Use of outdoor ranges by laying hens in different sized flocks.

In studies assessing outdoor range use of laying hens, the number of hens seen on outdoor ranges is inversely correlated to flock size. The aim of this study was to assess individual ranging behavior on a covered (veranda) and an uncovered outdoor run (free-range) in laying hen flocks varying in size. Five to ten percent of hens (aged 9–15 months) within 4 small (2–2500 hens), 4 medium (5–6000), and 4 large (≥9000) commercial flocks were fitted with radio frequency identification (RFID) tags. Antennas were placed at both sides of all popholes between the house and the veranda and the veranda and the free-range. Ranging behavior was directly monitored for approximately three weeks in combination with hourly photographs of the free-range for the distribution of hens and 6h long video recordings on two parts of the free-range during two days. Between 79 and 99% of the tagged hens were registered on the veranda at least once and between 47 and 90% were registered on the free-range at least once. There was no association between the percentage of hens registered outside the house (veranda or free-range) and flock size. However, individual hens in small and medium sized flocks visited the areas outside the house more frequently and spent more time there than hens from large flocks. Foraging behavior on the free-range was shown more frequently and for a longer duration by hens from small and medium sized flocks than by hens from large flocks. This difference in ranging behavior could account for the negative relationship between flock size and the number of hens seen outside at one point of time. In conclusion, our work describes individual birds’ use of areas outside the house within large scale commercial egg production.

Computer Assisted Diagnosis and Treatment Planning of Femoroacetabular Impingement (FAI)

Femoroacetabular impingement (FAI) is a dynamic conflict of the hip defined by a pathological, early abutment of the proximal femur onto the acetabulum or pelvis. In the past two decades, FAI has received increasing focus in both research and clinical practice as a cause of hip pain and prearthrotic deformity. Anatomical abnormalities such as an aspherical femoral head (cam-type FAI), a focal or general overgrowth of the acetabulum (pincer-type FAI), a high riding greater or lesser trochanter (extra-articular FAI), or abnormal torsion of the femur have been identified as underlying pathomorphologies. Open and arthroscopic treatment options are available to correct the deformity and to allow impingement-free range of motion. In routine practice, diagnosis and treatment planning of FAI is based on clinical examination and conventional imaging modalities such as standard radiography, magnetic resonance arthrography (MRA), and computed tomography (CT). Modern software tools allow three-dimensional analysis of the hip joint by extracting pelvic landmarks from two-dimensional antero-posterior pelvic radiographs. An object-oriented cross-platform program (Hip2Norm) has been developed and validated to standardize pelvic rotation and tilt on conventional AP pelvis radiographs. It has been shown that Hip2Norm is an accurate, consistent, reliable and reproducible tool for the correction of selected hip parameters on conventional radiographs. In contrast to conventional imaging modalities, which provide only static visualization, novel computer assisted tools have been developed to allow the dynamic analysis of FAI pathomechanics. In this context, a validated, CT-based software package (HipMotion) has been introduced. HipMotion is based on polygonal three-dimensional models of the patient’s pelvis and femur. The software includes simulation methods for range of motion, collision detection and accurate mapping of impingement areas. A preoperative treatment plan can be created by performing a virtual resection of any mapped impingement zones both on the femoral head-neck junction, as well as the acetabular rim using the same three-dimensional models. The following book chapter provides a summarized description of current computer-assisted tools for the diagnosis and treatment planning of FAI highlighting the possibility for both static and dynamic evaluation, reliability and reproducibility, and its applicability to routine clinical use.

UHP Metamorphism Documented in Ti-chondrodite- and Ti-clinohumite-bearing Serpentinized Ultramafic Rocks from Chinese Southwestern Tianshan

