Investigation on the Origin of Sperm DNA Fragmentation: Role of Apoptosis,Immaturity and Oxidative Stress

Sperm DNA fragmentation (sDF) represents a threat to male fertility, human reproduction and the health of the offspring. The causes of sDF are still unclear, even if apoptosis, oxidative assault and defects in chromatin maturation are hypothesized. Using multicolor flow cytometry and sperm sorting, we challenged the three hypothesized mechanisms by simultaneously evaluating sDF and signs of oxidative damage (8-hydroxy, 2′-deoxyguanosine [8-OHdG] and malondialdehyde [MDA]), apoptosis (caspase activity and cleaved poly[ADP-ribose] polymerase [cPARP]) and sperm immaturity (creatine phosphokinase [CK] and excess of residual histones). Active caspases and c-PARP were concomitant with sDF in a high percentage of spermatozoa (82.6% ± 9.1% and 53.5% ± 16.4%, respectively). Excess of residual histones was significantly higher in DNA-fragmented sperm versus sperm without DNA fragmentation (74.8% ± 17.5% and 37.3% ± 16.6%, respectively, p < 0.005), and largely concomitant with active caspases. Conversely, oxidative damage was scarcely concomitant with sDF in the total sperm population, at variance with live sperm, where 8-OHdG and MDA were clearly associated to sDF. In addition, most live cells with active caspase also showed 8-OHdG, suggesting activation of apoptotic pathways in oxidative-injured live cells. This is the first investigation on the origin of sDF directly evaluating the simultaneous presence of the signs of the hypothesized mechanisms with DNA breaks at the single cell level. The results indicate that the main pathway leading to sperm DNA breaks is a process of apoptosis, likely triggered by an impairment of chromatin maturation in the testis and by oxidative stress during the transit in the male genital tract. These findings are highly relevant for clinical studies on the effects of drugs on sDF and oxidative stress in infertile men and for the development of new therapeutic strategies.     

Characterization of acute phase proteins and oxidative stress response to road transportation in the dog

Haptoglobin (Hp), serum amyloid A (SAA), C-reactive protein (CRP), white blood cells (WBC), reactive oxygen metabolites (ROMs), the antioxidant barrier (Oxy-adsorbent) and thiol groups of plasma compounds (SHp) were measured in ten dogs that had been transported a distance of about 230 km within 2 h (experimental group) and in ten dogs that had not been subjected to road transportation (control group). Blood was collected via cephalic venipuncture before road transportation (T0), after road transportation (T1), and more than 6 (T6) and 24 (T24) hours after road transportation in the experimental group (Group A) and at the same time points in the control group (Group B). The GLM (general linear model) Repeated Measures procedure showed a significant difference between the two groups (P<0.0001) and a significant rise (P<0.0001) in the concentrations of Hp, SAA, CRP, WBC, ROMs, Oxy-adsorbent and SHp after road transportation in Group A, underlining that physiological and homeostatic mechanisms are modified differently at various sampling times.     

Long-chain polyunsaturated fatty acids profile in plasma phospholipids of hyperphenylalaninemic children on unrestricted diet

IntroductionThe aim of the present study was to examine whether hyperphenylalaninemic children on unrestricted diet (MHP) may exhibit a different LCPUFA profile from PKU or healthy children in plasma phospholipids.Patients and methodsForty-five MHP children (age 9–14 years) were age and sex matched with 45 PKU and 45 healthy children. Fatty acids were determined and expressed as % of total fatty acids.ResultsMHP children showed docosahexaenoic acid (DHA) levels higher than PKU children (mean difference, 0.2%; 95% confidence interval, 0.02%–0.38%), although difference was not significant after correction for multiple comparisons, and lower levels than healthy children (?0.8%; ?1.01% to ?0.59%). Concentration of n-3 PUFA was higher in MHP than PKU children (0.6%; 0.4% to 0.8%),ConclusionsThe results suggest that low DHA levels in plasma phospholipids not only are evident in PKU but also may occur in MHP children, who are on unrestricted diet, as compared to healthy children.     

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.     

