Testing Species Delimitations in Four Italian Sympatric Leuciscine Fishes in the Tiber River: A Combined Morphological and Molecular Approach

Leuciscine fishes represent an important component of freshwater ichthyofauna endemic to northern Mediterranean areas. This lineage shows high intra-specific morphological variability and exhibits high levels of hybridization, two characteristics that contribute to systematic uncertainties, misclassification of taxa and, potentially, the mismanagement of biodiversity. This study focused on brook chub, Squalius lucumonis, an endemic taxon of Central Italy. The taxonomic status of this species has long been questioned, and a hybrid origin from sympatric leusciscines (S. squalus x Rutilus rubilio, or S. squalus x Telestes muticellus) has been hypothesised. A phenotypic (evaluating shape and meristic counts) and genetic (using mitochondrial and nuclear markers) investigation of these four taxa was conducted to test species delimitation in sympatric areas and to evaluate the taxonomic status of S. lucumonis. One hundred and forty-five individuals of all four taxa were collected within streams of the lowest portion of the Tiber River basin and analysed; this region encompasses a large portion of the S. lucumonis distribution. The different morphological and genetic approaches were individually examined, compared, and then combined in a quantitative model to both investigate the limits of each approach and to identify cases of misclassification. The results obtained confirm the cladogenetic non-hybrid origin of S. lucumonis, highlight the need for immediate conservation actions and emphasise the value of an integrated approach in the study of leuciscines evolution.     

Reduction in wire tension caused by dynamic loading. An experimental Ilizarov frame study

Small diameter transosseous wires are main parts of the Ilizarov frame concept. Wire tensioning is essential to gain the necessary transversal stiffness, and the wire fixators are therefore important, coupling the wire to the ring. The ability of three different wire fixator designs to maintain wire tension under dynamic loading is described. The three fixator designs were combinations of two types of bolts and one washer. In a simplified frame set-up consisting of one ring and one wire, sixty wires were dynamically loaded in sequence with 200N for 450 times and wire tension was repeatedly measured. The tension loss employing the different fixator types at two wire pretension levels (883N, 1275N) was measured. Wire slippage in the fixators and the midpoint deflection angle of the wires were measured. Large differences in fixation ability were observed. Wire fixators holding a larger and rougher contact surface had significant smaller tension loss compared to fixators with a smaller and smother contact surface. Both plastic wire deformation and slippage causes tension loss with slippage as the main contributor. Wire fixators' design can be improved in order to maintain wire tension. Such improvement could be of clinical interest enhancing the mechanical stability of the fixator.     

Lower limb entheseal morphology in the Neandertal Krapina population (Croatia, 130,000 BP)

Although the Neandertal locomotor system has been shown to differ from Homo sapiens, characteristics of Neandertal entheses, the skeletal attachments for muscles, tendons, ligaments and joint capsules, have never been specifically investigated. Here, we analyse lower limb entheses of the Krapina Neandertal bones (Croatia, 130,000 BP) with the aim of determining how they compare with modern humans, using a standard developed by our research group for describing modern human entheseal variability. The entheses examined are those of the gluteus maximus, iliopsoas and vastus medialis on the femur, the quadriceps tendon on the patella, and soleus on the tibia. For the entheses showing a different morphological pattern from H. sapiens, we discuss the possibility of recognising genetic versus environmental causes. Our results indicate that only the gluteus maximus enthesis (the gluteal tuberosity), falls out of the modern human range of variation. It displays morphological features that could imply histological differences from modern humans, in particular the presence of fibrocartilage. In both H. sapiens and the Krapina Neandertals, the morphological pattern of this enthesis is the same in adult and immature femurs. These results can be interpreted in light of genetic differences between the two hominins. The possibility of functional adaptations to higher levels of mechanical load during life in the Neandertals seems less likely. The particular morphology and large dimensions of the Krapina enthesis, and perhaps its fibrocartilaginous nature, could have been selected for in association with other pelvic and lower limb characteristics, even if genetic drift cannot be ruled out.     

Docosahexaenoic acid reverts resistance to UV-induced apoptosis in human keratinocytes: involvement of COX-2 and HuR

The dramatic increase in the incidence of nonmelanoma skin cancer over the last decades has been related to the augmented exposure to ultraviolet (UV) radiation (UVR). It is known that apoptosis is induced as a protective mechanism after the acute irradiation of keratinocytes, whereas apoptotic resistance and carcinogenesis may follow the chronic exposure to UVR. We found that not all the human keratinocytes lines studied underwent apoptosis following acute exposure to UVR (10-60 mJ/cm²). Whereas UVR induced apoptosis in the HaCaT cells, NCTC 2544 and nr-HaCaT cells showed apoptosis resistance. The cytokeratin pattern of the apoptosis-resistant cells indicated that they possessed a degree of differentiation lower than that of HaCaT cells. They also showed an enhanced expression of cyclooxygenase-2 (COX-2), an early marker of carcinogenesis in various tissues, including skin. n-3 polyunsaturated fatty acids have drawn increasing interest as nutritional factors with the potential to reduce UVR carcinogenesis, and since they are apoptosis inducers and COX-2 inhibitors in cancer cells, we investigated the ability of n-3 polyunsaturated fatty acids to influence the resistance to UVR-induced apoptosis in keratinocytes. We observed that docosahexaenoic acid (DHA) reverted the resistance of nr-HaCaT cells to UVR-induced apoptosis, increasing the Bax/Bcl-2 ratio and caspase-3 activity, and reduced COX-2 levels by inhibiting the expression of the human antigen R (HuR), a known COX-2 mRNA stabilizer in keratinocytes. The transfection of nr-HaCaT cells with HuR siRNA mimicked the proapoptotic effect of DHA. Overall, our findings further support the role of DHA as a suitable anticarcinogenic factor against nonmelanoma skin cancers.     

The extent of whole‐genome copy number alterations predicts aggressive features in primary melanomas

Recent evidence indicates that melanoma comprises distinct types of tumors and suggests that specific morphological features may help predict its clinical behavior. Using a SNP‐array approach, we quantified chromosomal copy number alterations (CNA) across the whole genome in 41 primary melanomas and found a high degree of heterogeneity in their genomic asset. Association analysis correlating the number and relative length of CNA with clinical, morphological, and dermoscopic attributes of melanoma revealed that features of aggressiveness were strongly linked to the overall amount of genomic damage. Furthermore, we observed that melanoma progression and survival were mainly affected by a low number of large chromosome losses and a high number of small gains. We identified the alterations most frequently associated with aggressive melanoma, and by integrating our data with publicly available gene expression profiles, we identified five genes which expression was found to be necessary for melanoma cells proliferation. In conclusion, this work provides new evidence that the phenotypic heterogeneity of melanoma reflects a parallel genetic diversity and lays the basis to define novel strategies for a more precise prognostic stratification of patients.