Initial ontogeny of digestive enzymes in the early life stages of captive-bred European eels during fasting: A partial characterization.

The European eel has recently been included on the Red List of the International Union for Conservation of Nature (IUCN) as a critically endangered species. The rearing of Anguilla larvae is seen as a key bottleneck to the mass production of glass eels since very little ecological information is available regarding their natural nutrition. Studies of digestive physiology and ontogenetic development in eel larvae could provide useful information for solving some of the puzzles regarding larval fish culture. The aim of this study was to characterize the ontogeny of pancreatic enzymes (trypsin, lipase and amylase) and a peptide hormone regulator of pancreatic secretion (cholecystokinin) in terms of gene expression in European eel larvae from day 0 (P0) of hatching to 5, 10, 15 and 20 days post hatching during fasting. The results in the present study showed that all the genes selected were present, with different levels of expression and increasing trends, during larval development. At P0, the increase in the gene expression of lipase and amylase was higher than that of trypsin and cholecystokinin, confirming that enzymatic activity began before mouth opening and that larvae, provided with a complete enzymatic set, might have the capacity of digesting and absorbing various nutrients.

Relative abundance of heat shock proteins and clusterin transcripts in spermatozoa collected from boar routinely utilised in an artificial insemination centre: preliminary results.

It is widely accepted that mature sperm contains RNA. The first hypothesis was that sperm RNAs have no functions of their own but are simply residues of spermatogenesis reflecting the events that occurred during their formation in the testes. More recently new discoveries have essentially expanded these views, showing that sperm mRNAs constitute a population of stable full-length transcripts, many of which are selectively retained during spermatogenesis and delivered to oocytes contributing to early embryo development. It is well known that semen quality can be influenced by occasional physical stress, infection, and variation in temperature and the definition of new markers for evaluation of semen could offer knowledge about the fertility potential of a semen sample. The aim of the present study was to evaluate the presence and the relative quantity of transcripts and protein of heat shock protein 70 (HSP70), 90 (HSP90) and clusterin (CLU) in Percoll-selected spermatozoa collected from seven adult boars of proven fertility routinely employed for artificial insemination. Our results showed the presence of HSP70, HSP90 and CLU transcripts with different level of expression: high for HSPs and low for CLU transcripts. The transcript level of both HSPs are similar among selected spermatozoa derived from high quality sperm with the exception of one boar that showed a reduced content of HSP70 and HSP90 mRNA together with a lower semen quality. At protein level, both HSPs were detected with similar amount among all seven boars whilst no band was evidenced for CLU protein.

Embelin supplementation of in vitro maturation medium does not influence nuclear and cytoplasmic maturation of pig oocytes.

Oxidative stress caused from in vitro culture contributes to inadequate oocyte maturation which leads to a poor embryo development. Therefore, it is important to protect oocytes and embryos against oxidative stress. This study was aimed at evaluating the effect of Embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone), an antioxidant with various pharmacologic activities, on nuclear and cytoplasmic maturation of pig oocytes as well as on steroidogenesis of cumulus cells (CCs). Another objective was to determine the influence of Embelin on developmental competence of pig oocytes as well as the expression levels of three key genes (Nanog, Sox2 and Oct4) involved in the control of pluripotency in parthenogenetically activated embryos. Embelin (0, 10, 20 and 40 µM) was added during in vitro maturation of cumulus oocyte complexes; media of both the first and the second day of culture were collected and assayed for progesterone and estradiol-17ß. At the end of the maturation period, the oocytes were fixed (to determine nuclear maturation) or partenogenically activated to evaluate cytoplasmic maturation and genes expression. Embelin did not exert any effect on the proportion of MII oocytes, steroidogenesis of CCs, percentage of embryos that developed to blastocyst stage and the number of blastomeres/blastocyst. Moreover, no significant differences of Oct4, Nanog and Sox2 transcripts were detected in blastocyst stage embryos. In conclusion, Embelin did not influence the reproductive parameters assessed, confirming that it is not possible to predict whether the beneficial effect exerted by an antioxidant in a particular tissue could be present also in another one.

