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At least 50% of chronic disease patients don't follow their care plans, leading to lower health outcomes and higher medical costs. Providing Patient Education Materials (PEMs) to individuals living with a disease can help to overcome these problems. PEMs are especially beneficial for people suffering from multisystemic and underrecognized diseases, such as rare diseases. Congenital disorders of glycosylation (CDG) are ultra-rare diseases, where a need was identified for PEMs in plain language that can clearly explain complex information. Community involvement in the design of PEMs is extremely important for diseases whose needs are underserved, such as rare diseases; however, attempts to involve lay and professional stakeholders are lacking. This paper presents a community-based participatory framework to co-create PEMs for CDG, that is transferable to other diseases. A literature review and questionnaire were performed, and only four articles describing the development of PEMS for rare diseases have been found, which demonstrates a lack of standardized approaches. The framework and PEMs were co-developed with CDG families and will be crucial in increasing health literacy and empowering families. We will close a gap in the creation of PEMs for CDG by delivering these resources in lay language in several languages. ispartof: Int J Environ Res Public Health vol:20 issue:2 pages:968- ispartof: location:Switzerland status: Published online

Exposure to pollution is a worldwide societal challenge participating in the etiology and progression of different diseases. However, the scarce information hinders our understanding of the actual level of human exposure and its specific effects. Inadequate and excessive immune responses underlie diverse chronic diseases. Yet, it is unclear which and how toxicant exposures affect the immune system functions. There is a multiplicity of immunological outcomes and biomarkers being studied in human trials related to exposure to different toxicants but still without clear evidence of their value as biomarkers of exposure or effect. The main aim of this study was to collect scientific evidence and identify relevant immunological biomarkers used at the clinical level for toxicant exposures. We used the platform clinical trials.gov as a database tool. First, we performed a search combining research items related to toxicants and immunological parameters. The resulting117 clinical trials were examined for immune-related outcomes and specific biomarkers evaluated in subjects exposed to occupational and environmental toxicants. After categorization, relevant immunological outcomes and biomarkers were identified related to systemic and airway inflammation, modulation of immune cells, allergy and autoimmunity. In general, the immune markers related to inflammation are more frequently investigated for exposure to pollutants, namely IL-6, C-reactive protein (CRP) and nitric oxide (NO). Nevertheless, the data also indicated that prospective biomarkers of effect are gaining ground and a guiding representation of the established and novel biomarkers is suggested for upcoming trials. Finally, potential protective strategies to mitigate the adverse effects of specific toxicants are underlined for future studies. info:eu-repo/semantics/publishedVersion

Incorporation of bioactive natural compounds like polyphenols is an attractive approach for enhanced functionalities of biomaterials. In particular flavonoids have important pharmacological activities, and controlled release systems may be instrumental to realize the full potential of these phytochemicals. Alginate presents interesting attributes for dermal and other biomaterial applications, and studies were carried here to support the development of polyphenol-loaded alginate systems. Studies of capillary viscosity indicated that ionic medium is an effective strategy to modulate the polyelectrolyte effect and viscosity properties of alginates. On gelation, considerable differences were observed between alginate gels produced with Ca2+, Ba2+, Cu2+, Fe2+, Fe3+ and Zn2+ as crosslinkers, especially concerning shrinkage and morphological regularity. Stability assays with different polyphenols in the presence of alginate-gelling cations pointed to the choice of calcium, barium and zinc as safer crosslinkers. Alginate-based films loaded with epicatechin were prepared and the kinetics of release of the flavonoid investigated. The results with calcium, barium and zinc alginate matrices indicated that the release dynamics is dependent on film thicknesses, but also on the crosslinking metal used. On these grounds, an alginate-based system of convenient use was devised, so that flavonoids can be easily loaded at simple point-of-care conditions before dermal application. This epicatechin-loaded patch was tested on an ex-vivo skin model and demonstrated capacity to deliver therapeutically relevant concentrations on skin surface. Moreover, the flavonoid released was not modified and retained full antioxidant bioactivity. The alginate-based system proposed offers a multifunctional approach for flavonoid controllable delivery and protection of skin injured or under risk., Graphical abstract Image 1, Highlights • Production of alginate gels with diverse metal cations was compared. • Copper and iron ions may interfere in the production of polyphenol-loaded materials. • Epicatechin release rate by metal-alginates increased in the order Ba2+

