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Purpose To evaluate the correlation between the DNA Fragmentation Index (DFI) and sperm morphology in patients undergoing ICSI, as a predictive parameter in reproductive outcomes. Methods A retrospective study was conducted on 125 infertile patients enrolled in a fertility clinic. Seminal characteristics were measured following the WHO guidelines (2010) for the examination of the seminal fluid. After collecting motile sperm population by pellet swim up, DFI was calculated and simultaneously associated with sperm morphology using in situ TUNEL assay and an image analyzer software in at least 250 spermatozoa for each patient. Results All subjects were divided into two groups according to a cutoff established, by choice, of the sperm DFI (15%): group A (< 15%) consisting of 65 patients and group B (≥ 15%) of 60 patients. Data were analyzed using non-parametric statistical methods. The results demonstrate that there is no statistical difference between the two groups in seminal characteristics. The collective data show a high significant correlation, suggesting that spermatozoa with abnormal morphology are the best candidates to contain DNA damage (p < 0.001). Also, when group A is compared with group B, an increased percentage of morphologically normal spermatozoa with fragmented DNA was observed in patients, with DFI values ≥ 15% (p < 0.001). Conclusion These results are aimed at providing an exact value of DFI in morphologically normal spermatozoa, which will be helpful to the embryologist in evaluating the risk of transferring, during the ICSI procedure, a spermatozoon whit normal morphology but fragmented DNA.

To dissect the TBX5 regulatory circuit, we focused on microRNAs (miRNAs) that collectively contribute to make TBX5 a pivotal cardiac regulator. We profiled miRNAs in hearts isolated from wild-type, CRE, Tbx5lox/+and Tbx5del/+ mice using a Next Generation Sequencing (NGS) approach. TBX5 deficiency in cardiomyocytes increased the expression of the miR-183 cluster family that is controlled by Kruppel-like factor 4, a transcription factor repressed by TBX5. MiR-182-5p, the most highly expressed miRNA of this family, was functionally analyzed in zebrafish. Transient overexpression of miR-182-5p affected heart morphology, calcium handling and the onset of arrhythmias as detected by ECG tracings. Accordingly, several calcium channel proteins identified as putative miR-182-5p targets were downregulated in miR-182-5p overexpressing hearts. In stable zebrafish transgenic lines, we demonstrated that selective miRNA-182-5p upregulation contributes to arrhythmias. Moreover, cardiac-specific down-regulation of miR-182-5p rescued cardiac defects in a zebrafish model of Holt–Oram syndrome. In conclusion, miR-182-5p exerts an evolutionarily conserved role as a TBX5 effector in the onset of cardiac propensity for arrhythmia, and constitutes a relevant target for mediating the relationship between TBX5, arrhythmia and heart development. To dissect the TBX5 regulatory circuit, we focused on microRNAs (miRNAs) that collectively contribute to make TBX5 a pivotal cardiac regulator. We profiled miRNAs in hearts isolated from wild-type, CRE, Tbx5lox/+and Tbx5del/+ mice using a Next Generation Sequencing (NGS) approach. TBX5 deficiency in cardiomyocytes increased the expression of the miR-183 cluster family that is controlled by Kruppel-like factor 4, a transcription factor repressed by TBX5. MiR-182-5p, the most highly expressed miRNA of this family, was functionally analyzed in zebrafish. Transient overexpression of miR-182-5p affected heart morphology, calcium handling and the onset of arrhythmias as detected by ECG tracings. Accordingly, several calcium channel proteins identified as putative miR-182-5p targets were downregulated in miR-182-5p overexpressing hearts. In stable zebrafish transgenic lines, we demonstrated that selective miRNA-182-5p upregulation contributes to arrhythmias. Moreover, cardiac-specific down-regulation of miR-182-5p rescued cardiac defects in a zebrafish model of Holt–Oram syndrome. In conclusion, miR-182-5p exerts an evolutionarily conserved role as a TBX5 effector in the onset of cardiac propensity for arrhythmia, and constitutes a relevant target for mediating the relationship between TBX5, arrhythmia and heart development.