The southwestern Tianshan (China) metamorphic belt records high-pressure (HP) to ultrahigh-pressure (UHP) conditions corresponding to a cold oceanic subduction-zone setting. Serpentinites enclosing retrogressed eclogite and rodingite occur as lenses within metapelites in the UHP unit, which also hosts coesite-bearing eclogites. Based on the petrology and petrography of these serpentinites, five events are recognized: (1) formation of a wehrlite–harzburgite–dunite association in the mantle; (2) retrograde metamorphism and partial hydration during exhumation of the mantle rocks close to the seafloor; (3) oceanic metamorphism leading to the first serpentinization and rodingitization; (4) UHP metamorphism during subduction; (5) retrograde metamorphism during exhumation together with a second serpentinization. The peak metamorphic mineral assemblage of the serpentinized wehrlite comprises Ti-chondrodite + olivine + antigorite + chlorite + magnetite + brucite. A computed pseudosection for this serpentinized wehrlite shows that the Al content in antigorite is mostly sensititive to temperature but can also be used to constrain pressure. The average XAl = 0·204 ± 0·026 of antigorite (XAl = Al (a.p.f.u.)/8, where Al is in atoms per formula unit for a structural formula M48T34O85(OH)62, and M and T are octahedral and tetrahedral sites, respectively) included in Ti-chondrodite and average XAl = 0·203 ± 0·019 of antigorite in the matrix result in a well-constrained peak metamorphic temperature of 510–530°C. Peak pressures are less precisely constrained at 37 ± 7 kbar. The Tianshan serpentinites thus record UHP metamorphic conditions and represent the deepest subducted serpentinites discovered so far. The retrograde evolution occurs within the stability field of brucite + antigorite + olivine + chlorite and formation of Ti-clinohumite at the expense of Ti-chondrodite has been observed, suggesting isothermal decompression. The resulting P–T path is in excellent agreement with the metamorphic evolution of country rocks, indicating that the UHP unit in Tianshan was subducted and exhumed as a coherent block. To refine the metamorphic path of the ultramafic rocks, we have investigated the stability fields of Ti-chondrodite and Ti-clinohumite using piston-cylinder experiments. A total of 11 experiments were conducted at 25–55 kbar and 600–750°C in a F-free natural system. Combined with previous experiments and information from natural rocks we constructed a petrogenetic grid for the stability of Ti-chondrodite and Ti-clinohumite in F-free peridotite compositions. The formation of Ti-chondrodite in serpentinites requires a minimum pressure of about 26 kbar, whereas in Ti-rich systems it can form at considerably lower pressures. A key finding is that at UHP conditions, F-free Ti-chondrodite or Ti-clinohumite breaks down in the presence of orthopyroxene between 700 and 750°C, at temperatures that are significantly lower than those of the terminal breakdown reactions of these humite minerals. These breakdown reactions are an additional source of fluid during prograde subduction of serpentinites.

DNA-DAMAGING REACTIONS OF NEOCARZINOSTATIN (ANTIBIOTICS, DNA REPAIR, CYTOTOXICITY)

Sensitive assays utilizing a cell-free and an intracellular system were employed to study the molecular bases of the DNA-damaging reactions of neocarzinostatin (NCS). In the cell-free DNA system, super-helical form I DNA from the bacteriophage PM2 was used as the substrate. The three forms of DNA present after treatment with NCS were separated by agarose gel electrophoresis. When NCS-damaged DNA was assayed under neutral conditions, there was a progressive decrease in the amount of surviving form I DNA and a corresponding increase in form II (nicked, relaxed circular) DNA, but very little increase in form III (linear duplex) DNA. This indicates that NCS introduces primarily single-strand breaks. However later studies showed that there were some site-specific double-strand breaks mediated by NCS on PM2 DNA. Seven such specific sites were mapped on the PM2 genome. When the damage was assayed under nondenaturing alkaline conditions or with the apurinic/apyrimidinic endonuclease IV, there was a slightly greater decrease in the amount of surviving form I DNA compared with neutral conditions indicating the presence of some alkali-labile sites.^ NCS-mediated DNA damage and repair were examined with cultured Chinese hamster ovary (CHO) cells using either alkaline elution for analysis of single-strand breaks or neutral elution for analysis of double-strand breaks. Most of the strand breaks introduced by NCS were capable of being rejoined. However, there was a small amount of residual DNA damage remaining unrejoined at 24-hr after removal of the drug. The amount of residual DNA damage was higher in a CHO mutant cell line (EM9) having a higher sensitivity to killing by NCS than its parental strain (AA8). Other lesions, DNA-protein complexes and alkali-labile sites, were detected after NCS treatment but they constituted only a small fraction of the DNA damage.^ Based on the above information, it can be postulated that NCS introduces some very lethal DNA damage. It is likely that the lethal lesions are a subset of the total DNA lesions representing the residual DNA damage. This DNA damage may be composed of site-specific, unrejoinable double-strand breaks and are thus the primary lesion leading to NCS-mediated lethality.^

