hBD-1) in diabetes.This study assessed the association of hBD-1 gene (DEFB1) functional variations -52 G/A (rs1799946), -44 C/G (rs1800972) and -20 G/A (rs11362) with type 2 diabetes mellitus (T2DM) in order to investigate its effects on genetic susceptibility and progression to DKD in a Mexican population. A total of 214 T2DM patients with and without DKD (n = 102 and n = 112, respectively) and 117 healthy subjects participated in this case-control study. Genotyping was made by PCR-RFLPs. Clinical and biochemical parameters of all patients were measured. There was no statistically significant difference in genotype or allele frequencies between patients and healthy individuals. Nevertheless, compared with patients without DKD, DKD patients have a reduced prevalence of AA genotype of -52 G/A (OR = 0.307, 95% CI = 0.104-0.905, p =.026), as well as a higher frequency of GA genotype of -20 G/A variant (OR = 1.875, 95%CI = 1.031-3.409, p = .038). Our results suggest that rs1799946 and rs11362 could be useful variants to stratify T2DM Mexican patients in order to prescribe closer follow-up to prevent or retard DKD. Further tests in different ethnic groups are encouraged.

Face mask use during the COVID-19 pandemic: how risk perception, experience with COVID-19, and attitude towards government interact with country-wide policy stringency.

BACKGROUND: During the 2020 COVID-19 pandemic, governments imposed numerous regulations to protect public health, particularly the (mandatory) use of face masks. However, the appropriateness and effectiveness of face mask regulations have been widely discussed, as is apparent from the divergent measures taken across and within countries over time, including mandating, recommending, and discouraging their use. In this study, we analyse how country-level policy stringency and individual-level predictors associate with face mask use during the early stages of the global COVID-19 pandemic. METHOD: First, we study how (self and other-related) risk perception, (direct and indirect) experience with COVID-19, attitude towards government and policy stringency shape face mask use. Second, we study whether there is an interaction between policy stringency and the individual-level variables. We conduct multilevel analyses exploiting variation in face mask regulations across countries and using data from approximately 7000 students collected in the beginning of the pandemic (weeks 17 through 19, 2020). RESULTS: We show that policy stringency is strongly positively associated with face mask use. We find a positive association between self-related risk perception and mask use, but no relationship of mask use with experience with COVID-19 and attitudes towards government. However, in the interaction analyses, we find that government trust and perceived clarity of communication moderate the link between stringency and mask use, with positive government perceptions relating to higher use in countries with regulations and to lower use in countries without regulations. CONCLUSIONS: We highlight that those countries that aim for widespread use of face masks should set strict measures, stress self-related risks of COVID-19, and use clear communication.

Effectiveness of telemedicine psychoeducational interventions for adults with non-oncological chronic disease: A systematic review.

AIM: To evaluate the effectiveness of telemedicine psychoeducational interventions (PIs) in adult patients on the clinical management of chronic non-oncological diseases compared with another therapeutic option or no treatment. DESIGN: Systematic review of randomized controlled trials. DATA SOURCES: Six databases were searched between January 2011 and August 2021. REVIEW METHODS: A systematic review was reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Study selection, quality appraisal and data extraction were conducted independently by two reviewers. A third arbiter was available if discrepancies. RESULTS: A total of 719 articles were reviewed and 17 studies met the inclusion and quality criteria. The included studies related to smoking, chronic pain, obesity and mental illness. Most interventions were based on cognitive behavioural theory. Most of the included studies (12/17, 70.5%) showed improvements in health and significant reductions in anxiety, pain and depression with variable effect sizes. Patients reported a high satisfaction rate and indicated lectures or self-report writings as helpful in their recovery compared with more interactive items. Only financial incentives demonstrated greater adherence. A specific intervention format or complementary professional support was not associated with health outcomes. CONCLUSIONS: Telemedicine PIs are a safe and effective option for the clinical management of adults with chronic diseases. Future longitudinal studies are needed to assess the impact of these interventions on chronic physical and mental disease, evaluating the quality of life, morbidity and mortality. IMPACT: The results reinforce the telemedicine PIs with effects on clinical management similar to those of the face-to-face modality and can be carried out in a safe environment for patients at a lower cost to the health system. These conditions make them suitable for comprehensive care in the epidemiological COVID-19 context with the highest safety conditions for the patients and professionals.

