Association of Mycobacterium avium subsp. paratuberculosis with multiple sclerosis in Sardinian patients

Mycobacterium avium subsp. paratuberculosis (MAP) infection is highly spread in the ruminant herds of Sardinia, in the Western Mediterranean. The objective of this study was to investigate prevalence of MAP infection in association with Multiple Sclerosis (MS) using clinical specimen from patients and controls. We analyzed samples for the presence of MAP specific DNA and to demonstrate humoral response to a MAP protein (MAP2694), a predicted homologue of the T-cell receptor gamma-chain/complement component 1 of the host. We found presence of MAP DNA in 42% of the MS patients and an extremely significant humoral immune response revealed by the MS patients against the MAP protein. In our opinion, this is the first report that significantly associates MAP infection with MS. Further studies will be required to confirm if MAP could be one of the triggers of MS, according to the molecular mimicry theory, in susceptible (and genetically at risk) individuals.     

Kinetic and Structural Evidences on Human Prolidase Pathological Mutants Suggest Strategies for Enzyme Functional Rescue

Prolidase is the only human enzyme responsible for the digestion of iminodipeptides containing proline or hydroxyproline at their C-terminal end, being a key player in extracellular matrix remodeling. Prolidase deficiency (PD) is an intractable loss of function disease, characterized by mutations in the prolidase gene. The exact causes of activity impairment in mutant prolidase are still unknown. We generated three recombinant prolidase forms, hRecProl-231delY, hRecProl-E412K and hRecProl-G448R, reproducing three mutations identified in homozygous PD patients. The enzymes showed very low catalytic efficiency, thermal instability and changes in protein conformation. No variation of Mn(II) cofactor affinity was detected for hRecProl-E412K; a compromised ability to bind the cofactor was found in hRecProl-231delY and Mn(II) was totally absent in hRecProl-G448R. Furthermore, local structure perturbations for all three mutants were predicted by in silico analysis. Our biochemical investigation of the three causative alleles identified in perturbed folding/instability, and in consequent partial prolidase degradation, the main reasons for enzyme inactivity. Based on the above considerations we were able to rescue part of the prolidase activity in patients’ fibroblasts through the induction of Heath Shock Proteins expression, hinting at new promising avenues for PD treatment.     

miR669a and miR669q prevent skeletal muscle differentiation in postnatal cardiac progenitors

Postnatal heart stem and progenitor cells are a potential therapeutic tool for cardiomyopathies, but little is known about the mechanisms that control cardiac differentiation. Recent work has highlighted an important role for microribonucleic acids (miRNAs) as regulators of cardiac and skeletal myogenesis. In this paper, we isolated cardiac progenitors from neonatal β-sarcoglycan (Sgcb)-null mouse hearts affected by dilated cardiomyopathy. Unexpectedly, Sgcb-null cardiac progenitors spontaneously differentiated into skeletal muscle fibers both in vitro and when transplanted into regenerating muscles or infarcted hearts. Differentiation potential correlated with the absence of expression of a novel miRNA, miR669q, and with down-regulation of miR669a. Other miRNAs are known to promote myogenesis, but only miR669a and miR669q act upstream of myogenic regulatory factors to prevent myogenesis by directly targeting the MyoD 3' untranslated region. This finding reveals an added level of complexity in the mechanism of the fate choice of mesoderm progenitors and suggests that using endogenous cardiac stem cells therapeutically will require specially tailored procedures for certain genetic diseases.     

Recognition of Zinc Transporter 8 and MAP3865c Homologous Epitopes by Hashimoto's Thyroiditis Subjects from Sardinia: A Common Target with Type 1 Diabetes?

Mycobacterium avium subspecies paratuberculosis (MAP) asymptomatic infection has been previously linked to Type 1 diabetes (T1D) and Multiple Sclerosis. An association between MAP infection and Hashimoto''s thyroiditis (HT) was also proposed only in a case report. This study aimed to investigate the robustness of the latter association, testing a large cohort of HT and healthy control (HCs) subjects, all from Sardinia. Prevalence of anti-MAP3865c Abs was assessed by indirect enzyme-linked immunosorbent assay (ELISA). Moreover, given that human ZnT8 is specifically expressed in the pancreatic β-cells, in the follicle epithelial cells and in the parafollicular cells of the thyroid gland, we also tested ZnT8 epitopes homologues to the MAP3865c immunodominant peptides previously identified. Indeed, Abs targeting MAP3865c and ZnT8 homologous regions display similar frequencies in patients and controls, thus suggesting that Abs recognizing these epitopes could be cross-reactive. A statistically significant difference was found between HT patients and HCs when analyzing the humoral response mounted against MAP3865c/ZnT8 homologues epitopes. To our knowledge, this is the first report, which provides statistically significant evidence sustaining the existence of an association between MAP sero-reactivity and HT. Further studies are required to investigate the relevance of MAP to HT, aimed at deciphering if this pathogen can be at play in triggering this autoimmune disease. Likewise, genetic polymorphism of the host, and other environmental factors need to be investigated.     