A current literature review of the social and economic ramifications of pharmacogenomic research

Objective. To explore issues in current literature concerning possible social and economic ramifications of pharmacogenomic research. Design. Review of the literature. Data sources: Academic Search Premier, Blackwell Synergy, PUBMED and Social Sciences Citation Index. Review methods. Articles dealing with the social and economic ramifications of pharmacogenomic research were selected. The articles discussed at least one of 5 areas (race, privacy/confidentiality, ethics, insurance, and research and development). Some restrictions were placed on the articles chosen to narrow down the number of articles to a relevant, manageable amount. Results. Approximately 219 articles were selected for review; 159 were fully reviewed and found to be relevant to the issues; and 33 were cited. Conclusion. Insurance and research and development decisions are led by the free-market system with limited intervention from government. Race/ethnicity, privacy/confidentiality, and ethics continue to be debated with no clear answer. However, some compromise is regulated by government based upon current laws involving these issues. ^

Efecto del sistema de alimentación sobre los resultados técnicos, calidad de la canal, de la carne y de la grasa de cerdos ibéricos

La presente tesis doctoral pretende ampliar conocimientos de los sistemas de producción y tecnología asociada del cerdo Ibérico. Los trabajos de campo fueron llevados a cabo en el CIA ‘El Dehesón del Encinar’ (Oropesa, Toledo, España) con cerdos Ibéricos de la estirpe Torbiscal. En los dos primeros experimentos, utilizando dos grupos de cerdos, se estudió el efecto de la restricción de la alimentación en premontanera sobre las características de la canal y sobre la composición de la grasa al sacrificio de los cerdos posteriormente cebados en montanera. Los animales restringidos en premontanera crecieron significativamente menos que los no restringidos durante dicho periodo, y manifestaron un crecimiento compensatorio durante la montanera. Este crecimiento compensatorio no fue suficiente, y los animales restringidos llegaron con pesos significativamente inferiores al sacrificio, aunque esto no supuso diferencias para las características de la canal entre ambos grupos de animales. También al sacrificio, los animales restringidos durante la premontanera tuvieron concentraciones significativamente diferentes de ácidos grasos, sobre todo en la capa interna de la grasa subcutánea y en la fracción de lípidos polares de la grasa intramuscular; asimismo, tuvieron mayores concentraciones de tocoferoles en el músculo Longissimus dorsi. En un tercer experimento, utilizando cuatro grupos de animales, se estudió el efecto del tiempo de estancia de los cerdos en montanera exclusiva sobre las características de la canal y de la grasa al sacrificio. La duración de la montanera afectó significativamente al crecimiento, a los pesos y porcentajes de los productos del despiece y al perfil de ácidos grasos de la grasa subcutánea. Los animales cebados con pienso en confinamiento tuvieron mayor crecimiento, mejor calidad de la canal, y peor calidad de grasa que los otros tres grupos de animales que se acabaron en montanera exclusiva durante 46, 83 y 111 días. En el último experimento se evaluó si la impedancia bioeléctrica es un método adecuado para realizar un estudio tisular de los jamones de cerdos Ibéricos, aspecto especialmente relevante para la industria. La impedancia bioeléctrica resultó ser un método no invasivo adecuado, que puede ser aplicado para predecir la composición tisular de los jamones, aunque se necesitan más ensayos antes de llevar este método a la práctica cotidiana de la industria. ABSTRACT This Thesis aims to expand knowledge on production systems and technology associated to Iberian pigs. Fieldwork was conducted in the CIA ‘El Dehesón del Encinar’ (Oropesa, Toledo, Spain) with Iberian pigs Torbiscal line. In first two experiments, using two groups of pigs, the effect of feed restriction during the period previous to free-range fattening on growth and carcass characteristics in Iberian pigs finished under free-range conditions was studied. The pigs fed low feeding levels during the period previous to free-range finishing period, had significantly lower growth than those feed with a high level during that period, and showed a compensatory growth during free-range period. However, this compensatory growth was not enough to reach the same weight than the animals fed with a high level, and the slaughter weights were significantly different, although no differences were observed for the carcass characteristics between both groups of pigs. At slaughter, animals fed low feeding levels during the period previous to free-range finishing period, had significantly different fatty acid proportions, especially in the inner backfat layer and in polar lipids fraction of the intramuscular fat; also, the tocopherol concentration found in Longissimus dorsi muscle from pigs fed low feeding levels was higher than those fed high feeding levels. In a third experiment, using four groups of animals, the effect of duration under freerange exclusive conditions on carcass and fat characteristics at slaughter was studied. The growth, weights and percentages of carcass cuts, and fatty acid profile of the backfat were significantly affected by the duration under free-range exclusively conditions. The animals fed in confinement with concentrate diets during the whole fattening period had high growth, better carcass quality and worse fat quality than the other three groups of animals fed under free-range exclusive conditions during 46, 83 and 111 days. In the last experiment, it assessed whether the bioelectrical impedance is a suitable method for perform a tissue study of Iberian pig hams, particularly important aspect for industry. The bioelectrical impedance has proved to be a non-invasive suitable method, which can be applied to predict the tissues composition of the hams, but more trials are needed before carrying this method to everyday practice in the industry.

