ANALYSIS OF A RARE GENETIC DISEASE DUE TO PROTEIN INSTABILITY: STUDY AND CHARACTERIZATION OF CONGENITAL DISORDER OF GLYCOSYLATION TYPE IA

Congenital disorder of glycosylation type Ia (CDG-Ia ) is an autosomal recessive disease caused by mutations in PMM2 gene encoding phosphomannomutase 2. In humans two PMM enzymes exist, PMM1 and PMM2; both of them catalyze the interconversion of mannose-6-phosphate into mannose-1-phosphate but only PMM1 can also hydrolyze bis-phosphate hexoses after stimulation with inosine monophosphate (IMP). Although no mutation in PMM1 gene has been associated with the disease, the role of the paralogous enzyme in CDG-Ia should be clarified. PMM1 and PMM2 were analyzed by conventional enzymatic assays as well as by novel techniques such as 31P-NMR and thermal shift assay. In order to evaluate the role of IMP in regulating mannose-1-phosphate production and ultimately protein glycosylation, it was performed the characterization of a triple mutant of PMM1 that retains mutase and phosphatase activity, but not sensitive to IMP. For the purpose of developing a therapeutic approach for CDG-Ia, for which there is no cure at the moment, it was generated a new cell model in order to identify biomarkers for cell-based screening, such as glycosylated proteins. A cancer cell line HepG2 was edited by Crispr/Cas9 system to introduce the hypomorphic PMM2 mutation, F119L, that has been observed in CDG-Ia patients in homozygosity. Hypo-glycosylated forms of α-1-anti-trypsin (AAT) was detected by Western blot in HepG2-F119L similarly to the serum from CDG-Ia patients. Furthermore, a difference in the amount of a secreted protein, alpha-fetoprotein (AFP), which is N-glycosylated, was found in HepG2-F119L secretome compared to the control cells. These results suggest that HepG2-F119L represent a good cell model for CDG-Ia and a useful tool for drug screening since they are defective in N-glycosylation of serum proteins that are found altered in patients. Further studies will be performed to propose AFP as a suitable biomarker

E-Learning for Rare Diseases: An Example Using Fabry Disease

Rare diseases represent a challenge for physicians because patients are rarely seen, and they can manifest with symptoms similar to those of common diseases. In this work, genetic confirmation of diagnosis is derived from DNA sequencing. We present a tutorial for the molecular analysis of a rare disease using Fabry disease as an example

Enzyme Replacement Therapy for FABRY Disease: Possible Strategies to Improve Its Efficacy

Enzyme replacement therapy is the only therapeutic option for Fabry patients with completely absent AGAL activity. However, the treatment has side effects, is costly, and requires conspicuous amounts of recombinant human protein (rh-AGAL). Thus, its optimization would benefit patients and welfare/health services (i.e., society at large). In this brief report, we describe preliminary results paving the way for two possible approaches: i. the combination of enzyme replacement therapy with pharmacological chaperones; and ii. the identification of AGAL interactors as possible therapeutic targets on which to act. We first showed that galactose, a low-affinity pharmacological chaperone, can prolong AGAL half-life in patient-derived cells treated with rh-AGAL. Then, we analyzed the interactomes of intracellular AGAL on patient-derived AGAL-defective fibroblasts treated with the two rh-AGALs approved for therapeutic purposes and compared the obtained interactomes to the one associated with endogenously produced AGAL (data available as PXD039168 on ProteomeXchange). Common interactors were aggregated and screened for sensitivity to known drugs. Such an interactor-drug list represents a starting point to deeply screen approved drugs and identify those that can affect (positively or negatively) enzyme replacement therapy

Discovery of an Italian slow worm (Anguis veronensis Pollini, 1818) population on a Western Mediterranean Island confirmed by genetic analysis

The genus Anguis is known to be mainly continental in the Mediterranean area, and accordingly it has never been recorded in Western Mediterranean islands. Here we report for the first time the presence of the slow worm in a Western Mediterranean island, the Ile Sainte-Marguerite from Lérins archipelago (southeastern France). The molecular analysis of ND2 and PRLR genes assigned the specimens to A. veronensis Pollini, 1818 and showed that they are genetically related to the mainland population from Les Mayons, in mainland France.