Aims: Laccases and peroxidases have attracted great interest for industrial and environmental applications. These enzymes have a broad substrate range and a robust oxidizing ability. Moreover, using mediators or co-oxidants makes it possible to increase their catalytic activity and extend their substrate scope to more resistant chemical structures. Background: Fungal laccases and ligninolytic peroxidases, mainly lignin and manganese peroxidases, are the privileged oxidoreductases for bioremediation processes. Nonetheless, an increasing diversity of laccases and peroxidase-type enzymes has been proposed for environmental technologies. Objective: This article aims to provide an overview of these enzymes and compare their applicability in the degradation of organic pollutants. Method: Fundamental properties of the proteins are covered and applications towards polycyclic aromatic hydrocarbons (PAHs) and pesticides are specially focused. Result: Laccases are multicopper oxidases initially studied for applications in the pulp and paper industry but able to oxidize a variety of environmentally concerning compounds. Relying on O2, laccases do not require peroxides nor auxiliary agents, like Mn2+, although suitable redox mediators are needed to attack the more recalcitrant pollutants (e.g., PAHs). True and pseudo-peroxidases use a stronger oxidant (H2O2) and the redox chemistry at the heme site generates high potential species that allow the oxidation of dyes and some pesticides. Conclusion: Lately, research efforts have been directed to enzyme discovery, testing with micropollutants, and improving biocatalysts’ stability by immobilization and protein engineering. Further understanding of the effects of natural media components and solvents on the enzymes might lead to competitive enzymatic treatments of highly toxic media.

Funding Information: This research was funded by the FUNDAÇÃO PARA A CIÊNCIA e TECNOLOGIA (FCT—Portugal), grant number PTDC/BIA-MIB/31864/2017 and by LA/P/0045/2020 (ALiCE), UIDB/50020/2020 and UIDP/50020/2020 (LSRE-LCM), funded by national funds through FCT/MCTES (PIDDAC) and by FEDER funding CENTRO-01-0246-FEDER-000044. Publisher Copyright: © 2022 by the authors. Organic pollutants (OPs) and heavy metals are environmental toxicants associated with great concerns. Decontamination processes are urgent for both, and the possibility to achieve their simultaneous removal from polluted waters is highly interesting. Additionally, in many cases, the effect of organic matter in the removal process is overlooked and must be considered. This work aimed to study the potential of alginate-based and polycaprolactone (PCL) materials to remove OPs and copper ions in the absence and presence of organic matter. The OPs investigated were the polycyclic aromatic hydrocarbons anthracene and benzo[a]pyrene, and the pesticide chlorpyrifos, both hydrophobic compounds. Copper (II) ions were used as a model of heavy metals. Alginate-based spheres were prepared by gelation, and PCL microparticles were obtained by oil/water emulsion solvent evaporation. The materials with the highest efficiencies for OP removal from aqueous solutions were those with activated carbon and PCL. Furthermore, the spheres with activated carbon could remove anthracene and copper simultaneously, even in the presence of humic acid. This work points to activated carbon–alginate spheres as a multifunctional adsorbent able to remove different pollutants and to PCL for potential applications in OP decontamination processes. publishersversion published

Congenital disorders of glycosylation (CDG) are a rapidly growing family comprising >100 genetic diseases. Some 25 CDG are pure O-glycosylation defects. Even among this CDG subgroup, phenotypic diversity is broad, ranging from mild to severe poly-organ/system dysfunction. Ophthalmic manifestations are present in 60% of these CDG. The ophthalmic manifestations in N-glycosylation-deficient patients have been described elsewhere. The present review documents the spectrum and incidence of eye disorders in patients with pure O-glycosylation defects with the aim of assisting diagnosis and management and promoting research.

Recently, a global analysis of the structure-activity-relationship of a series of polyoxometalates (POMs) revealed that the most active POMs were ascribed to be polyoxovanadates (POVs), especially decavanadate (V-10), which was very active against certain bacteria (Bijelic et al., Chem. Commun., 2018). The present study explores this observation and compares the effects of three POVs namely MnV11, MnV13 and V-10 against Escherichia coli growth. It was observed that MnV11 presents the lowest growth inhibition (GI(50)) value for Escherichia coli followed by the MnV13 compound, being about 2 times lower than that of V-10 respectively, the values obtained were 0.21, 0.27 and 0.58 mM. All three compounds were more effective than vanadate alone (GI(50) = 1.1 mM) and also than decaniobate, Nb-10 (GI(50) > 10 mM), an isostructural POM of V-10. However, the POVs exhibiting the highest antibacterial activity (MnV11) were shown to have the lowest Ca2+-ATPase inhibitor capacity (IC50 = 58 mM) whereas decavanadate, which was also very active against this membranar ATPase (IC50 = 15 mM), was less active against bacterial growth, suggesting that POV inhibition of ion pumps might not be associated with the inhibition of Escherichia coli growth. Council of Scientific & Industrial Research (CSIR)Council of Scientific & Industrial Research (CSIR) - India [01(2906)/17/EMR-II]

Background The cannabinoid receptor type-1 (CB1R) is a major regulator of metabolism, growth and inflammation. Yet, its potential role in the skin is not well understood. Our aim was to evaluate the role of CB1R in aging-like diabetic skin changes by using a CB1R knockout mouse model. Methods We evaluated several signals of skin aging in wild-type control (WT), WT streptozotocin-induced type 1 diabetic mice (WT DM), CB1R knockout (CB1RKO) and CB1RKO DM mice. We quantified markers of inflammation, angiogenesis, antioxidant enzymes and collagen content. Moreover, we evaluate reactive oxygen species (ROS) levels and macrophage phenotype, M1 and M2. Results CB1R expression is decreased in the skin of WT DM mice and collagen levels are decreased in the skin of WT DM, CB1RKO and CB1RKO DM mice. Additionally, the absence of CB1R correlated with higher expression of pro-inflammatory markers, also evident in WT DM or CB1RKO DM mice. Moreover, the M1/M2 macrophage ratio and ROS levels were significantly elevated but in the diabetic WT and the CB1RKO mice, consistent with a significant decrease in the antioxidant capacity of the skin. Conclusions Our results indicate that CB1R deficiency in the skin may lead to accelerated skin aging due to the increased production of ROS, a decrease in the antioxidant defenses and a higher pro-inflammatory environment. A significant decrease in the CB1R expression may be a significant contributing factor to the early aging-like changes in diabetes.