Mutations in SLC30A10 cause parkinsonism and dystonia with hypermanganesemia, polycythemia, and chronic liver disease

Manganese is essential for several metabolic pathways but becomes toxic in excessive amounts. Manganese levels in the body are therefore tightly regulated, but the responsible protein(s) remain incompletely known. We studied two consanguineous families with neurologic disorders including juvenile-onset dystonia, adult-onset parkinsonism, severe hypermanganesemia, polycythemia, and chronic hepatic disease, including steatosis and cirrhosis. We localized the genetic defect by homozygosity mapping and then identified two different homozygous frameshift SLC30A10 mutations, segregating with disease. SLC30A10 is highly expressed in the liver and brain, including in the basal ganglia. Its encoded protein belongs to a large family of membrane transporters, mediating the efflux of divalent cations from the cytosol. We show the localization of SLC30A10 in normal human liver and nervous system, and its depletion in liver from one affected individual. Our in silico analyses suggest that SLC30A10 possesses substrate specificity different from its closest (zinc-transporting) homologs. We also show that the expression of SLC30A10 and the levels of the encoded protein are markedly induced by manganese in vitro. The phenotype associated with SLC30A10 mutations is broad, including neurologic, hepatic, and hematologic disturbances. Intrafamilial phenotypic variability is also present. Chelation therapy can normalize the manganesemia, leading to marked clinical improvements. In conclusion, we show that SLC30A10 mutations cause a treatable recessive disease with pleomorphic phenotype, and provide compelling evidence that SLC30A10 plays a pivotal role in manganese transport. This work has broad implications for understanding of the manganese biology and pathophysiology in multiple human organs.     

Targeting the X-linked inhibitor of apoptosis protein through 4-substituted azabicyclo[5.3.0]alkane smac mimetics. Structure, activity, and recognition principles

The X-linked inhibitor of apoptosis protein (XIAP) is overexpressed in several malignant cells where it prevents apoptosis by binding to, and blocking, the activation of caspase-3, -7, and -9. Human XIAP (479 residues) is composed of three tandem-repeated baculoviral IAP repeat (BIR) domains (BIR1-3), and by a C-terminal RING domain. Smac-DIABLO [second mitochondria-derived activator of caspases (Smac)-direct IAP binding protein with low pI (DIABLO)], the natural antagonist of XIAP, binds through its N-terminal sequence AVPI to the same surface groove, in the BIR domains, that binds caspases. Synthetic compounds mimicking such tetrapeptide motif effectively block the interaction between IAP and active caspases, thus triggering apoptosis. Peptidomimetics based on an azabicyclo[x.y.0]alkane scaffolds, have been shown to bind the BIR3 domain of XIAP with micromolar to nanomolar affinities, thus presenting attractive features for drug lead optimization. Here we report a study on three newly synthesized Smac mimetics, which have been characterized in their complexes with XIAP BIR3 domain through X-ray crystallography and molecular modelling/docking simulations. Based on analysis of the crystal structures, we show that specific substitutions at the 4-position of the azabicyclo[5.3.0]alkane scaffold results in sizeable effects on the peptidomimetic-BIR3 domain affinity. By means of functional, biophysical and simulative approaches we also propose that the same Smac mimetics can bind XIAP BIR2 domain at a location structurally related to the BIR3 domain AVPI binding groove. Details of the XIAP-Smac mimetic recognition principles highlighted by this study are discussed in light of the drug-like profile of the three (potentially proapoptotic) compounds developed that show improved performance in ADMET (adsorption, distribution, metabolism, excretion and toxicity) tests.     