Protein phosphatase 2A is required for the initiation of chromosomal DNA replication

Protein phosphatase 2A (PP2A) is an abundant, multifunctional serine/threonine-specific phosphatase that stimulates simian virus 40 DNA replication. The question as to whether chromosomal DNA replication also depends on PP2A was addressed by using a cell-free replication system derived from Xenopus laevis eggs. Immunodepletion of PP2A from Xenopus egg extract resulted in strong inhibition of DNA replication. PP2A was required for the initiation of replication but not for the elongation of previously engaged replication forks. Therefore, the initiation of chromosomal DNA replication depends not only on phosphorylation by protein kinases but also on dephosphorylation by PP2A.

Multiple benefits of multiple mating in guppies

The rewards of promiscuity for males are undisputed. But why should a female mate promiscuously, particularly when her partners offer no resources other than sperm and increase her chances of succumbing to predation or disease? This question has been hotly debated but at present remains largely unresolved [Jennions, M. D. & Petrie, M. (2000) Biol. Rev. 75, 21–64]. One possibility is that females exploit postcopulatory mechanisms, such as sperm competition, to increase both the quality and quantity of their offspring. In this paper, we use the Trinidadian guppy, a species with a resource-free mating system, to test the hypothesis that females gain multiple benefits from multiple mating. Our results indicate that multiply mated females secure substantive advantages: They have shorter gestation times and larger broods, and they produce offspring with better developed schooling abilities and escape responses than their singly mated counterparts.

DNA polymerase ɛ is required for coordinated and efficient chromosomal DNA replication in Xenopus egg extracts

DNA polymerase ɛ (Polɛ) is thought to be involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the requirement of other replicative DNA polymerases, DNA polymerases α and δ (Polα and δ), for chromosomal DNA replication has been well documented by genetic and biochemical studies, the precise role, if any, of Polɛ in chromosomal DNA replication is still obscure. Here we show, with the use of a cell-free replication system with Xenopus egg extracts, that Xenopus Polɛ is indeed required for chromosomal DNA replication. In Polɛ-depleted extracts, the elongation step of chromosomal DNA replication is markedly impaired, resulting in significant reduction of the overall DNA synthesis as well as accumulation of small replication intermediates. Moreover, despite the decreased DNA synthesis, excess amounts of Polα are loaded onto the chromatin template in Polɛ-depleted extracts, indicative of the failure of proper assembly of DNA synthesis machinery at the fork. These findings strongly suggest that Polɛ, along with Polα and Polδ, is necessary for coordinated chromosomal DNA replication in eukaryotic cells.

Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis.