Human exposure to polycyclic aromatic hydrocarbons (PAHs) and organophosphorus pesticides (OPPs) by dermal route is a continuing concern in environmental and occupational toxicology. Diverse authors have measured in vitro the absorption flux and permeability coefficient (kP) of those compounds delivered on skin surface using volatile solvents. However, there isn't a harmonized method to obtain kP when the test substance is deposited on the skin as a solid. Consequently, varied experimental kPs have been reported for PAHs and OPPs, most in clear disagreement with the values predicted by well-established mathematical models. In this work, we collected the permeation fluxes reported for these toxicants through human skin and calculated the (aqueous) kPs using a method based on the maximum flux and water solubility. The reanalyzed fluxes and recalculated kPs show improved consistency between the different experimental works and mathematical models. Notably, the recalculated kP of benzo[a]pyrene, among others, was approximately 100 times higher than it had been previously considered. Suggestions are given to generalize the method in studies with other solvent-deposited toxicants and drugs.

Alterations in mitochondrial function, cellular redox signaling, and metabolism participate in the etiology and progression of different pathological conditions, such as diabetes, cancer, cardiovascular, and neurodegenerative diseases. Epidemiological, preclinical, and clinical studies indicate that anthocyanins afford therapeutic benefits for these pathologies. It is thus important to revise the effects of anthocyanins on mitochondria and metabolism that hold potential for the development of novel health protective methods. The studies available support the ability of certain anthocyanins to prevent damage to mitochondria and to sustain its function. Evidence is more compelling for cyanidin and delphinidin compounds, but promising data could also be found for malvidin, pelargonidin, and protocatechuic acid. Additionally, berry extracts also demonstrated positive outcomes in different models of neurodegeneration, endothelial dysfunction, myocardial damage, metabolic disorders, longevity, and cancer. At the molecular level, major anthocyanins can modulate the expression and activity of mitochondrial proteins, apoptotic and biogenesis factors, antioxidant defenses, inflammation, and the AMPK pathway. Noteworthy, anthocyanins could balance abnormalities in ROS production, respiration, and mitochondrial fragmentation in cells exposed to toxicants or oxidizing agents.

Delivery systems carrying natural bioactive compounds for enhanced targeting and controlled release are capturing increasing attention. High loadings and sustained release are common design goals. However, in the case of compounds naturally present in human nutrition and physiology, further efforts are justified to optimize their bioactivity and promote clinical success. In this work, it is proposed a specific attention to the regulation of drug temporal presentation as important factor to obtain novel multifunctional delivery systems meeting higher therapeutic efficiencies. Case studies on the relation between drug release dynamics and biological responses are presented for some major delivery strategies and different bioactive molecules. Pharmacokinetic essential concepts and issues concerning the multi-target mode of action typical of the pharmacological properties of natural compounds are discussed in the perspective of improving the development of efficient drug formulations. Several classes of controlled release systems are considered through the chapter, and laboratory setups for testing films and particulate delivery systems are detailed, as well as the application of models for kinetic analysis. Descriptions are illustrated with experimental results obtained with caffeine and epicatechin in our laboratory. Future investigations will benefit from preclinical and clinical evaluation of the new formulations developed by emerging approaches and tools that are being suggested by diverse authors. info:eu-repo/semantics/publishedVersion

Phosphoglucomutase 1 (PGM1) deficiency is a rare genetic disorder that affects glycogen metabolism, glycolysis, and protein glycosylation. Previously known as GSD XIV, it was recently reclassified as a congenital disorder of glycosylation, PGM1-CDG. PGM1-CDG usually manifests as a multisystem disease. Most patients present as infants with cleft palate, liver function abnormalities and hypoglycemia, but some patients present in adulthood with isolated muscle involvement. Some patients develop life-threatening cardiomyopathy. Unlike most other CDG, PGM1-CDG has an effective treatment option, d-galactose, which has been shown to improve many of the patients' symptoms. Therefore, early diagnosis and initiation of treatment for PGM1-CDG patients are crucial decisions. In this article, our group of international experts suggests diagnostic, follow-up, and management guidelines for PGM1-CDG. These guidelines are based on the best available evidence-based data and experts' opinions aiming to provide a practical resource for health care providers to facilitate successful diagnosis and optimal management of PGM1-CDG patients. ispartof: Journal Of Inherited Metabolic Disease vol:s44 issue:1 pages:148-163 ispartof: location:United States status: published

