XHas2 activity is required during somitogenesis and precursor cell migration in Xenopus development

In vertebrates, hyaluronan biosynthesis is regulated by three transmembrane catalytic enzymes denoted Has1, Has2 and Has3. We have previously cloned the Xenopus orthologues of the corresponding genes and defined their spatiotemporal distribution during development. During mammalian embryogenesis, Has2 activity is known to be crucial, as its abrogation in mice leads to early embryonic lethality. Here, we show that, in Xenopus, morpholino-mediated loss-of-function of XHas2 alters somitogenesis by causing a disruption of the metameric somitic pattern and leads to a defective myogenesis. In the absence of XHas2, early myoblasts underwent apoptosis, failing to complete their muscle differentiation programme. XHas2 activity is also required for migration of hypaxial muscle cells and trunk neural crest cells (NCC). To approach the mechanism whereby loss of HA, following XHas2 knockdown, could influence somitogenesis and precursor cell migration, we cloned the orthologue of the primary HA signalling receptor CD44 and addressed its function through an analogous knockdown approach. Loss of XCD44 did not disturb somitogenesis, but strongly impaired hypaxial muscle precursor cell migration and the subsequent formation of the ventral body wall musculature. In contrast to XHas2, loss of function of XCD44 did not seem to be essential for trunk NCC migration, suggesting that the HA dependence of NCC movement was rather associated with an altered macromolecular composition of the ECM structuring the cells' migratory pathways. The presented results, extend our knowledge on Has2 function and, for the first time, demonstrate a developmental role for CD44 in vertebrates. On the whole, these data underlie and confirm the emerging importance of cell-ECM interactions and modulation during embryonic development.     

Q-Elastography in the Presurgical Diagnosis of Thyroid Nodules with Indeterminate Cytology

Quantitative ultrasound (US) elastography (Q-USE), able to evaluate tissue stiffness has been indicated as a new diagnostic tool to differentiate benign from malignant thyroid lesions. Aim of this prospective study, conducted at the Department of Surgical Sciences, of the “Sapienza” University of Rome, was to evaluate the diagnostic accuracy of Q-USE, compared with US parameters, in thyroid nodules with indeterminate cytology (Thy3).The case study included 140 nodules from 140 consecutive patients. Patient’s thyroid nodules were evaluated by Q-USE, measuring the strain ratio (SR) of stiffness between nodular and surrounding normal thyroid tissue, and conventional US parameters prior fine-needle aspiration cytology. Those with Thy3 diagnosis were included in the study. Forty of the nodules analyzed harbored a malignant lesion. Q-USE demonstrated that malignant nodules have a significant higher stiffness with respect to benign one and an optimun SR cut-off value of 2.05 was individuated following ROC analysis. Univariate analysis showed that hypoechogenicity, irregular margins and SR >2.05 associated with malignancy, with an accuracy of 67.2%, 81,0% and 89.8%, respectively. Data were unaffected by nodule size or thyroiditis. These findings were confirmed in multivariate analysis demonstrating a significant association of the SR and the irregular margins with thyroid nodule’s malignancy. In conclusion, we demonstrated the diagnostic utility of Q-USE in the differential diagnosis of thyroid nodules with indeterminate cytology that, if confirmed, could be of major clinical utility in patients’ presurgical selection.     

Synthesis physicochemical profile and PAMPA study of new NO-donor edaravone co-drugs

A new class of co-drugs were synthesised by joining antioxidant edaravone with a vasodilating substructure containing NO-donor nitrooxy functions, and characterised for their stability in different media, lipophilicity and permeability profile. The products display good stability in water/co-solvent at different pH. Conversely, they are rapidly metabolised into edaravone and NO-donor moieties when incubated in human serum or rat-liver homogenates. In the latter conditions time dependent production of nitrite/nitrate (NO(x)) occurs. The compounds display wide-ranging lipophilicity. PAMPA studies predict good gastrointestinal absorption for a number of these compounds. The title products are potentially useful for treating ROS-related conditions accompanied by decreased NO availability.     

A PCR-based diagnostic tool for distinguishing grape skin color mutants

Berry skin color mutants are phenotypically different from their original cultivars, but they show identical molecular profile if analysed by using microsatellite markers. This work gives an easy, inexpensive and quick diagnostic tool to discriminate these somatic variants. We distinguished some grape (Vitis vinifera L.) skin color mutants from white to red or pink and from black to grey, pink or white and we investigated their molecular bases by single-strand conformational polymorphism (SSCP), single base primer extension and coding sequence analysis of anthocyanin biosynthetic enzyme genes and by polymerase chain reaction (PCR) analysis of VvmybA1 regulatory gene. Analyses of structural genes did not reveal polymorphisms between wild type and mutant cultivars but only among different varieties, whereas the study of VvmybA1 regulatory gene has given important outcomes for color mutants characterisation. The discrimination between white wild type and its derived colored mutant and between black wild type and white mutant has been obtained through a simple test of amplification for presence/absence. The discrimination between black wild type and less colored mutant has occurred through a quantitative result on agarose gel confirmed by real-time PCR analysis: the amount of functional allele in less colored somatic variants genome was about one-fourth of the correspondent quantity in original black cultivars genome.     

Population genetic structure of the melon fly, <Emphasis Type="Italic">Bactrocera cucurbitae</Emphasis> (Diptera: Tephritidae), from China and Southeast Asia

The melon fly, Bactrocera cucurbitae Coquillett, is a species of fruit flies of significant agricultural interest. Of supposed Indian origin, the melon fly is now widely distributed throughout South East Asia up to China, while it has been recently eradicated from Japan. The population structure of seven geographic populations from coastal China, as well as samples from other regions of South East Asia and Japan, including lab colonies, have been studied using a 782 bp fragment of mitochondrial cytochrome oxidase I (COI) gene sequence. The observed genetic diversity was exceedingly low, considering the geographic scale of the sampling, and one single haplotype was found to be predominant from Sri Lanka to China. We confirm that Bactrocera cucurbitae exists in South East Asia as a single phyletic lineage, that Chinese populations are genetically uniform, and that no apparent genetic differentiation exists between these and three available Japanese melon fly sequences.     

Platelet fragmentation requires a specific structural conformation of human monoclonal antibody against beta3 integrin

We have described an autoantibody against beta3 (GPIIIa49-66), a region of platelet integrin alphaIIbbeta3 that is unique. It induces platelet fragmentation in the absence of complement via antibody activation of platelet NADPH oxidase and 12-lipoxygenase to release reactive oxygen species, which destroy platelets. To study the mechanism of anti-GPIIIa antibody-induced platelet fragmentation, we screened a human single chain Fv antibody library with the GPIIIa49-66 peptide. Nine monoclonal antibodies were identified that were capable of binding to GPIIIa49-66. Surprisingly, binding avidity for GPIIIa49-66 did not correlate with activity of induction of platelet fragmentation. We therefore investigated the requirements for platelet fragmentation. Mutations were introduced into the heavy chain complementary-determining region-3 of clones 11, 43, and 54 by site-directed mutagenesis. The capability of these clones to induce platelet fragmentation or bind to GPIIIa49-66 subsequently changed. Molecular modeling of these clones with their mutants revealed that the ability to induce platelet fragmentation is affected by the side chain orientation of positively charged amino acids in the heavy chain of residues 99-102. Thus, a structural change in the conformation of anti-GPIIIa49-66 antibody contributes to its binding to the beta3 integrin and subsequent antibody-induced platelet fragmentation and aggregate dissolution.