Expression of a highly differentiated phenotype and hepatic functionality markers in gilthead seabream (Sparus aurata L.) long-cultured hepatocytes: first morphological and functional in vitro characterization

The development of primary cultures and cell lines from aquatic organisms is a valuable tool for a wide range of research activities applied to aquaculture. Despite several efforts, derivation and long-term culturing of primary hepatocytes from marine vertebrates are still rare and unsuccessful. This is the first report to fully characterize long-term cultures of primary hepatocytes from the European seabream, Sparus aurata L. (Osteichthyes, Sparidae) (SaHePs). In this new model, hepatocyte cells were long-term viable, active proliferating, and fully retained liver function up to 3 weeks. SaHePs expressed a differentiated phenotype, owing to the reacquisition of the peculiar cytoarchitecture with the complete assembly of cytoskeletal and junctional network, as shown by the production and immunolocalization of several polarity markers and cytoskeletal proteins (MDR1, ZO-2, C-CAM1, Vimentin, Cadherin, ??-Tubulin, ??-Catenin, ??-Actin). Cytostructural analysis to identify polarized expression and bile canaliculi formation was performed by immunofluorescence and contrast phase microscopy. Long cultured SaHePs also demonstrated evidence of Albumin, ??1-Antitrypsin (AAT) and ??-Fetoprotein (AFP) synthesis, expression of the detoxifying metabolic enzyme cytochrome P-4501A (CYP 1A), and production of hepatocyte specific cytoskeleton proteins, such as Cytokeratin 8 (CK8) and Cytokeratin 18 (CK 18). The presence of specific markers for hepatic phenotype, detected by immunocytochemistry and Western blot analysis, is suggestive of the full maintenance of a highly differentiated phenotype and hepatic maturation. These data demonstrate that SaHePs can be long cultured without losing the hepatic functionality. This study provides a useful tool for innovative research applications in fish toxicological, pathological, and physiological studies, as one of the few hepatic, functionally active, in vitro model from marine fish.     

Studying the effects of laccase-catalysed grafting of ferulic acid on sisal pulp fibers

Functionalization of sisal specialty pulp fibers by laccase-catalysed grafting of ferulic acid (FRC) was investigated. To this end, the extent of phenol coupling to fibers under different reaction conditions (laccase and FRC rates, and time) was evaluated in terms of pulp properties including kappa number (expressed as the combined contributions of lignin and hexenuronic acids), brightness, Klason lignin and surface anionic charge after Soxhlet extraction of acetone-treated pulp. The specific treatment resulting in the highest degree of grafting was then used in a comparative study of the effects of applying the laccase-FRC system to refined and unrefined pulp with a view to confirming whether the increased surface area obtained by effect of fibrillation would lead to enhanced grafting. Based on the results, refining the pulp prior to the enzyme treatment resulted in increased grafting which in turn led to handsheets with improved strength-related properties (particularly wet tensile strength) relative to control samples.     

High throughput MLVA-16 typing for <Emphasis Type="Italic">Brucella</Emphasis> based on the microfluidics technology

Background  

Brucellosis, a zoonosis caused by the genus Brucella, has been eradicated in Northern Europe, Australia, the USA and Canada, but remains endemic in most areas of the world. The strain and biovar typing of Brucella field samples isolated in outbreaks is useful for tracing back source of infection and may be crucial for discriminating naturally occurring outbreaks versus bioterrorist events, being Brucella a potential biological warfare agent. In the last years MLVA-16 has been described for Brucella spp. genotyping. The MLVA band profiles may be resolved by different techniques i.e. the manual agarose gels, the capillary electrophoresis sequencing systems or the microfluidic Lab-on-Chip electrophoresis. In this paper we described a high throughput system of MLVA-16 typing for Brucella spp. by using of the microfluidics technology.     

Adhesion molecule signalling: not always a sticky business

The signalling activity of cell adhesion molecules (CAMs) such as cadherins, immunoglobulin-like CAMs or integrins has long been considered to be a direct consequence of their adhesive properties. However, there are physiological and pathological processes that reduce or even abrogate the adhesive properties of CAMs, such as cleavage, conformational changes, mutations and shedding. In some cases these 'adhesion deficient' CAMs still retain signalling properties through their cytoplasmic domains and/or their mutated or truncated extracellular domains. The ability of CAMs to activate signal transduction cascades in the absence of cell adhesion significantly extends their range of biological activities.