Can Microfiltered Seminal Plasma Preserve the Morphofunctional Characteristics of Porcine Spermatozoa in the Absence of Antibiotics? A Preliminary Study.

Artificial insemination is extensively performed in pig farms in Europe, the United States and Canada. Antibiotics are typically added to the inseminating dose to limit bacterial growth during liquid phase storage at 16°C, as bacterial contamination is unavoidable. The World Organization for Animal Health (OIE) and the Food and Agriculture Organization (FAO) take action to control and reduce antibiotic use in animals as more bacteria are becoming resistant to antimicrobials. To avoid the use of antibiotics, we prepared inseminating doses using microfiltered seminal plasma (SP). Microfiltration is a common technology used to reduce bacterial contamination but may retain seminal substances, influencing sperm quality during storage. Therefore, the aim of this study was to evaluate the morphofunctional parameters of spermatozoa during storage at 16°C in doses prepared with or without microfiltered SP, with or without the addition of antibiotics, in a Latin square design. Artificial insemination doses with microfiltered SP and without antibiotic addition preserved spermatozoa viability, mitochondrial membrane potential, acrosome integrity and objective motility, with absolute values equal or even better than those observed in conventional doses. In conclusion, although the results could be considered preliminary due to the small sample size, this study suggests that microfiltration of SP can be a simple method, feasible on farms, to replace antibiotic use in extended doses stored in the liquid phase at 16°C for up to 7 days.

Life cycle assessment and economic analysis of a low concentrating photovoltaic system.

Many new photovoltaic (PV) applications, such as the concentrating PV (CPV) systems, are appearing on the market. The main characteristic of CPV systems is to concentrate sunlight on a receiver by means of optical devices and to decrease the solar cells area required. A low CPV (LCPV) system allows optimizing the PV effect with high increase of generated electric power as well as decrease of active surface area. In this paper, an economic analysis and a life cycle assessment (LCA) study of a particular LCPV scheme is presented and its environmental impacts are compared with those of a PV traditional system. The LCA study was performed with the software tool SimaPro 8.0.2, using the Econinvent 3.1 database. A functional unit of 1 kWh of electricity produced was chosen. Carbon Footprint, Ecological Footprint and ReCiPe 2008 were the methods used to assess the environmental impacts of the LCPV plant compared with a corresponding traditional system. All the methods demonstrated the environmental convenience of the LCPV system. The innovative system allowed saving 16.9% of CO2 equivalent in comparison with the traditional PV plant. The environmental impacts saving was 17% in terms of Ecological Footprint, and, finally, 15.8% with the ReCiPe method.

Histological Classification and Immunohistochemical Evaluation of MDM2 and CDK4 Expression in Canine Liposarcoma.

Canine liposarcoma is an uncommon soft tissue sarcoma usually arising in the subcutis. While liposarcoma classification in dogs is based solely on histology, in humans it depends on the detection of genetic abnormalities that can lead to specific protein overexpression. This study is an immunohistochemical evaluation of MDM2 and CDK4 expression in canine liposarcoma designed to assess the correlation of these proteins with histologic type, grade, mitotic index and Ki67 labeling index and evaluate their utility in improving tumor classification. Fifty-three liposarcomas were retrospectively collected: 24 were well differentiated liposarcomas (WDL), 16 of which expressed MDM2 and 21 CDK4; 7 were myxoid liposarcomas (ML), 1 of which expressed MDM2 and 5 expressed CDK4; 18 were pleomorphic liposarcomas (PL), all were MDM2 negative and 12 expressed CDK4. Four tumors were morphologically consistent with dedifferentiated liposarcoma (DDL) a subtype described only in humans: 3 expressed MDM2 and 4 expressed CDK4. MDM2 expression correlated with histotype (highly expressed in WDL and DDL) and grade (highly expressed in grade 1 tumors). Histotype correlated with the Ki67 labeling index (lowest in WDL and highest in DDL). A revised classification, considering MDM2 expression, allowed 8 WDL to be reclassified as PL and correlated significantly with mitotic and Ki67 labeling index (both significantly lower in WDL and progressively higher in ML and DDL). These results partially parallel data reported for human liposarcomas, suggesting that WDL and DDL are distinct neoplastic entities characterized by MDM2 expression, which may represent a useful diagnostic and potentially prognostic marker for canine liposarcoma.