Large-Scale Spatio-Temporal Patterns of Mediterranean Cephalopod Diversity

Species diversity is widely recognized as an important trait of ecosystems’ functioning and resilience. Understanding the causes of diversity patterns and their interaction with the environmental conditions is essential in order to effectively assess and preserve existing diversity. While diversity patterns of most recurrent groups such as fish are commonly studied, other important taxa such as cephalopods have received less attention. In this work we present spatio-temporal trends of cephalopod diversity across the entire Mediterranean Sea during the last 19 years, analysing data from the annual bottom trawl survey MEDITS conducted by 5 different Mediterranean countries using standardized gears and sampling protocols. The influence of local and regional environmental variability in different Mediterranean regions is analysed applying generalized additive models, using species richness and the Shannon Wiener index as diversity descriptors. While the western basin showed a high diversity, our analyses do not support a steady eastward decrease of diversity as proposed in some previous studies. Instead, high Shannon diversity was also found in the Adriatic and Aegean Seas, and high species richness in the eastern Ionian Sea. Overall diversity did not show any consistent trend over the last two decades. Except in the Adriatic Sea, diversity showed a hump-shaped trend with depth in all regions, being highest between 200–400 m depth. Our results indicate that high Chlorophyll a concentrations and warmer temperatures seem to enhance species diversity, and the influence of these parameters is stronger for richness than for Shannon diversityVersión del editor4,411

A network approach to identify bioregions in the distribution of Mediterranean amphipods associated with Posidonia oceanica meadows

Although amphipods are key components of the macro-fauna associated with Posidonia oceanica meadows, to date no studies focused on the structure and diversity of their assemblages across the whole Mediterranean Sea. Here, we applied a network approach based on modularity on a dataset mined from literature to identify biogeographic modules and to assess the biogeographic roles of associated localities. We also correlated the patterns evidenced with the biogeographic distribution of amphipod groups by means of a multivariate analysis. Modularity analysis highlighted four biogeographic modules bounded by the main Mediterranean biogeographic divides and evidenced a decrease in species diversity along a NW-SE gradient. Assemblages associated with Central-Western Mediterranean and, to a lesser extent, Tunisian modules showed the highest species richness and were identified as hubs, characterized by species with regional distributions that behave as source in a biogeographic context. The paleogeographic history of the host seagrass and the ecology of associated amphipods, both suggest the joint effect of species persistence and post-Last Glacial Maximum expansion in explaining the pattern of amphipod distribution in the Mediterranean Sea.n

Reactive species in progeroid syndromes and ageing-related processes

International audienceReactive species have been classically considered causative of age-related degenerative processes, but the scenario appears considerably more complex and to some extent counterintuitive than originally anticipated. The impact of reactive species in precocious ageing syndromes is revealing new clues to understand and perhaps challenge the resulting degenerative processes. Recent Advances. Our understanding of reactive species has considerably evolved, including their hormetic effect (beneficial at a certain level, harmful beyond this level), the occurrence of diverse hormetic peaks in different cell types and organisms, and the extended type of reactive species that are relevant in biological processes. Our understanding of the impact of reactive species has also expanded from the dichotomic damaging/signaling role to modulation of gene expression

E-Learning for Rare Diseases: An Example Using Fabry Disease

Background: Rare diseases represent a challenge for physicians because patients are rarely seen, and they can manifest with symptoms similar to those of common diseases. In this work, genetic confirmation of diagnosis is derived from DNA sequencing. We present a tutorial for the molecular analysis of a rare disease using Fabry disease as an example. Methods: An exonic sequence derived from a hypothetical male patient was matched against human reference data using a genome browser. The missense mutation was identified by running BlastX, and information on the affected protein was retrieved from the database UniProt. The pathogenic nature of the mutation was assessed with PolyPhen-2. Disease-specific databases were used to assess whether the missense mutation led to a severe phenotype, and whether pharmacological therapy was an option. Results: An inexpensive bioinformatics approach is presented to get the reader acquainted with the diagnosis of Fabry disease. The reader is introduced to the field of pharmacological chaperones, a therapeutic approach that can be applied only to certain Fabry genotypes. Conclusion: The principle underlying the analysis of exome sequencing can be explained in simple terms using web applications and databases which facilitate diagnosis and therapeutic choices