Congenital disorders of glycosylation (CDG) are rare diseases with variable phenotypes and severity. Immunological involvement remains a largely uncharted topic in CDG, mainly due to lack of robust data. To better characterize immune-related manifestations&rsquo, prevalence, relevance, and quality-of-life (QoL) impact, we developed electronic questionnaires targeting (1) CDG patients and (2) the general &ldquo, healthy&rdquo, population. Two-hundred and nine CDG patients/caregivers and 349 healthy participants were included in this study. PMM2-CDG was the most represented CDG (n = 122/209). About half of these participants (n = 65/122) described relevant infections with a noteworthy prevalence of those affecting the gastrointestinal tract (GI) (63.1%, n = 41/65). Infection burden and QoL impact were shown as infections correlated with more severe clinical phenotypes and with a set of relevant non-immune PMM2-CDG signs. Autoimmune diseases had only a marginal presence in PMM2-CDG (2.5%, n = 3/122), all being GI-related. Allergy prevalence was also low in PMM2-CDG (33%, n = 41/122) except for food allergies (26.8%, n = 11/41, of PMM2-CDG and 10.8%, n = 17/158, of controls). High vaccination compliance with greater perceived ineffectiveness (28.3%, n = 17/60) and more severe adverse reactions were described in PMM2-CDG. This people-centric approach not only confirmed literature findings, but created new insights into immunological involvement in CDG, namely by highlighting the possible link between the immune and GI systems in PMM2-CDG. Finally, our results emphasized the importance of patient/caregiver knowledge and raised several red flags about immunological management.

Nitric oxide (NO) is well established as a regulator of neurogenesis. NO increases the proliferation of neural stem cells (NSC), and is essential for hippocampal injury-induced neurogenesis following an excitotoxic lesion. One of the mechanisms underlying non-classical NO cell signaling is protein S-nitrosylation. This post-translational modification consists in the formation of a nitrosothiol group (R–SNO) in cysteine residues, which can promote formation of other oxidative modifications in those cysteine residues. S-nitrosylation can regulate many physiological processes, including neuronal plasticity and neurogenesis. In this work, we aimed to identify S-nitrosylation targets of NO that could participate in neurogenesis. In NSC, we identified a group of proteins oxidatively modified using complementary techniques of thiol redox proteomics. S-nitrosylation of some of these proteins was confirmed and validated in a seizure mouse model of hippocampal injury and in cultured hippocampal stem cells. The identified S-nitrosylated proteins are involved in the ERK/MAPK pathway and may be important targets of NO to enhance the proliferation of NSC., Graphical abstract Image 1, Highlights • Nitric oxide (NO) regulates neurogenesis, and increases proliferation of neural stem cells (NSC). • S-nitrosylation is a cysteine oxidation that acts as a relevant non-classical mechanism of NO signaling. • Cysteine oxidation targets were screened in NSC, confirming S-nitrosylation of several proteins. • S-nitrosylation of some of these targets was confirmed in cultured hippocampal stem cells. • S-nitrosylation of several proteins was observed in an in vivo model of hippocampal neurogenesis.

Human exposure to environmental toxicants with diverse mechanisms of action is a growing concern. In addition to well-recognized carcinogens, various chemicals in environmental and occupational settings have been sug-gested to impact health, increasing susceptibility to cancer by inducing genetic and epigenetic changes. Accordingly, in this review, we have discussed recent insights into the pathological mechanisms of these chemicals, namely their effects on cell redox and calcium homeostasis, mitochondria and inflammatory signaling, with a focus on the possible implications for multi-stage carcinogenesis and its reversal by poly- phenols. Plant-derived polyphenols, such as epigallocatechin-gallate, resveratrol, curcumin and anthocyanins reduce the incidence of cancer and can be useful nutraceuticals for alleviating the detrimental outcomes of harmful pollutants. However, development of therapies based on polyphenol administration requires further studies to validate the biological efficacy, identifying effective doses, mode of action and new delivery forms. Innovative microphysiological testing models are presented and specific proposals for future trials are given. Merging the current knowledge of multifactorial actions of specific polyphenols and chief environmental toxi- cants, this work aims to potentiate the delivery of phytochemical-based protective treatments to individuals at high-risk due to environmental exposure. info:eu-repo/semantics/publishedVersion

