In vitro effect of myo-inositol on sperm motility in normal and oligoasthenospermia patients undergoing in vitro fertilization.

It is a known fact that abnormal seminal liquid specimens contain abnormal amounts of oxygen free radicals and reactive oxygen species (ROS), and that the use of antioxidant molecules both in vivo and in vitro leads to improvement of semen quality in terms of motility, reduction in DNA damage, with obvious consequences on the fertilization potential. Myo-inositol has been observed to have anti-oxidant properties and be present in much greater concentrations specifically in seminal liquid than in the blood. Moreover, there seems to be a direct relationship between myo-inositol and mitochondrial membrane potential (MMP) and sperm motility. Studies performed in vivo have demonstrated that a dietary supplementation with myo-inositol in men undergoing assisted reproduction techniques may improve sperm quality and motility in oligoasthenospermia (OAT) patients. In the following study we utilized myo-inositol in vitro to verify its effect on semen quality in both normal and OAT patients undergoing in vitro fertilization (IVF) with respect to standard sperm medium. In vitro incubation of seminal liquid carried out using myo-inositol (Andrositol-Lab, Lo.Li. Pharma-Roma, Italy) at a concentration of 15 µl/ml improved progressive motility in both normospermia and OAT subjects. In our opinion, myo-inositol may prove to be a useful strategy to improve sperm preparation for clinical use in IVF.

A novel miR-371a-5p-mediated pathway, leading to BAG3 upregulation in cardiomyocytes in response to epinephrine, is lost in Takotsubo cardiomyopathy.

Molecular mechanisms protecting cardiomyocytes from stress-induced death, including tension stress, are essential for cardiac physiology and defects in these protective mechanisms can result in pathological alterations. Bcl2-associated athanogene 3 (BAG3) is expressed in cardiomyocytes and is a component of the chaperone-assisted autophagy pathway, essential for homeostasis of mechanically altered cells. BAG3 ablation in mice results in a lethal cardiomyopathy soon after birth and mutations of this gene have been associated with different cardiomyopathies including stress-induced Takotsubo cardiomyopathy (TTC). The pathogenic mechanism leading to TTC has not been defined, but it has been suggested that the heart can be damaged by excessive epinephrine (epi) spillover in the absence of a protective mechanism. The aim of this study was to provide more evidence for a role of BAG3 in the pathogenesis of TTC. Therefore, we sequenced BAG3 gene in 70 TTC patients and in 81 healthy donors with the absence of evaluable cardiovascular disease. Mutations and polymorphisms detected in the BAG3 gene included a frequent nucleotide change g2252c in the BAG3 3'-untranslated region (3'-UTR) of Takotsubo patients (P<0.05), resulting in loss of binding of microRNA-371a-5p (miR-371a-5p) as evidenced by dual-luciferase reporter assays and argonaute RNA-induced silencing complex catalytic component 2/pull-down assays. Moreover, we describe a novel signaling pathway in cardiomyocytes that leads to BAG3 upregulation on exposure to epi through an ERK-dependent upregulation of miR-371a-5p. In conclusion, the presence of a g2252c polymorphism in the BAG3 3'-UTR determines loss of miR-371a-5p binding and results in an altered response to epi, potentially representing a new molecular mechanism that contributes to TTC pathogenesis.

Influence of scaffold properties on the inter-relationship between human bone marrow derived stromal cells and endothelial cells in pro-osteogenic conditions.