Profiles of adult people in a Spanish sample with chronic pain: Cluster analysis.

AIM: To establish groups of people with chronic non-cancer pain according to the impairment caused by pain and to identify factors associated with the group with a higher level of impairment. BACKGROUND: Knowing the profiles of people who suffer from chronic non-cancer pain could make it possible to direct their treatment and to detect associated risks. DESIGN: A cross-sectional study. METHODS: A sample of 395 people with chronic non-cancer pain was collected in Pain Units and Primary Healthcare Centres in southern Spain (January to March 2020). A cluster analysis was performed to divide the population into groups and a binary logistic regression model was established to determine factors associated with the group with a higher level of impairment. RESULTS: Two groups were identified: lower level of impairment due to pain, characterized by being 45-65 years old, not medicated with opioids or anxiolytics, employed and with a mild level of impact on daily life; and higher level of impairment characterized by being older than 65 years old, medicated with opioids and anxiolytics, retired or on medical leave and with a severe impact on daily life. In addition, among women, being widowed, single or a smoker are risk factors for belonging to the group with a higher level of impairment; being smokers or consuming alcohol three or less times a week would be risk factors in men. CONCLUSIONS: Age, chronic non-cancer pain impact on daily life, work situation and the consumption of opioid drugs and/or anxiolytics are factors that appear to influence the level of impairment due to chronic pain. IMPACT: These findings could help detect impairment due to pain in its early stages, determining the specific needs of each person.

Shedding Light on the Role of Na,K-ATPase as a Phosphatase during Matrix-Vesicle-Mediated Mineralization.

Matrix vesicles (MVs) contain the whole machinery necessary to initiate apatite formation in their lumen. We suspected that, in addition to tissue-nonspecific alkaline phosphatase (TNAP), Na,K,-ATPase (NKA) could be involved in supplying phopshate (Pi) in the early stages of MV-mediated mineralization. MVs were extracted from the growth plate cartilage of chicken embryos. Their average mean diameters were determined by Dynamic Light Scattering (DLS) (212 ± 19 nm) and by Atomic Force Microcopy (AFM) (180 ± 85 nm). The MVs had a specific activity for TNAP of 9.2 ± 4.6 U·mg-1 confirming that the MVs were mineralization competent. The ability to hydrolyze ATP was assayed by a colorimetric method and by 31P NMR with and without Levamisole and SBI-425 (two TNAP inhibitors), ouabain (an NKA inhibitor), and ARL-67156 (an NTPDase1, NTPDase3 and Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) competitive inhibitor). The mineralization profile served to monitor the formation of precipitated calcium phosphate complexes, while IR spectroscopy allowed the identification of apatite. Proteoliposomes containing NKA with either dipalmitoylphosphatidylcholine (DPPC) or a mixture of 1:1 of DPPC and dipalmitoylphosphatidylethanolamine (DPPE) served to verify if the proteoliposomes were able to initiate mineral formation. Around 69-72% of the total ATP hydrolysis by MVs was inhibited by 5 mM Levamisole, which indicated that TNAP was the main enzyme hydrolyzing ATP. The addition of 0.1 mM of ARL-67156 inhibited 8-13.7% of the total ATP hydrolysis in MVs, suggesting that NTPDase1, NTPDase3, and/or NPP1 could also participate in ATP hydrolysis. Ouabain (3 mM) inhibited 3-8% of the total ATP hydrolysis by MVs, suggesting that NKA contributed only a small percentage of the total ATP hydrolysis. MVs induced mineralization via ATP hydrolysis that was significantly inhibited by Levamisole and also by cleaving TNAP from MVs, confirming that TNAP is the main enzyme hydrolyzing this substrate, while the addition of either ARL-6715 or ouabain had a lesser effect on mineralization. DPPC:DPPE (1:1)-NKA liposome in the presence of a nucleator (PS-CPLX) was more efficient in mineralizing compared with a DPPC-NKA liposome due to a better orientation of the NKA active site. Both types of proteoliposomes were able to induce apatite formation, as evidenced by the presence of the 1040 cm-1 band. Taken together, the findings indicated that the hydrolysis of ATP was dominated by TNAP and other phosphatases present in MVs, while only 3-8% of the total hydrolysis of ATP could be attributed to NKA. It was hypothesized that the loss of Na/K asymmetry in MVs could be caused by a complete depletion of ATP inside MVs, impairing the maintenance of symmetry by NKA. Our study carried out on NKA-liposomes confirmed that NKA could contribute to mineral formation inside MVs, which might complement the known action of PHOSPHO1 in the MV lumen.