Background Health-related Quality of Life (HrQoL) is a multidimensional measure, which has gained clinical and social relevance. Implementation of a patient-centred approach to both clinical research and care settings, has increased the recognition of patient and/or observer reported outcome measures (PROMs or ObsROMs) as informative and reliable tools for HrQoL assessment. Inherited Metabolic Diseases (IMDs) are a group of heterogeneous conditions with phenotypes ranging from mild to severe and mostly lacking effective therapies. Consequently, HrQoL evaluation is particularly relevant. Objectives We aimed to: (1) identify patient and/or caregiver-reported HrQoL instruments used among IMDs; (2) identify the main results of the application of each HrQoL tool and (3) evaluate the main limitations of HrQoL instruments and study design/methodology in IMDs. Methods A scoping review was conducted using methods outlined by Arksey and O’Malley. Additionally, we critically analysed each article to identify the HrQoL study drawbacks. Results Of the 1954 studies identified, 131 addressed HrQoL of IMDs patients using PROMs and/or ObsROMs, both in observational or interventional studies. In total, we identified 32 HrQoL instruments destined to self- or proxy-completion; only 2% were disease-specific. Multiple tools (both generic and disease-specific) proved to be responsive to changes in HrQoL; the SF-36 and PedsQL questionnaires were the most frequently used in the adult and pediatric populations, respectively. Furthermore, proxy data often demonstrated to be a reliable approach complementing self-reported HrQoL scores. Nevertheless, numerous limitations were identified especially due to the rarity of these conditions. Conclusions HrQoL is still not frequently assessed in IMDs. However, our results show successful examples of the use of patient-reported HrQoL instruments in this field. The importance of HrQoL measurement for clinical research and therapy development, incites to further research in HrQoL PROMs’ and ObsROMs’ creation and validation in IMDs. Electronic supplementary material The online version of this article (10.1186/s13023-018-0953-9) contains supplementary material, which is available to authorized users.

Congenital disorders of glycosylation (CDG) are inborn errors of metabolism due to protein and lipid hypoglycosylation. This rapidly growing family of genetic diseases comprises 103 CDG types, with a broad phenotypic diversity ranging from mild to severe poly-organ -system dysfunction. This literature review summarizes cardiac involvement, reported in 20% of CDG. CDG with cardiac involvement were divided according to the associated type of glycosylation: N-glycosylation, O-glycosylation, dolichol synthesis, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, COG complex, V-ATPase complex, and other glycosylation pathways. The aim of this review was to document and interpret the incidence of heart disease in CDG patients. Heart disorders were grouped into cardiomyopathies, structural defects, and arrhythmogenic disorders. This work may contribute to improved early management of cardiac complications in CDG.

Bioactive films encapsulating antioxidant compounds are increasingly used in skin patches, mucoadhesive drug release systems, nutraceutical coatings and engineered tissue substitutes. However, the loading dosage incorporated into the film remains a highly empirical issue concerning the design of these devices, since there are no methods to determine optimal or effective antioxidant loads. In this work, alginate and epicatechin were selected as prototypical components in the development of bioactive-loaded materials for biomedical and food applications. After testing, a methodology is proposed to predict effective antioxidant dosages. Studies with the biopolymer showed that formation of calcium alginate pre-gel can be followed by simple turbidity measurements, and this matrix was used as model system to investigate epicatechin bioactivity in the presence of the polysaccharide. The antioxidant activities of different concentrations of the flavonoid were studied by two assays, the cytochrome c reduction in alginate pre-gel and the prevention of oil oxidation. The results converged to 100 ppm concentration as efficacious load of epicatechin to use. Alginate films loaded with this antioxidant dosage were tested in pine nuts and the films were able to slow down lipid oxidation, as indicated by peroxide value and conjugated dienes parameters. The experimental approach here presented may be useful to further systematize the design of multifunctional antioxidant systems with different applications.

Contains fulltext : 203022.pdf (Publisher’s version ) (Closed access) Phosphomannomutase 2 (PMM2-CDG) is the most common congenital disorder of N-glycosylation and is caused by a deficient PMM2 activity. The clinical presentation and the onset of PMM2-CDG vary among affected individuals ranging from a severe antenatal presentation with multisystem involvement to mild adulthood presentation limited to minor neurological involvement. Management of affected patients requires a multidisciplinary approach. In this article, a systematic review of the literature on PMM2-CDG was conducted by a group of international experts in different aspects of CDG. Our managment guidelines were initiated based on the available evidence-based data and experts' opinions. This guideline mainly addresses the clinical evaluation of each system/organ involved in PMM2-CDG, and the recommended management approach. It is the first systematic review of current practices in PMM2-CDG and the first guidelines aiming at establishing a practical approach to the recognition, diagnosis and management of PMM2-CDG patients.

Cytochrome b5 reductase (Cb5R) and cytochrome b5 (Cb5) are coupled redox systems with a high potential as biomarkers of health and disease in the brain because they regulate metabolic pathways that are essential to maintain normal neuronal function, like lipid biosynthesis, steroid and xenobiotics metabolism, neuronal bioenergetics and production of reactive oxygen species. Mutations of the Cb5R reported in humans produce recessive congenital methemoglobinemia of type II, a disease with severe clinical neurological dysfunctions. The isoform 3 of Cb5R (Cb5R3) and Cb5 are highly expressed in pyramidal neurons of the primary and secondary motor areas of frontoparietal cerebral cortex, hippocampus, vestibular, reticular and motor nuclei of the cerebellum and brain stem, and also in Purkinje and granule neurons of the cerebellum cortex. These brain areas are highly prone to undergo oxidative stress-induced neurodegeneration and their functional impairment can account for neurological deficits reported in type II congenital methemoglobinemia.