In this study, the carotenoid astaxanthin was obtained by supercritical fluid extraction (SFE) from shrimp by-products (SBP). Its bioactive properties were evaluated in vitro in human normal and cancerous cells lines. The antioxidant activity of the extracted astaxanthin of the SFE fraction (ASTA) was tested in fibroblast cells (HS-68), by inducing oxidative stress and by evaluating the protective effect of the pre-treatment with different levels of ASTA against toxicity. The anti-proliferative activity was evaluated in hepatoma cells (HEP-G2), treated with increased concentrations of ASTA and measuring the effects on vitality and on some biomolecular markers related to oxidative stress, cell cycle, and apoptosis. It was found that pre-treating normal fibroblast cells with ASTA resulted in a marked increase in cell viability in a dose-dependent manner (P < 0.05) attesting its antioxidant power; in cancer cell line, increased concentrations of ASTA caused a time-dose-dependent decrease in the vitality, attesting its anti-proliferative activity (P < 0.05). The increased levels of the protein p-53 and the reduced levels of the proteins c-Jun and c-Fos at higher concentrations of ASTA, as well as, suggest the pro-apoptotic and anti-cancerous effects that this extract has on hepatocellular carcinomas, confirmed also by caspase-3 activation. These findings suggest biotechnological utilisation of marine by-products for nutraceutical and pharmaceutical applications avoiding the employment of organic solvents for extraction.

Distribution of aquaporin-4 (AQP4) was studied by western analysis and immunofluorescence in rat astrocytes exposed to either hypothermic (30 °C) or hyperosmolar (0.45 M sucrose) stress, and in the cerebrospinal fluid (CSF) of patients who suffered traumatic brain injury (TBI). CSF was obtained from 5 healthy subjects and from 20 patients suffering from severe TBI. CSF samples were taken at admission and on days 3 and 5-7. Here we report that, in response to both hypothermia and hyperosmolar stress, AQP4 was markedly reduced in cultured astrocytes. We also found that AQP4 significantly increased in patients with severe brain injury in respect to healthy subjects (P < 0.002). AQP4 in CSF remained unchanged in patients with elevated intracranial pressure (ICP), whereas there was a clear tendency to further increase in those patients whose ICP could be controlled within the normal range. We conclude that AQP4 levels in CSF are elevated after TBI and it might serve as a useful biochemical marker to assess brain water metabolism in clinical settings.

It has been proposed that the apoptosis is an essential requirement for the evolution of all animals, in fact the apoptotic program is highly conserved from nematodes to mammals. Throughout development, apoptosis is employed by multicellular organisms to eliminate damaged or unnecessary cells. Here, we will discuss both developmental programmed cell death (PCD) under normal conditions and stress induced apoptosis, in sea urchin embryos. Sea urchin represent an excellent model system for studying embryogenesis and cellular processes involved in metamorphosis. PCD plays an essential role in sculpting and remodelling the embryos and larvae undergoing metamorphosis. Moreover, this marine organism directly interacts with its environment, and is susceptible to effects of several aquatic contaminants. Apoptosis can be adopted as a defence mechanism against any environmental chemical, physical and mechanical stress, for removing irreversibly damaged cells. This review, while not comprehensive in its reporting, aims to provide an overview of current knowledge on mechanisms to regulate physiological and the induced apoptotic program in sea urchin embryos.

Emerging evidence seems to indicate that the heavy metal cadmium (Cd) is able to regulate gene expression, drastically affecting the pattern of transcriptional activity in normal and pathological eukaryotic cells, also affecting intracellular signalization events. Human p38 is a family of mitogen-activated protein kinases consisting of four isoforms (alpha, beta, gamma and delta) which mediate signal transduction cascades controlling several aspects of cell physiology. In this study we examined whether exposure of MDA-MB231 tumor cells from the human breast to Cd may exert some effect on p38 isoform expression and accumulation, as well as on p38 activation. Employing a combination of proliferation tests, conventional and semiquantitative multiplex (SM)-polymerase chain reaction (PCR) and Western blot assays, we report that the treatment of breast cancer cells with 5 microM CdCl(2) induces a diversified modulation of the transcription patterns of p38 isoform genes and of the accumulation of the related protein products, which are, on the other hand, also affected by alpha and beta isoform functional inactivation induced by SB203580. Our findings suggest the existence of so far unexplored mechanisms of gene regulation in our model system and validate that MDA-MB231 cell line is a suitable in vitro model for further and more detailed studies on the intracellular mechanisms underlying the control of p38 expression, synthesis and activation in mammary tumor cells exposed to different stresses.

Mitochondria, responsible for the energy-generating process essential for the cell metabolism, differ for the number, localization and activity in animal cells and tissues in relation to the energetic needs. Using fluorescent probes specific for mitochondria, Mitotracker Green (MTG) and Orange (MTO), and Confocal Laser-Scanning Microscope (CLSM), we elaborated a method to measure in vivo the mitochondrial mass and activity, in sea urchin Paracentrotus lividus eggs and embryos. The analysis of captured images, revealed a variation of mitochondrial distribution and an increase of activity after fertilization.