Main roads and land cover shaped the genetic structure of a Mediterranean island wild boar population

Patterns of genetic differentiation within and among animal populations might vary due to the simple effect of distance or landscape features hindering gene flow. An assessment of how landscape connectivity affects gene flow can help guide management, especially in fragmented landscapes. Our objective was to analyze population genetic structure and landscape genetics of the native wild boar (Sus scrofa meridionalis) population inhabiting the island of Sardinia (Italy), and test for the existence of Isolation‐by‐Distance (IBD), Isolation‐by‐Barrier (IBB), and Isolation‐by‐Resistance (IBR). A total of 393 Sardinian wild boar samples were analyzed using a set of 16 microsatellite loci. Signals of genetic introgression from introduced non‐native wild boars or from domestic pigs were revealed by a Bayesian cluster analysis including 250 reference individuals belonging to European wild populations and domestic breeds. After removal of introgressed individuals, genetic structure in the population was investigated by different statistical approaches, supporting a partition into five discrete subpopulations, corresponding to five geographic areas on the island: north‐west (NW), central west (CW), south‐west (SW), north‐central east (NCE), and south‐east (SE). To test the IBD, IBB, and IBR hypotheses, we optimized resistance surfaces using genetic algorithms and linear mixed‐effects models with a maximum likelihood population effects parameterization. Landscape genetics analyses revealed that genetic discontinuities between subpopulations can be explained by landscape elements, suggesting that main roads, urban settings, and intensively cultivated areas are hampering gene flow (and thus individual movements) within the Sardinian wild boar population. Our results reveal how human‐transformed landscapes can affect genetic connectivity even in a large‐sized and highly mobile mammal such as the wild boar, and provide crucial information to manage the spread of pathogens, including the African Swine Fever virus, endemic in Sardinia.     

Acetylcholine receptor channels are present in undifferentiated satellite cells but not in embryonic myoblasts in culture

The expression and the physiological properties of acetylcholine receptors (AChRs) of mononucleated myogenic cells, isolated from either embryonic or adult muscle of the mouse, have been investigated using the gigaohm seal patch-clamp technique in combination with immunocytochemistry (with an anti-myosin antibody) and alpha-bungarotoxin binding techniques. Undifferentiated (myosin-negative) embryonic myoblasts, grown either in mass culture or under clonal conditions, were found to be unresponsive to ACh and did not bind alpha-bungarotoxin. On the contrary, undifferentiated satellite cells (from adult muscle) exhibited channels activated by ACh and alpha-bungarotoxin binding sites similar to those observed in differentiated (myosin-positive) embryonic myoblasts and myotubes. Two classes of ACh-activated channels with different opening frequencies were identified. The major class of channels had a conductance of about 42 pS and mean open time of 3.1-8.2 msec. The minor class of channels had smaller conductance (about 17 pS) and similar open time. During differentiation, the conductance of the two channels did not change significantly, while channel lifetime became shorter in myotubes derived from satellite cells but not in myotubes derived from embryonic myoblasts. The relative proportion of small over large channels was significantly larger in embryonic than in adult myogenic cells.     

Emergence of TPA-resistant ‘satellite’ cells during muscle histogenesis of human limb

Human satellite cells, obtained by surgical biopsies of traumatized legs of healthy individuals, were grown in culture in the presence of different concentrations of the phorbol ester tetradecanoyl-phorbol 12 acetate (TPA). Satellite cells, after an initial duplicative period, fused into large multinucleated myotubes which readily synthesized myosin and acetylcholine receptor (AChR). The presence of TPA at concentrations up to 10(-7) M did not affect the differentiation pattern, while higher concentrations were toxic. Thus human satellite cells are capable of differentiating in the presence of phorbol esters which block differentiation of embryonic myoblasts [1]. We then examined the appearance of TPA-resistant cells during human muscle histogenesis, since we had observed that differentiation of human myoblasts from a 6-week-old limb was completely and reversibly inhibited by 10(-7) M TPA. Differentiation of myoblasts from 6-, 7- and 8-week-old fetuses was completely inhibited by TPA. Myoblasts from 10-week-old limbs did not form myotubes in the presence of TPA; however, immunohistochemical staining with an antimyosin antibody revealed the presence of a few mononucleated myosin-positive cells which escaped the TPA-induced block of differentiation. At 12 weeks of development, a few oligonucleated, myosin-positive myotubes developed in cultures treated with TPA, and the level of AChR expressed (measured as [125I] alpha-bungarotoxin bound) reached 20% of controls. At 14 weeks of development, about half of the cells in culture were TPA-resistant and by 16 weeks of development no major differences could be detected between control and treated cells. We conclude from these data that a population of TPA-resistant myogenic cells emerges between the 10th and 14th week of human limb development and suggest that this population represents satellite cells.