The apoptosis of cerebellar granule neurons (CGN) induced by low-potassium in serum free medium in vitro has become a widely used model for neuronal apoptosis during in vivo brain development. In this review we shall summarize first the basic features of this model for neuronal apoptosis. Next, we shall focus on the L-type calcium channels (LTCC) inactivation as the primary pro-apoptotic signal in low K+-induced CGN death. This apoptotic process can be split into two major and sequential cellular signaling phases: one reversible phase that offers a temporal window for therapeutic interventions to prevent neuronal death, and an irreversible later phase. Therefore, we shall comment next the critical role of reactive oxygen species (ROS) production and major ROS sources triggering the entry of CGN in the irreversible stages of low K+-induced apoptosis. Then, we shall present the experimental evidences showing clustering of LTCC and ROS producing enzymes in plasma membrane lipid rafts of CGN matured in vitro, which have opened new perspectives for cell signaling in the early and reversible phase of this apoptosis. The role of lipid rafts nanodomains as fast response calcium/nitric oxide transducers of the switch of CGN to low K+ medium will be discussed next. The two major conclusions drawn from this review are: (1) deregulation of the pool of cytochrome b5 reductase associated to plasma membrane-lipid rafts, at least in part due to overexpression of cytochrome b5, can account for the critical superoxide anion overshot which triggers the entry in the irreversible phase of low K+ apoptosis of CGN, and (2) LTCC inactivation is rapidly transduced by lipid rafts nanodomains into a large drop of cytosolic calcium, a switch-off of nitric oxide production and subsequent inactivation of survival signaling pathways dependent on the activity of CaMKII, PKA and Akt/PKB kinases.

Congenital disorders of glycosylation (CDG) are a rapidly growing family of genetic diseases that currently includes some 130 different types. CDG diagnosis is a challenge, not only because of this large number but also because of the huge clinical heterogeneity even within a number of CDG. In addition, the classical screening test, serum transferrin isoelectrofocusing, is only positive in about 60% of CDG, and can even become negative in some CDG particularly in PMM2-CDG, the most frequent N-glycosylation defect. In order to facilitate CDG diagnosis, we hereby provide some practical tools: (1) a list of clinical features strongly suggestive of a distinctive CDG; (2) a table of clinical, biochemical and laboratory findings reported in CDG, arranged per organ/system; (3) an overview of the affected organs/systems in each CDG; and (4) a diagnostic decision tree in face of a patient with a suspicion of CDG. Most important is to keep in mind a CDG in any unexplained syndrome, in particular when there is neurological involvement. This mini-review enumerates clinical and biochemical hallmarks of these diseases and the biochemical and genetic testing available, and provides an updated list and information on identified CDG. The main aim is to act as a CDG diagnosis simplified guide for healthcare professionals and, additionally, as an awareness and lobbying tool to help in the effectiveness and promptness of CDG diagnosis.

Pattern recognition, using a group of characteristic, or discriminating features, is a powerful tool in metabolic diagnostic. A classic example of this approach is used in biochemical analysis of urine organic acid analysis, where the reporting depends more on the correlation of pertinent positive and negative findings, rather than on the absolute values of specific markers. Similar uses of pattern recognition in the field of biochemical genetics include the interpretation of data obtained by metabolomics, like glycomics, where a recognizable pattern or the presence of a specific glycan sub-fraction can lead to the direct diagnosis of certain types of congenital disorders of glycosylation. Another indispensable tool is the use of clinical pattern recognition–or syndromology–relying on careful phenotyping. While genomics might uncover variants not essential in the final clinical expression of disease, and metabolomics could point to a mixture of primary but also secondary changes in biochemical pathways, phenomics describes the clinically relevant manifestations and the full expression of the disease. In the current review we apply phenomics to the field of congenital disorders of glycosylation, focusing on recognizable differentiating findings in glycosylation disorders, characteristic dysmorphic features and malformations in PMM2-CDG, and overlapping patterns among the currently known glycosylation disorders based on their pathophysiological basis.

Congenital disorders of glycosylation (CDG) are a group of genetic disorders that affect protein and lipid glycosylation and glycosylphosphatidylinositol synthesis. More than 100 different disorders have been reported and the number is rapidly increasing. Since glycosylation is an essential post-translational process, patients present a large range of symptoms and variable phenotypes, from very mild to extremely severe. Only for few CDG, potentially curative therapies are being used, including dietary supplementation (e.g., galactose for PGM1-CDG, fucose for SLC35C1-CDG, Mn2+ for TMEM165-CDG or mannose for MPI-CDG) and organ transplantation (e.g., liver for MPI-CDG and heart for DOLK-CDG). However, for the majority of patients, only symptomatic and preventive treatments are in use. This constitutes a burden for patients, care-givers and ultimately the healthcare system. Innovative diagnostic approaches, in vitro and in vivo models and novel biomarkers have been developed that can lead to novel therapeutic avenues aiming to ameliorate the patients’ symptoms and lives. This review summarizes the advances in therapeutic approaches for CDG.