Effect of tributyltin on mammalian endothelial cell integrity.

Tributyltin (TBT), is a man-made pollutants, known to accumulate along the food chain, acting as an endocrine disruptor in marine organisms, with toxic and adverse effects in many tissues including vascular system. Based on the absence of specific studies of TBT effects on endothelial cells, we aimed to evaluate the toxicity of TBT on primary culture of porcine aortic endothelial cells (pAECs), pig being an excellent model to study human cardiovascular disease. pAECs were exposed for 24h to TBT (100, 250, 500, 750 and 1000nM) showing a dose dependent decrease in cell viability through both apoptosis and necrosis. Moreover the ability of TBT (100 and 500nM) to influence endothelial gene expression was investigated at 1, 7 and 15h of treatment. Gene expression of tight junction molecules, occludin (OCLN) and tight junction protein-1 (ZO-1) was reduced while monocyte adhesion and adhesion molecules ICAM-1 and VCAM-1 (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) levels increased significantly at 1h. IL-6 and estrogen receptors 1 and 2 (ESR-1 and ESR-2) mRNAs, after a transient decrease, reached the maximum levels after 15h of exposure. Finally, we demonstrated that TBT altered endothelial functionality greatly increasing monocyte adhesion. These findings indicate that TBT deeply alters endothelial profile, disrupting their structure and interfering with their ability to interact with molecules and other cells.

Effect of photoperiod on endocrine profiles and vitellogenin expression in European eels Anguilla anguilla during artificially induced ovarian development.

The aim of this work was to determine the effects of dark and light conditions on the E2, testosterone and thyroid hormones levels and on the gene expression levels (vitellogenin 1, vitellogenin 2, and estradiol receptor one) in European eels (Anguilla anguilla) during ovarian development induced by increasing doses of carp pituitary extracts (CPEs). The subjects were divided into 2 groups: 14-hour light:10-hour dark (Light Group) and 24-hour darkness (Dark Group). All the eels received intramuscular injections with CPE at a dosage of 10 mg/kg body weight (BW) once a week for the first 3 weeks, 20 mg/kg BW fourth-sixth week, 30 mg/kg BW seventh-ninth week, and 40 mg/kg up to the end of the experiment (13th week). Vitellogenin and estradiol receptor expression levels did not show significant differences between the two housing conditions whereas in both groups vitellogenin mRNA increased starting from first CPE injection. Testosterone and 17-beta estradiol plasma levels were significantly greater in the Dark Group compared with the Light Group starting from the ninth and the 13th week, respectively. These results suggest that darkness could be a useful variable for standardizing gonadal maturation in eels kept in captivity.

A study on some welfare-related parameters of hDAF transgenic pigs when compared with their conventional close relatives.

Pigs are increasingly used in medical research as transgenic laboratory animals; however, little knowledge is presently available concerning their welfare assessment. The aim of the present study was to investigate some welfare-related parameters of transgenic pigs intended for xenotrasplantation (human decay-accelerating factor (hDAF)) when compared with their conventional (i.e. not transgenic) close relatives (full sibs and half sibs). A total of 14 Large White female transgenic pigs and 10 female non-transgenic (conventional) pigs from four litters were used. All pigs were from the same conventional boar, donor of the semen treated for sperm-mediated gene transfer. During the experiment, BW ranged from 50 to about 80 kg and pigs were weighed at the beginning and at the end of the experiment. Animals were subjected to a set of behavioural tests: a human approach test (HAT), a novel object test (NOT) and an open-door test (ODT). Food preferences were tested through the offer of different foods (banana, apple, carrot, cracker and lemon). During a 4-day period, pigs were diurnally videotaped to study the prevalence of the different behaviours and social interactions (aggressive and non-aggressive interactions). At the end of the trial, cortisol level had been assessed on bristles. No significant differences (P>0.05) were observed between hDAF transgenic and conventional pigs with respect to growth traits, reactivity towards unexpected situations (HAT, NOT, ODT), food preferences, main behavioural traits, social interactions and hair cortisol.