Although proteases related to the interleukin 1 beta-converting enzyme (ICE) are known to be essential for apoptotic execution, the number of enzymes involved, their substrate specificities, and their specific roles in the characteristic biochemical and morphological changes of apoptosis are currently unknown. These questions were addressed using cloned recombinant ICE-related proteases (IRPs) and a cell-free model system for apoptosis (S/M extracts). First, we compared the substrate specificities of two recombinant human IRPs, CPP32 and Mch2 alpha. Both enzymes cleaved poly-(ADP-ribose) polymerase, albeit with different efficiencies. Mch2 alpha also cleaved recombinant and nuclear lamin A at a conserved VEID decreases NG sequence located in the middle of the coiled-coil rod domain, producing a fragment that was indistinguishable from the lamin A fragment observed in S/M extracts and in apoptotic cells. In contrast, CPP32 did not cleave lamin A. The cleavage of lamin A by Mch2 alpha and by S/M extracts was inhibited by millimolar concentrations of Zn2+, which had a minimal effect on cleavage of poly (ADP-ribose) polymerase by CPP32 and by S/M extracts. We also found that N-(acetyltyrosinylvalinyl-N epsilon-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone, which derivatizes the larger subunit of active ICE, can affinity label up to five active IRPs in S/M extracts. Together, these observations indicate that the processing of nuclear proteins in apoptosis involves multiple IRPs having distinct preferences for their apoptosis-associated substrates.

Translational regulation of mammalian and Drosophila citric acid cycle enzymes via iron-responsive elements.

The posttranscriptional control of iron uptake, storage, and utilization by iron-responsive elements (IREs) and iron regulatory proteins (IRPs) provides a molecular framework for the regulation of iron homeostasis in many animals. We have identified and characterized IREs in the mRNAs for two different mitochondrial citric acid cycle enzymes. Drosophila melanogaster IRP binds to an IRE in the 5' untranslated region of the mRNA encoding the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (SDH). This interaction is developmentally regulated during Drosophila embryogenesis. In a cell-free translation system, recombinant IRP-1 imposes highly specific translational repression on a reporter mRNA bearing the SDH IRE, and the translation of SDH-Ip mRNA is iron regulated in D. melanogaster Schneider cells. In mammals, an IRE was identified in the 5' untranslated regions of mitochondrial aconitase mRNAs from two species. Recombinant IRP-1 represses aconitase synthesis with similar efficiency as ferritin IRE-controlled translation. The interaction between mammalian IRPs and the aconitase IRE is regulated by iron, nitric oxide, and oxidative stress (H2O2), indicating that these three signals can control the expression of mitochondrial aconitase mRNA. Our results identify a regulatory link between energy and iron metabolism in vertebrates and invertebrates, and suggest biological functions for the IRE/IRP regulatory system in addition to the maintenance of iron homeostasis.

Simian virus 40 late gene expression is regulated by members of the steroid/thyroid hormone receptor superfamily.

Transcription of the late genes of simian virus 40 (SV40) is repressed during the early phase of the lytic cycle of infection of binding of cellular factors, called IBP-s, to the SV40 late promoter; repression is relieved after the onset of viral DNA replication by titration of these repressors. Preliminary data indicated that one of the major components of IBP-s was human estrogen-related receptor 1 (hERR1). We show here that several members of the steroid/thyroid hormone receptor superfamily, including testis receptor 2, thyroid receptor alpha 1 in combination with retinoid X receptor alpha, chicken ovalbumin upstream promoter transcription factors 1 and 2 (COUP-TF1 and COUP-TF2), as well as hERR1, possess the properties of IBP-s. These receptors bind specifically to hormone receptor binding sites present in the SV40 major late promoter. Recombinant COUP-TF1 specifically represses transcription from the SV40 major late promoter in a cell-free transcription system. Expression of COUP-TF1, COUP-TF2, or hERR1 in monkey cells results in repression of the SV40 late promoter, but not the early promoter, in the absence of the virally encoded large tumor antigen. Overexpression of COUP-TF1 leads to a delay in the early-to-late switch in SV40 gene expression during the lytic cycle of infection. Thus, members of this superfamily can play major direct roles in regulating expression of SV40. Possibly, natural or synthetic ligands to these receptors can serve as antiviral drugs. Our findings also provide the basis for the development of assays to screen for the ligands to testis receptor 2 and hERR1.

