A MANBA mutation resulting in residual beta-mannosidase activity associated with severe leukoencephalopathy: a possible pseudodeficiency variant

<p>Abstract</p> <p>Background</p> <p>β-Mannosidosis (OMIM 248510) is a rare inborn lysosomal storage disorder caused by the deficient activity of β-mannosidase, an enzyme encoded by a single gene (<it>MANBA</it>) located on chromosome 4q22-25. To date, only 20 cases of this autosomal recessive disorder have been described and 14 different <it>MANBA </it>mutations were incriminated in the disease. These are all null mutations or missense mutations that abolish β-mannosidase activity. In this study, we characterized the molecular defect of a new case of β-mannosidosis, presenting with a severe neurological disorder.</p> <p>Methods</p> <p>Genomic DNA was isolated from peripheral blood leukocytes of the patient to allow <it>MANBA </it>sequencing. The identified mutation was engineered by site-directed mutagenesis and the mutant protein was expressed through transient transfection in HEK293T cells. The β-mannosidase expression and activity were respectively assessed by Western blot and fluorometric assay in both leukocytes and HEK293T cells.</p> <p>Results</p> <p>A missense disease-associated mutation, c.1922G>A (p.Arg641His), was identified for which the patient was homozygous. In contrast to previously described missense mutations, this substitution does not totally abrogate the enzyme activity but led to a residual activity of about 7% in the patient's leukocytes, 11% in lymphoblasts and 14% in plasma. Expression studies in transfected cells also resulted in 7% residual activity.</p> <p>Conclusion</p> <p>Correlations between MANBA mutations, residual activity of β-mannosidase and the severity of the ensuing neurological disorder are discussed. Whether the c.1922G>A mutation is responsible for a yet undescribed pseudodeficiency of β-mannosidase is also discussed.</p

SANI definition of Clinical Remission in Severe Asthma: a Delphi consensus

: Severe Asthma affects about 10% of the asthmatic population, and it is characterized by a low lung function and a higher count of blood leucocytes, mainly eosinophils. To date, various definitions are used in clinical practice and in the literature to identify asthma remission: clinical remission, inflammatory remission, and complete remission. The aim of this work is to highlight a consensus for asthma remission using a Delphi method. In the context of SANI (Severe Asthma Network Italy), accounting for 57 Severe Asthma Centers and more then 2200 patients, a Board of six expert drafted a list of candidate statements in a questionnaire, which has been revised to minimize redundancies and ensure clear and consistent wording for the first round (R1) of the analysis. 32 statements have been included in the R1 questionnaire, and then submitted to a panel of 80 experts, which used a 5-points Likert scale to measure their agreement to each statement. Then, an Interim Analysis of R1 data have been performed, items were discussed and considered to produce a consistent questionnaire for the round 2 (R2) of the analysis. After this, the Board set the R2 questionnaire, which included only the important key topics. Panelists have been asked to vote the statements in the R2 questionnaire afterwards. During R2, the criteria of complete clinical remission (the absence of need for OCS, symptoms, exacerbations/attacks, and a pulmonary function stability) and those of partial clinical remission (the absence of need for OCS, and 2 out of 3 criteria: the absence of symptoms, exacerbations/attacks, and a pulmonary stability) were confirmed. This SANI Delphi Analysis defined a valuable, independent and easy to use tool to test the efficacy of different treatments in patients with severe asthma enrolled into the SANI registry

Conserved BK Channel-Protein Interactions Reveal Signals Relevant to Cell Death and Survival

The large-conductance Ca2+-activated K+ (BK) channel and its β-subunit underlie tuning in non-mammalian sensory or hair cells, whereas in mammals its function is less clear. To gain insights into species differences and to reveal putative BK functions, we undertook a systems analysis of BK and BK-Associated Proteins (BKAPS) in the chicken cochlea and compared these results to other species. We identified 110 putative partners from cytoplasmic and membrane/cytoskeletal fractions, using a combination of coimmunoprecipitation, 2-D gel, and LC-MS/MS. Partners included 14-3-3γ, valosin-containing protein (VCP), stathmin (STMN), cortactin (CTTN), and prohibitin (PHB), of which 16 partners were verified by reciprocal coimmunoprecipitation. Bioinformatics revealed binary partners, the resultant interactome, subcellular localization, and cellular processes. The interactome contained 193 proteins involved in 190 binary interactions in subcellular compartments such as the ER, mitochondria, and nucleus. Comparisons with mice showed shared hub proteins that included N-methyl-D-aspartate receptor (NMDAR) and ATP-synthase. Ortholog analyses across six species revealed conserved interactions involving apoptosis, Ca2+ binding, and trafficking, in chicks, mice, and humans. Functional studies using recombinant BK and RNAi in a heterologous expression system revealed that proteins important to cell death/survival, such as annexinA5, γ-actin, lamin, superoxide dismutase, and VCP, caused a decrease in BK expression. This revelation led to an examination of specific kinases and their effectors relevant to cell viability. Sequence analyses of the BK C-terminus across 10 species showed putative binding sites for 14-3-3, RAC-α serine/threonine-protein kinase 1 (Akt), glycogen synthase kinase-3β (GSK3β) and phosphoinositide-dependent kinase-1 (PDK1). Knockdown of 14-3-3 and Akt caused an increase in BK expression, whereas silencing of GSK3β and PDK1 had the opposite effect. This comparative systems approach suggests conservation in BK function across different species in addition to novel functions that may include the initiation of signals relevant to cell death/survival