Orexin system expression in the gastrointestinal tract of pigs.

The aim of the present study was to characterize the expression of both proteins and gene transcripts for orexins (OXA and OXB) and their cognate receptors (OX1R and OX2R) in the different gastrointestinal sections of pigs. Using immunohistochemistry, OXA and OXB were found to be co-expressed in the same endocrine cells localized in the basal third of the glands of the body portion of the stomach. Using double immunostaining technique, these orexin-immunoreactive (IR) cells co-stored ghrelin and gastrin. Apparently, OX1R was also expressed within the same cells, forming the tubular gastric gland which displayed positive immunostaining for orexins and the other peptides. Neurons of the enteric nervous system of the stomach were not immunolabeled. We did not find any definite OXA- or OXB-IR cells as well as any immunosignal for orexin receptors in sections of the duodenum, ileum, cecum and rectum. PPOX, OX1R, OX2R mRNA were similarly expressed in all the gastrointestinal tracts. Gastrin and ghrelin showed the highest levels of expression in the gastric mucosa, but their abundance decreased along the subsequent tracts. Thus, in pigs, orexins do not play any role in the local control of intestinal motility and secretion but may rather be involved as endocrine modulators for the regulation of feeding and metabolic homeostasis. However, the co-localization of ghrelin and gastrin with both orexins in the same endocrine cells of the gastric glands suggests that these gut peptides may collaborate in the regulation of gastric secretion, energy homeostasis, body weight and food intake.

Differential expression of CD90 and CD14 stem cell markers in malignant breast cancer cell lines.

The recently emerged concept of cancer stem cell (CSC) has led to a new hypothesis on the basis for tumor progression. Basically, the CSC theory hypothesizes the presence of a hierarchically organized and relatively rare cell population, which is responsible for tumor initiation, self-renewal, and maintenance, in addition to accumulation of mutation and resistance to chemotherapy. CSCs have recently been described in breast cancer. Different genetic markers have been used to isolate breast CSCs, none of which have been correlated with the tumorigenicity or metastatic potential of the cells, limiting their precise characterization and clinical application in the development of therapeutic protocols. Here, we sought for subpopulations of CSCs by analyzing 10 judiciously chosen stem cell markers in a normal breast cell line (MCF10-A) and in four human breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-435, and Hs578-T) displaying different degrees of metastatic and invasiveness potential. We were able to identify two markers, which are differentially expressed in nontumorigenic versus tumor cells. The CD90 marker was highly expressed in the malignant cell lines. Interestingly, the CD14 molecule displayed higher expression levels in the nontumorigenic cell line. Therefore, we demonstrated that these two markers, which are more commonly used to isolate and characterize stem cells, are differentially expressed in breast tumor cells, when compared with nontumorigenic breast cells.

In vitro production of multigene transgenic blastocysts via sperm-mediated gene transfer allows rapid screening of constructs to be used in xenotransplantation experiments.

Multigene transgenic pigs would be of benefit for large animal models and in particular for xenotransplantation, where extensive genetic manipulation of donor pigs is required to make them suitable for organ grafting to humans. We have previously produced multitransgenic pigs via sperm-mediated gene transfer (SMGT) using integrative constructs expressing 3 different reporter genes. The aim of the present work was to evaluate the efficacy and safety of using 3 integrative constructs carrying 3 different human genes involved in the modulation of inflammatory responses. We developed an in vitro fertilization system to demonstrate that SMGT can be used to efficiently produce multigene transgenic embryos through a 1-step genetic modification using multiple integrative constructs each carrying a different human gene involved in the modulation of inflammatory processes (hHO1, hCD39, and hCD73). The results suggest that this system allowed an effective preliminary test of transgenesis optimization, greatly reducing the number of animals used in the experiments and fulfilling important ethical issues. We performed 5 in vitro fertilization experiments using sperm cells preincubated with all 3 integrative constructs. A total of 1,498 oocytes were fertilized to obtain 775 embryos, among which 340 further developed into blastocysts. We did not observe any toxicity related to the transgenesis procedure that affected normal embryo development. We observed 68.5% transgenesis efficiency. Blastocysts were 48% single, 31% double, and 21% triple transgenic.