One of the significant challenges in bone tissue engineering is the integration of biomaterials designed to facilitate and stimulate mineralization with a simultaneously rapid rate of angiogenesis and vascularization of the tissue construct, a challenge complicated by our lack of knowledge of the interactions among key cell types and scaffold properties. This study compared functional activity of human bone marrow-derived stromal cells (hMSC) seeded onto a porous salt-leached poly(D,L-lactic acid) (PDLLA) scaffolds, with and without the incorporation of silk fibroin fibers and then further investigated their co-culture with human umbilical vein endothelial cells (HUVECs). Cell viability, proliferation, and alkaline phosphatase activity were measured for a range of time points in culture, with osteogenic and angiogenic marker immunolocalization and gene expression at selected stages. Our findings suggest that, despite similar porosity and pore size distribution exhibited by the PDLLA and PDLLA plus silk fibroin scaffolds, there were marked differences in cell distribution and function. In the absence of fibers, a highly osteogenic response was observed in hMSCs in the scaffolds co-cultured with endothelial cells, greater than that observed with hMSCs alone or in either of the scaffolds with fibers added. However, fiber presence clearly better supported endothelial cell cultures, as determined by greater levels of endothelial marker expression at both the gene and protein level after 3 weeks of culture. The design of composite scaffolds integrating beneficial components of differing structures and materials to facilitate appropriate biological responses appears a promising yet challenging avenue of research. STATEMENT OF SIGNIFICANCE: A significant challenge in bone tissue engineering is to promote a rapid vascularization of the tissue construct in parallel to the extracellular matrix mineralization. The design of composite scaffolds integrating beneficial components of differing structures and materials to facilitate appropriate biological responses appears a promising yet challenging avenue of research. Here we investigated cultures of hMSCs and HUVECs on a silk fibroin enhanced PDLLA scaffold, showing that the final output of this in vitro system is not the linear sum of the effects of the single variables. These results are of interest as they demonstrate how the addition of endothelial cells can affect hMSC phenotype and that the output can be further modulated by the introduction of silk fibroin fibers.

Functional role of scaffold geometries as a template for physiological ECM formation: evaluation of collagen 3D assembly.

Bone tissue regeneration involves different healing stages and the resulting final hard tissue is formed from natural templates such as fibrous collagen, soft and hard callus and capillary bed. This work aims to evaluate the efficiency of different scaffold geometries with a novel approach: exploring the relationships among scaffold morphologies, cell activity and collagen 3D organization, which serves as a natural template for subsequent mineralization. Among the possible systems to fabricate scaffolds, solvent casting with particulate leaching and microfabrication were used to produce random vs ordered structures from poly(D,L-lactic acid). In vitro biological testing was carried out by culturing a human osteosarcoma-derived osteoblast cell line (MG63) and measuring material cytotoxicity, cell proliferation and migration. Assemblage of collagen fibres was evaluated. A preliminary study of collagen distribution over the two different matrices was performed by confocal laser microscopy after direct red 80 staining. Both of the scaffolds were seen to be a good substrate for cell attachment, growth and proliferation. However, it seems that random, rather than regular, well-ordered porosity induces a more proper collagen fibre distribution and organization, similar to the natural one formed in the early stages of bone repair.

FLASH is essential during early embryogenesis and cooperates with p73 to regulate histone gene transcription.

Replication-dependent histone gene expression is a fundamental process occurring in S-phase under the control of the cyclin-E/CDK2 complex. This process is regulated by a number of proteins, including Flice-Associated Huge Protein (FLASH) (CASP8AP2), concentrated in specific nuclear organelles known as HLBs. FLASH regulates both histone gene transcription and mRNA maturation, and its downregulation in vitro results in the depletion of the histone pull and cell-cycle arrest in S-phase. Here we show that the transcription factor p73 binds to FLASH and is part of the complex that regulates histone gene transcription. Moreover, we created a novel gene trap to disrupt FLASH in mice, and we show that homozygous deletion of FLASH results in early embryonic lethality, owing to arrest of FLASH(-/-) embryos at the morula stage. These results indicate that FLASH is an essential, non-redundant regulator of histone transcription and cell cycle during embryogenesis.

Microfabrication of PDLLA scaffolds.