Conservation of Mediterranean oak woodlands: understorey dynamics under different shrub management

The effect of experimental disturbances on the dynamics of a shrub community was studied on a ‘Montado’ ecosystem, in southern Portugal. The evolution of the community physiognomy, composition and diversity were monitored after shrub clearing followed by biomass removal, deposition on soil surface and incorporation with the soil, over a 9-year period. Maximum shrub density was recorded in the first year after the disturbances, excepting in mulched plots which showed the greatest number of individuals 1 year later. The increment of shrub leaf biomass was very fast in the first 3 years, whereas wood production was slower but occurred along the whole study period. At the end of the study, leaf and wood biomass was still significantly lower than in the predisturbance situation. The variation pattern of leaf area index was similar to that of leaf biomass. The evolution of total plant cover and diversity was similar across treatments. The highest species richness and diversity were recorded 2 years after cutting, decreasing afterwards with the increasing dominance of shrubs. Thus it seems likely that, although a 9 year period is too short for these communities to reach steady equilibrium, they are very resistant and resilient to disturbances, as regeneration was fast and vegetation dynamics was not influenced by differences among treatments. We can conclude that shrub clearing promotes biodiversity and the time of permanence of shrub patches depends on the particular goal we want to achieve

Conservation of Mediterranean oak woodlands: understorey dynamics under different shrub management

The effect of experimental disturbances on the dynamics of a shrub community was studied on a ‘Montado’ ecosystem, in southern Portugal. The evolution of the community physiognomy, composition and diversity were monitored after shrub clearing followed by biomass removal, deposition on soil surface and incorporation with the soil, over a 9-year period. Maximum shrub density was recorded in the first year after the disturbances, excepting in mulched plots which showed the greatest number of individuals 1 year later. The increment of shrub leaf biomass was very fast in the first 3 years, whereas wood production was slower but occurred along the whole study period. At the end of the study, leaf and wood biomass was still significantly lower than in the predisturbance situation. The variation pattern of leaf area index was similar to that of leaf biomass. The evolution of total plant cover and diversity was similar across treatments. The highest species richness and diversity were recorded 2 years after cutting, decreasing afterwards with the increasing dominance of shrubs. Thus it seems likely that, although a 9 year period is too short for these communities to reach steady equilibrium, they are very resistant and resilient to disturbances, as regeneration was fast and vegetation dynamics was not influenced by differences among treatments. We can conclude that shrub clearing promotes biodiversity and the time of permanence of shrub patches depends on the particular goal we want to achieve

Molecular Mechanisms Associated with Nicotine Pharmacology and Dependence.

Tobacco dependence is a leading cause of preventable disease and death worldwide. Nicotine, the main psychoactive component in tobacco cigarettes, has also been garnering increased popularity in its vaporized form, as derived from e-cigarette devices. Thus, an understanding of the molecular mechanisms underlying nicotine pharmacology and dependence is required to ascertain novel approaches to treat drug dependence. In this chapter, we review the field's current understanding of nicotine's actions in the brain, the neurocircuitry underlying drug dependence, factors that modulate the function of nicotinic acetylcholine receptors, and the role of specific genes in mitigating the vulnerability to develop nicotine dependence. In addition to nicotine's direct actions in the brain, other constituents in nicotine and tobacco products have also been found to alter drug use, and thus, evidence is provided to highlight this issue. Finally, currently available pharmacotherapeutic strategies are discussed, along with an outlook for future therapeutic directions to achieve to the goal of long-term nicotine cessation

Nicotinic Receptors Underlying Nicotine Dependence: Evidence from Transgenic Mouse Models.

Nicotine underlies the reinforcing properties of tobacco cigarettes and e-cigarettes. After inhalation and absorption, nicotine binds to various nicotinic acetylcholine receptor (nAChR) subtypes localized on the pre- and postsynaptic membranes of cells, which subsequently leads to the modulation of cellular function and neurotransmitter signaling. In this chapter, we begin by briefly reviewing the current understanding of nicotine's actions on nAChRs and highlight considerations regarding nAChR subtype localization and pharmacodynamics. Thereafter, we discuss the seminal discoveries derived from genetically modified mouse models, which have greatly contributed to our understanding of nicotine's effects on the reward-related mesolimbic pathway and the aversion-related habenulo-interpeduncular pathway. Thereafter, emerging areas of research focusing on modulation of nAChR expression and/or function are considered. Taken together, these discoveries have provided a foundational understanding of various genetic, neurobiological, and behavioral factors underlying the motivation to use nicotine and related dependence processes, which are thereby advancing drug discovery efforts to promote long-term abstinence