Pulmonary kinetic expression of the endothelin system in a swine model of endotoxic shock.

Endothelin (ET)-1 is a potent vasoconstrictor peptide involved in the derangement of respiratory mechanics during endotoxic shock. We measured the kinetics of pulmonary mRNA expression of the key components of the ET system [i.e., ET-1, ET-converting enzyme (ECE), and ETA and ETB receptors] by quantitative real-time reverse transcriptase-polymerase chain reaction in a swine model of endotoxic shock (0, 1, 2, 3, and 4 h of continuous LPS infusion at 40 microg/kg/hour; sham group, 4 hour saline infusion). A significant increase in mRNA expression levels was observed for ET-1 in LPS-treated piglets; the increase began as early as 1 hour. In contrast, no significant variations were observed for the ECE, ETA, or ETB genes. Small gene expression differences observed with respect to our previous results suggest a possible effect of the anesthesia or surgical protocol on ET system regulation.

Sperm-mediated gene transfer-treated spermatozoa maintain good quality parameters and in vitro fertilization ability in swine.

A simple and efficient method for producing multitransgenic animals is required for medical and veterinary applications. Sperm-mediated gene transfer (SMGT) is an effective method for introducing multiple genes into pigs (Sus, Sus scrofa). The major benefits of this technique are the high efficiency, low cost, and ease of use compared with that of other methods: Sperm-mediated gene transfer does not require embryo handling or expensive equipment. The aim of this study was to investigate the influence of SMGT treatment and exogenous DNA uptake on sperm quality. Even after a coincubation with a 20-fold larger amount (100 microg/mL) of DNA than usual (5 microg/mL), sperm quality parameters were not significantly affected, confirming the hypothesis that the SMGT protocol itself or the amount of bound DNA do not compromise the possibility of an extended employment of SMGT. More importantly, we found that semen used for in vitro fertilization 24h after DNA uptake gave good cleavage (60% vs. 58%, treated vs. control) and developmental rates definitely positive (41% vs. 48%, treated vs. control). These good results are connected to a competitive efficiency of transformation (62%) due to the numerous improvements in SMGT technique. We demonstrate that SMGT-treated spermatozoa retain good quality and fertilization potential for at least 24h, expanding the possibility to apply transgenesis in field conditions in swine, where the greatest hurdles are fertilization timing and plain procedure.

Ixolaris, a tissue factor inhibitor, blocks primary tumor growth and angiogenesis in a glioblastoma model.

BACKGROUND: The expression levels of the clotting initiator protein Tissue Factor (TF) correlate with vessel density and the histological malignancy grade of glioma patients. Increased procoagulant tonus in high grade tumors (glioblastomas) also indicates a potential role for TF in progression of this disease, and suggests that anticoagulants could be used as adjuvants for its treatment. OBJECTIVES: We hypothesized that blocking of TF activity with the tick anticoagulant Ixolaris might interfere with glioblastoma progression. METHODS AND RESULTS: TF was identified in U87-MG cells by flow-cytometric and functional assays (extrinsic tenase). In addition, flow-cytometric analysis demonstrated the exposure of phosphatidylserine in the surface of U87-MG cells, which supported the assembly of intrinsic tenase (FIXa/FVIIIa/FX) and prothrombinase (FVa/FXa/prothrombin) complexes, accounting for the production of FXa and thrombin, respectively. Ixolaris effectively blocked the in vitro TF-dependent procoagulant activity of the U87-MG human glioblastoma cell line and attenuated multimolecular coagulation complexes assembly. Notably, Ixolaris inhibited the in vivo tumorigenic potential of U87-MG cells in nude mice, without observable bleeding. This inhibitory effect of Ixolaris on tumor growth was associated with downregulation of VEGF and reduced tumor vascularization. CONCLUSION: Our results suggest that Ixolaris might be a promising agent for anti-tumor therapy in humans.