Dorinda Marques-da-Silva acknowledges the support from the Rare Disease Foundation’s microgrant and ‘Liliana Scientific Scholarship’; Rita Francisco acknowledges Fundação para a Ciência e Tecnologia for the grant SFRH/BD/124326/2016 awarded to her. Congenital disorders of glycosylation (CDG) are ultra-rare diseases showing a great phenotypic diversity ranging from mono-to multi-organ/multisystem involvement. Liver involvement, mostly nonprogressive, is often reported in CDG patients. The main objectives of this work were (1) to better understand liver involvement in CDG patients through a liver electronic questionnaire targeting CDG families (LeQCDG) and (2) to compare responses from LeQCDG participants with literature review regarding the prevalence of liver disease and the occurrence of liver symptoms in CDG patients. The network of patient advocacy groups, families and professionals (CDG & Allies – PPAIN) developed the LeQCDG by adapting validated published questionnaires. The LeQCDG was approved by an ethics committee, and the recruitment of patients and caregivers proceeded through social media platforms. Participants were asked to report past or present liver-related symptoms (e.g. hepatomegaly, liver fibrosis and cirrhosis) and laboratory results (e.g. biochemical and/ or radiological). From 11 December 2016 to 22 January 2017, 155 questionnaires were completed. Liver disease was present in 29.9% of CDG patients. Main symptoms reported included hepatomegaly, increased levels of serum transaminases, fibrosis, steatosis and cirrhosis. The data obtained in this online survey confirm findings from a recent literature review of 25 years of published evidence (r ¼ 0.927, P ¼ 0.02). Our questionnaire collected large amounts of meaningful, clinical and patient-oriented data in a short period of time without geographic limitations. Internet-based approaches are especially relevant in the context of ultra-rare diseases such as CDG. publishersversion published

Cytochrome b 5 reductase (Cb 5R) and cytochrome b 5 (Cb 5) form an enzymatic redox system that plays many roles in mammalian cells. In the last 15 years, it has been proposed that this system is involved in the recycling of ascorbate, a vital antioxidant molecule in the brain and that its deregulation can lead to the production of reactive oxygen species that play a major role in oxidative-induced neuronal death. In this work, we have performed a regional and cellular distribution study of the expression of this redox system in adult rat brain by anti-Cb 5R isoform 3 and anti-Cb 5 antibodies. We found high expression levels in cerebellar cortex, labeling heavily granule neurons and Purkinje cells, and in structures such as the fastigial, interposed and dentate cerebellar nuclei. A large part of Cb 5R isoform 3 in the cerebellum cortex was regionalized in close proximity to the lipid raft-like nanodomains, labeled with cholera toxin B, as we have shown by fluorescence resonance energy transfer imaging. In addition, vestibular, reticular and motor nuclei located at the brain stem level and pyramidal neurons of somatomotor areas of the brain cortex and of the hippocampus have been also found to display high expression levels of these proteins. All these results point out the enrichment of Cb 5R isoform 3/Cb 5 system in neuronal cells and structures of the cerebellum and brain stem whose functional impairment can account for neurological deficits reported in type II congenital methemoglobinemia, as well as in brain areas highly prone to undergo oxidative stress-induced neurodegeneration.

Rotenone is a neurotoxin that is an active component of many pesticides which has been shown to induce Parkinsonism in animal models. We show that the cytotoxicity of exposure to nanomolar concentrations of rotenone in cultures of mature cerebellar granule neurons (CGN) in serum-free medium is not due to phagocytosis by glial contamination. A concentration as low as 5.65 ± 0.51 nM of rotenone was enough to trigger 50% cell death of mature CGN in culture after 12 h. The addition of serum proteins to the culture medium attenuated rotenone neurotoxicity, and this can account at least in part for the requirement of higher rotenone concentrations to elicit neuronal cytotoxicity reported in previous works. Creatine partial protection against CGN death promoted by 5 nM rotenone correlated with creatine protection against rotenone-induced mitochondrial depolarization and oxidative stress. Furthermore, creatine largely attenuated the early dysregulation of cytosolic Ca2+ concentration after acute rotenone treatment. Noteworthy, our results also revealed that the sustained alteration of Ca2+ homeostasis induced by rotenone takes place at the onset of the enhancement of intracellular oxidative stress and before mitochondrial depolarization, pointing out that cytosolic Ca2+ dysregulation is a very early event in the rotenone toxicity to CGN.

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Congenital disorders of glycosylation (CDG) are a rapidly growing family of genetic diseases caused by defects in glycosylation. Nearly 100 CDG types are known so far. Patients present a great phenotypic diversity ranging from poly- to mono-organ/system involvement and from very mild to extremely severe presentation. In this literature review, we summarize the liver involvement reported in CDG patients. Although liver involvement is present in only a minority of the reported CDG types (22 %), it can be debilitating or even life-threatening. Sixteen of the patients we collated here developed cirrhosis, 10 had liver failure. We distinguish two main groups: on the one hand, the CDG types with predominant or isolated liver involvement including MPI-CDG, TMEM199-CDG, CCDC115-CDG, and ATP6AP1-CDG, and on the other hand, the CDG types associated with liver disease but not as a striking, unique or predominant feature, including PMM2-CDG, ALG1-CDG, ALG3-CDG, ALG6-CDG, ALG8-CDG, ALG9-CDG, PGM1-CDG, and COG-CDG. This review aims to facilitate CDG patient identification and to understand CDG liver involvement, hopefully leading to earlier diagnosis, and better management and treatment.