This study aimed to comprehend the potentialities of the microfabrication to produce tissue-engineering scaffolds. Structures presenting homogeneously distributed pores of size 100 and 200 µm were fabricated through layer-by-layer deposition of filaments of poly(D,L-lactic acid) (PDLLA) prepared from dichloromethane/dimethylformamide solutions. Rheological tests on the solution and molecular weight distributions of PDLLA, solvent cast films and microfabricated scaffolds were performed to determine which material conditions are optimal for the microfabricated system and to identify any possible material modification induced by the process. In vitro qualitative preliminary cell culture studies were conducted using MG63 osteoblast cell lines after assuring the non-cytotoxicity of the scaffold material by the lactate dehydrogenase in vitro toxicology assay; biological evaluations were initially performed using scaffolds with the smaller (100 µm) pore size. Scanning electron microscopy imaging was used to determine cell morphology distribution. A second cell culture test was performed, using the scaffold with the higher (200 µm) porosity. Confocal laser microscopy (CLM) was utilized to examine cell morphology and growth behaviour. Cellular metabolic activity and viability were also examined using Alamar Blue assay and further verifications were performed using CLM. Cell culture studies indicated homogeneous distribution, high viability and metabolic activity. Pore dimension affects cell distribution: pores < 100 µm acted as barrier structures for the MG63 osteoblast cell line; penetration inside the matrix was hindered and cells grew on the outer part. Increasing pore size resulted in a more homogeneous cell distribution and penetration of cells inside the structure was achieved.

[Nursing documentation in the Stroke Unit]. / Cartella infermieristica nelle stroke units.

The organization of Stroke Unit improved prognosis of stroke patient. The randomized studies emphasized the importance of nursing in such a structure. In order to improve nursing process in our Stroke Unit, we designed a nurse record which includes a neurological scale, to define dependence classes and guidelines based on Virgina Henderson's theoretical model. In this paper, we present this nurse record and we describe the various steps of its design.

Functional analysis of the evolutionarily conserved proline 53 residue in Proteus mirabilis glutathione transferase B1-1.

The role of the evolutionarily conserved residue Pro-53 in Proteus mirabilis glutathione transferase B1-1 has been examined by replacing it with a serine residue using site-directed mutagenesis. The effect of the replacement on the activity, thermal stability and antibiotic binding capacity of the enzyme was examined. The results presented support the view that Pro-53 participates in the maintenance of the proper conformation of the enzyme fold rather than playing a direct role in the catalytic reaction. Furthermore, this residue appears to be an important determinant of the antibiotic binding to the enzyme. Experiments with wild type and mutated enzymes provide evidence that glutathione transferases may play an important role in antibiotic resistance exhibited by bacteria.

Indirect radiographic and angiographic signs of isolated left superior vena cava in congenital heart disease.

Blood that drains into the right atrium through an isolated left superior vena cava (LSVC) can be a complicating condition during cardiac surgery. In cases of LSVC, a double shadow can be present on standard chest X-ray over the left mediastinum. A notch along the lower contour of left atrium has been previously reported by Owen et al. in 4 cases as an indirect sign of this LSVC anomaly. These two indirect signs were evaluated in 135 (4.5%) cases of LSVC, viewing 89 chest radiographs and 40 angiograms. Their incidence, separately (17-45%) or in combination (9-20%), was observed to be frequent enough to justify a systematic search of these signs in open-heart surgery candidates.

Interruption of the aortic arch in an infant with left subclavian steal syndrome. Case report.

An unusual case of interruption of the aortic arch with flow to the descending aorta through the left vertebral and left subclavian arteries is described. The ductus arteriosus was functionally closed. The aortic arch was reconstructed with a PTFE graft, but the patient died because of lung problems, related to a high left-to-right shunt through a ventricular septal defect. A new surgical approach is postulated in view of the unusual hemodynamic features. Surgical control of pulmonary congestion could be the initial step, followed by aortic arch reconstruction later when insertion of a non-restrictive aortic prosthesis is possible.