Intermediate hosts of Toxoplasma gondii and food safety:epidemiology, genetics and parasite survival in meat-producing animals in Italy

Toxoplasma gondii is a coccidian parasite with a global distribution. The definitive host is the cat (and other felids). All warm-blooded animals can act as intermediate hosts, including humans. Sexual reproduction (gametogony) takes place in the final host and oocysts are released in the environment, where they then sporulate to become infective. In intermediate hosts the cycle is extra-intestinal and results in the formation of tachyzoites and bradyzoites. Tachyzoites represent the invasive and proliferative stage and on entering a cell it multiplies asexually by endodyogeny. Bradyzoites within tissue cysts are the latent form. T. gondii is a food-borne parasite causing toxoplasmosis, which can occur in both animals and humans. Infection in humans is asymptomatic in more than 80% of cases in Europe and North-America. In the remaining cases patients present fever, cervical lymphadenopathy and other non-specific clinical signs. Nevertheless, toxoplasmosis is life threatening if it occurs in immunocompromised subjects. The main organs involved are brain (toxoplasmic encephalitis), heart (myocarditis), lungs (pulmonary toxoplasmosis), eyes, pancreas and parasite can be isolated from these tissues. Another aspect is congenital toxoplasmosis that may occur in pregnant women and the severity of the consequences depends on the stage of pregnancy when maternal infection occurs. Acute toxoplasmosis in developing foetuses may result in blindness, deformation, mental retardation or even death. The European Food Safety Authority (EFSA), in recent reports on zoonoses, highlighted that an increasing numbers of animals resulted infected with T. gondii in EU (reported by the European Member States for pigs, sheep, goats, hunted wild boar and hunted deer, in 2011 and 2012). In addition, high prevalence values have been detected in cats, cattle and dogs, as well as several other animal species, indicating the wide distribution of the parasite among different animal and wildlife species. The main route of transmission is consumption of food and water contaminated with sporulated oocysts. However, infection through the ingestion of meat contaminated with tissue cysts is frequent. Finally, although less frequent, other food products contaminated with tachyzoites such as milk, may also pose a risk. The importance of this parasite as a risk for human health was recently highlighted by EFSA’s opinion on modernization of meat inspection, where Toxoplasma gondii was identified as a relevant hazard to be addressed in revised meat inspection systems for pigs, sheep, goats, farmed wild boar and farmed deer (Call for proposals -GP/EFSA/BIOHAZ/2013/01). The risk of infection is more highly associated to animals reared outside, also in free-range or organic farms, where biohazard measure are less strict than in large scale, industrial farms. Here, animals are kept under strict biosecurity measures, including barriers, which inhibit access by cats, thus making soil contamination by oocysts nearly impossible. A growing demand by the consumer for organic products, coming from free-range livestock, in respect of animal-welfare, and the desire for the best quality of derived products, have all led to an increase in the farming of free-range animals. The risk of Toxoplasma gondii infection increases when animals have access to environment and the absence of data in Italy, together with need for in depth study of both the prevalence and genotypes of Toxoplasma gondii present in our country were the main reasons for the development of this thesis project. A total of 152 animals have been analyzed, including 21 free-range pigs (Suino Nero race), 24 transhumant Cornigliese sheep, 77 free-range chickens and 21 wild animals. Serology (on meat juice) and identification of T. gondii DNA through PCR was performed on all samples, except for wild animals (no serology). An in-vitro test was also applied with the aim to find an alternative and valid method to bioassay, actually the gold standard. Meat samples were digested and seeded onto Vero cells, checked every day and a RT-PCR protocol was used to determine an eventual increase in the amount of DNA, demonstrating the viability of the parasite. Several samples were alos genetically characterized using a PCR-RFLP protocol to define the major genotypes diffused in the geographical area studied. Within the context of a project promoted by Istituto Zooprofilattico of Pavia and Brescia (Italy), experimentally infected pigs were also analyzed. One of the aims was to verify if the production process of cured “Prosciutto di Parma” is able to kill the parasite. Our contribution included the digestion and seeding of homogenates on Vero cells and applying the Elisa test on meat juice. This thesis project has highlighted widespread diffusion of T. gondii in the geographical area taken into account. Pigs, sheep, chickens and wild animals showed high prevalence of infection. The data obtained with serology were 95.2%, 70.8%, 36.4%, respectively, indicating the spread of the parasite among numerous animal species. For wild animals, the average value of parasite infection determined through PCR was 44.8%. Meat juice serology appears to be a very useful, rapid and sensitive method for screening carcasses at slaughterhouse and for marketing “Toxo-free” meat. The results obtained on fresh pork meat (derived from experimentally infected pigs) before (on serum) and after (on meat juice) slaughter showed a good concordance. The free-range farming put in evidence a marked risk for meat-producing animals and as a consequence also for the consumer. Genotyping revealed the diffusion of Type-II and in a lower percentage of Type-III. In pigs is predominant the Type-II profile, while in wildlife is more diffused a Type-III and mixed profiles (mainly Type-II/III). The mixed genotypes (Type-II/III) could be explained by the presence of mixed infections. Free-range farming and the contact with wildlife could facilitate the spread of the parasite and the generation of new and atypical strains, with unknown consequences on human health. The curing process employed in this study appears to produce hams that do not pose a serious concern to human health and therefore could be marketed and consumed without significant health risk. Little is known about the diffusion and genotypes of T. gondii in wild animals; further studies on the way in which new and mixed genotypes may be introduced into the domestic cycle should be very interesting, also with the use of NGS techniques, more rapid and sensitive than PCR-RFLP. Furthermore wildlife can become a valuable indicator of environmental contamination with T. gondii oocysts. Other future perspectives regarding pigs include the expansion of the number of free-range animals and farms and for Cornigliese sheep the evaluation of other food products as raw milk and cheeses. It should be interesting to proceed with the validation of an ELISA test for infection in chickens, using both serum and meat juice on a larger number of animals and the same should be done also for wildlife (at the moment no ELISA tests are available and MAT is the reference method for them). Results related to Parma ham do not suggest a concerning risk for consumers. However, further studies are needed to complete the risk assessment and the analysis of other products cured using technological processes other than those investigated in the present study. For example, it could be interesting to analyze products such as salami, produced with pig meat all over the Italian country, with very different recipes, also in domestic and rural contexts, characterized by a very short period of curing (1 to 6 months). Toxoplasma gondii is one of the most diffuse food-borne parasites globally. Public health safety, improved animal production and protection of endangered livestock species are all important goals of research into reliable diagnostic tools for this infection. Future studies into the epidemiology, parasite survival and genotypes of T. gondii in meat producing animals should continue to be a research priority.