Cytosolic calcium plays a leading role in the control of neuronal excitability, plasticity and survival. This work aims to experimentally assess the possibility that lipid rafts of the plasma membrane can provide a structural platform for a faster and tighter functional coupling between calcium and nitric-oxide signaling in neurons. Using primary cerebellar granule neurons (CGN) in culture this hypothesis has been experimentally assessed with fluorescence resonance energy transfer imaging, preparations of lipid rafts-enriched membrane fragments and western blotting. The results obtained in this work demonstrated that major calcium entry systems of the plasma membrane of CGN (L-type calcium channels and N-methyl-D-aspartate receptors) and nitric-oxide synthase are separated by less than 80 nm from each other within lipid rafts-associated sub-microdomains, suggesting a new role of lipid rafts as neuronal calcium/redox nano-transducers.

Cytoskeleton damage is a frequent feature in neuronal cell death and one of the early events in oxidant-induced cell injury. This work addresses whether actin cytoskeleton reorganization is an early event of SIN-1-induced extracellular nitrosative/oxidative stress in cultured cerebellar granule neurons (CGN). The actin polymerization state, i.e. the relative levels of G-/F-actin, was quantitatively assessed by the ratio of the fluorescence intensities of microscopy images obtained from CGN double-labelled with Alexa594-DNase-I (for actin monomers) and Bodipy-FL-phallacidin (for actin filaments). Exposure of CGN to a flux of peroxynitrite as low as 0.5-1μM/min during 30min (achieved with 0.1mM SIN-1) was found to promote alterations of the actin cytoskeleton dynamics as it increases the G-actin/F-actin ratio. Because L-type voltage-operated Ca(2+) channels (L-VOCC) are primary targets in CGN exposed to SIN-1, the possible role of Ca(2+) dynamics on the perturbation of the actin cytoskeleton was also assessed from the cytosolic Ca(2+) concentration response to the L-VOCC's agonist FPL-64176 and to the L-VOCC's blocker nifedipine. The results showed that SIN-1 induced changes in the actin polymerization state correlated with its ability to decrease Ca(2+) influx through L-VOCC. Combined analysis of cytosolic Ca(2+) concentration and G-actin/F-actin ratio alterations by SIN-1, cytochalasin D, latrunculin B and jasplakinolide support that disruption of the actin cytoskeleton is linked to cytosolic calcium concentration changes.

The activation of L-type calcium channels (LTCCs) prevents cerebellar granule neurons (CGNs) from entering low-K+-induced apoptosis. In previous works, we showed that LTCCs are largely associated with caveolin-1-rich lipid rafts in the CGN plasma membrane. In this work, we show that protein kinase A (PKA) and calmodulin-dependent protein kinase II (CaMK-II) are associated with caveolin-1-rich lipid rafts of mature CGNs, and we further show that treatment with the cholesterol-trapping and lipid raft-disrupting agent methyl-&beta, cyclodextrin decreases the phosphorylation level of the LTCC &beta, 2 subunit and the steady-state calcium concentration in neuronal somas ([Ca2+]i) to values close to those measured in 5 mM KCl proapoptotic conditions. These effects correlate with the effects produced by a short (15 min) treatment of CGNs with H-89 and KN-93&mdash, inhibitors of PKA and CaMK-II, respectively&mdash, in 25 mM KCl medium. Moreover, only a 15 min incubation of CGNs with H-89 produces about a 90% inhibition of the calcium entry that would normally occur through LTCCs to increase [Ca2+]i upon raising the extracellular K+ from 5 to 25 mM, i.e., from proapoptotic to survival conditions. In conclusion, the results of this work suggest that caveolin-1-rich lipid rafts play a major role in the control of the PKA- and CaMK-II-induced phosphorylation level of the LTCC &beta, 2 subunit, thus preventing CGNs from entering apoptosis.

The presence of cytosolic calcium microcompartments in neurons is well established. L-type voltage calcium channels play a leading role in the rise of cytosolic calcium in the neuronal soma and are sensitive to redox modulation. In a recent work [Samhan-Arias, A.K., García-Bereguiaín, M.A., Martín-Romero, F.J. and Gutiérrez-Merino, C. (2009) Mol. and Cell. Neurosci. 40, 14-26], we have shown that cytochrome b(5) reductase, whose deregulation leads to an overshot of superoxide anion production at the neuronal plasma membrane that triggers apoptosis in primary cultures of cerebellar granule neurons in culture, forms a large mesh of redox centres associated with lipid rafts in these neurons. In this work, we have implemented the use of fluorescent antibodies as reagents for quantitative Förster resonance energy transfer measurements and analysis using fluorescence microscopy images of cerebellar granule neurons in culture. The results of this study show that L-type voltage-operated calcium channels are also enriched in lipid rafts associated protein microdomains at a distance between 10 and 100 nm from cytochrome b(5) reductase. The methodological improvements done in this work can be also valuable for the study of proteins compartmentalization within other subcellular microdomains in any cell type in culture.