Generation of the First Human In Vitro Model for McArdle Disease Based on iPSC Technology.

McArdle disease is a rare autosomal recessive disorder caused by mutations in the PYGM gene. This gene encodes for the skeletal muscle isoform of glycogen phosphorylase (myophosphorylase), the first enzyme in glycogenolysis. Patients with this disorder are unable to obtain energy from their glycogen stored in skeletal muscle, prompting an exercise intolerance. Currently, there is no treatment for this disease, and the lack of suitable in vitro human models has prevented the search for therapies against it. In this article, we have established the first human iPSC-based model for McArdle disease. For the generation of this model, induced pluripotent stem cells (iPSCs) from a patient with McArdle disease (harbouring the homozygous mutation c.148C>T; p.R50* in the PYGM gene) were differentiated into myogenic cells able to contract spontaneously in the presence of motor neurons and generate calcium transients, a proof of their maturity and functionality. Additionally, an isogenic skeletal muscle model of McArdle disease was created. As a proof-of-concept, we have tested in this model the rescue of PYGM expression by two different read-through compounds (PTC124 and RTC13). The developed model will be very useful as a platform for testing drugs or compounds with potential pharmacological activity.

Mineralization Profile of Annexin A6-Harbouring Proteoliposomes: Shedding Light on the Role of Annexin A6 on Matrix Vesicle-Mediated Mineralization.

The biochemical machinery involved in matrix vesicles-mediated bone mineralization involves a specific set of lipids, enzymes, and proteins. Annexins, among their many functions, have been described as responsible for the formation and stabilization of the matrix vesicles' nucleational core. However, the specific role of each member of the annexin family, especially in the presence of type-I collagen, remains to be clarified. To address this issue, in vitro mineralization was carried out using AnxA6 (in solution or associated to the proteoliposomes) in the presence or in the absence of type-I collagen, incubated with either amorphous calcium phosphate (ACP) or a phosphatidylserine-calcium phosphate complex (PS-CPLX) as nucleators. Proteoliposomes were composed of 1,2-dipalmitoylphosphatidylcholine (DPPC), 1,2-dipalmitoylphosphatidylcholine: 1,2-dipalmitoylphosphatidylserine (DPPC:DPPS), and DPPC:Cholesterol:DPPS to mimic the outer and the inner leaflet of the matrix vesicles membrane as well as to investigate the effect of the membrane fluidity. Kinetic parameters of mineralization were calculated from time-dependent turbidity curves of free Annexin A6 (AnxA6) and AnxA6-containing proteoliposomes dispersed in synthetic cartilage lymph. The chemical composition of the minerals formed was investigated by Fourier transform infrared spectroscopy (FTIR). Free AnxA6 and AnxA6-proteoliposomes in the presence of ACP were not able to propagate mineralization; however, poorly crystalline calcium phosphates were formed in the presence of PS-CPLX, supporting the role of annexin-calcium-phosphatidylserine complex in the formation and stabilization of the matrix vesicles' nucleational core. We found that AnxA6 lacks nucleation propagation capacity when incorporated into liposomes in the presence of PS-CPLX and type-I collagen. This suggests that AnxA6 may interact either with phospholipids, forming a nucleational core, or with type-I collagen, albeit less efficiently, to induce the nucleation process.