Evaluation of swine fertilisation medium (SFM) efficiency in preserving spermatozoa quality during long-term storage in comparison to four commercial swine extenders.

In pig production, artificial insemination is widely carried out and the use of fresh diluted semen is predominant. For this reason, there are increasing interests in developing new extenders and in establishing the optimal storage conditions for diluted spermatozoa. In the last few decades, we utilised a homemade diluent (swine fertilisation medium (SFM)) for spermatozoa manipulation and biotechnological application as the production of transgenic pigs utilising the sperm-mediated gene transfer technique. The purpose of the present study is therefore to analyse the ability of SFM, in comparison to four commercial extenders, in preserving the quality of diluted boar semen stored at 16.5°C till 15 days. We utilised some of the main predictive tests as objectively measured motility, acrosome and sperm membrane integrity, high mitochondrial membrane potential and pH. Based on our in vitro study, SFM could be declared as a good long-term extender, able to preserve spermatozoa quality as well as Androhep Enduraguard for up to 6 to 9 days and more.

Laboratory animal science: a resource to improve the quality of science.

The contribution of animal experimentation to biomedical research is of undoubted value, nevertheless the real usefulness of animal models is still being hotly debated. Laboratory Animal Science is a multidisciplinary approach to humane animal experimentation that allows the choice of the correct animal model and the collection of unbiased data. Refinement, Reduction and Replacement, the "3Rs rule", are now widely accepted and have a major influence on animal experimentation procedures. Refinement, namely any decrease in the incidence or severity of inhumane procedures applied to animals, has been today extended to the entire lives of the experimental animals. Reduction of the number of animals used to obtain statistically significant data may be achieved by improving experimental design and statistical analysis of data. Replacement refers to the development of validated alternative methods. A Laboratory Animal Science training program in biomedical degrees can promote the 3Rs and improve the welfare of laboratory animals as well as the quality of science with ethical, scientific and economic advantages complying with the European requirement that "persons who carry out, take part in, or supervise procedures on animals, or take care of animals used in procedures, shall have had appropriate education and training".

Anaplastic large cell lymphomas: a study of 75 pediatric patients.

In this article, we describe the morphologic and immunophenotypic features of 75 cases of pediatric anaplastic large cell lymphoma (ALCL). According to the World Health Organization classification, 49 cases were common subtype ALCL, and respectively, 3, 6, and 17 cases were small cell, lymphohistiocytic, or mixed histologic variants. Anaplastic lymphoma kinase positivity was detected in 90.7% of the tumors and, using a panel of 9 T-cell surface markers, 88% could be assigned to the T-cell lineage. A molecular analysis for the T-cell receptor gamma (TCR- gamma) and the heavy chain of the immunoglobulin H rearrangements was performed on 6/9 ALCLs with a null immunophenotype, and a TCR clonal pattern was detected in 5/6 cases. In addition, 94.1% were immunoreactive for 1 or more cytotoxic proteins (Tia1, granzyme B, or perforin), and 15% expressed CD56. Clusterin, CD83, and Pax5, respectively, expressed in 91.3%, 1.7%, and 0% of the ALCLs, were useful biomarkers for the differential diagnosis with Hodgkin's lymphomas.

Recent increase of breast cancer incidence among women under the age of forty.

Using data from the Geneva Cancer Registry, we found that in 2002-2004, breast cancer incidence in women aged 25-39 years increased by 46.7% per year (95% CI: 7.1-74.0, P=0.015), which surveillance or detection bias may not fully explain.