Neural Stem cell Isolation and Characterization

Throughout the process of development and continuing into adulthood, stem cells function as a reservoir of undifferentiated cell types, whose role is to underpin cell genesis in a variety of tissues and organs. In the adult, they play an essential homeostatic role by replacing differentiated tissue cells "worn off" by physiological turnover or lost to injury or disease. As such, the discovery of such cells in the adult mammalian central nervous system (CNS), an organ traditionally thought to have little or no regenerative capacity, was most unexpected. Nonetheless, by employing a novel serum-free culture system termed the neurosphere assay, Reynolds and Weiss demonstrated the presence of neural stem cells in both the adult (Reynolds and Weiss, 1992) and embryonic mouse brain (Reynolds et al., 1992). Here we describe how to generate, serially passage, and differentiate neurospheres derived from both the developing and adult brain, and provide more technical details that will enable one to achieve reproducible cultures, which can be passaged over an extended period of time.

Fatty acid composition, antioxidants and lipid oxidation in chicken breasts from different production regimes

Chicken breast from nine products and from the following production regimes: conventional (chilled and frozen), organic and free range, were analysed for fatty acid composition of total lipids, preventative and chain breaking antioxidant contents and lipid oxidation during 5 days of sub-ambient storage following purchase. Total lipids were extracted with an optimal amount of a cold chloroform methanol solvent. Lipid compositions varied, but there were differences between conventional and organic products in their contents of total polyunsaturated fatty acids and n-3 and n-6 fatty acids and n-6:n-3 ratio. Of the antioxidants, a-tocopherol content was inversely correlated with lipid oxidation. The antioxidant enzyme activities of catalase, glutathione peroxidase and glutathione reductase varied between products. Modelling with partial least squares regression showed no overall relationship between total antioxidants and lipid data, but certain individual antioxidants showed a relationship with specific lipid fractions.