Longitudinal effects of ketamine on cell proliferation and death in the CNS of zebrafish.

Zebrafish is known for its widespread neurogenesis and regenerative capacity, as well as several biological advantages, which turned it into a relevant animal model in several areas of research, namely in toxicological studies. Ketamine is a well-known anesthetic used both in human as well as veterinary medicine, due to its safety, short duration and unique mode of action. However, ketamine administration is associated with neurotoxic effects and neuronal death, which renders its use on pediatric medicine problematic. Thus, the evaluation of ketamine effects administration at early stages of neurogenesis is of pivotal importance. The 1-41-4 somites stage of zebrafish embryo development corresponds to the beginning of segmentation and formation of neural tube. In this species, as well as in other vertebrates, longitudinal studies are scarce, and the evaluation of ketamine long-term effects in adults is poorly understood. This study aimed to assess the effects of ketamine administration at the 1-4 somites stage, both in subanesthetic and anesthetic concentrations, in brain cellular proliferation, pluripotency and death mechanisms in place during early and adult neurogenesis. For that purpose, embryos at the 1-4 somites stage (10.5 h post fertilization - hpf) were distributed into study groups and exposed for 20 min to ketamine concentrations at 0.2/0.8 mg/mL. Animals were grown until defined check points, namely 50 hpf, 144 hpf and 7 months adults. The assessment of the expression and distribution patterns of proliferating cell nuclear antigen (PCNA), of sex-determining region Y-box 2 (Sox 2), apoptosis-inducing factor (AIF) and microtubule-associated protein 1 light chain 3 (LC3) was performed by Western-blot and immunohistochemistry. The results evidenced the main alterations in 144 hpf larvae, namely in autophagy and in cellular proliferation at the highest concentration of ketamine (0.8 mg/mL). Nonetheless, in adults no significant alterations were seen, pointing to a return to a homeostatic stage. This study allowed clarifying some of the aspects pertaining the longitudinal effects of ketamine administration regarding the CNS capacity to proliferate and activate the appropriate cell death and repair mechanisms leading to homeostasis in zebrafish. Moreover, the results indicate that ketamine administration at 1-4 somites stage in the subanesthetic and anesthetic concentrations despite some transitory detrimental effects at 144 hpf, is long-term safe for CNS, which are newly and promising results in this research field.

Decellularized human colorectal cancer matrices polarize macrophages towards an anti-inflammatory phenotype promoting cancer cell invasion via CCL18.

Macrophages are frequently identified in solid tumors, playing important roles in cancer progression. Their remarkable plasticity makes them very sensitive to environmental factors, including the extracellular matrix (ECM). In the present work, we investigated the impact of human colorectal tumor matrices on macrophage polarization and on macrophage-mediated cancer cell invasion. Accordingly, we developed an innovative 3D-organotypic model, based on the decellularization of normal and tumor tissues derived from colorectal cancer patients' surgical resections. Extensive characterization of these scaffolds revealed that DNA and other cell constituents were efficiently removed, while native tissue characteristics, namely major ECM components, architecture and mechanical properties, were preserved. Notably, normal and tumor decellularized matrices distinctly promoted macrophage polarization, with macrophages in tumor matrices differentiating towards an anti-inflammatory M2-like phenotype (higher IL-10, TGF-ß and CCL18 and lower CCR7 and TNF expression). Matrigel invasion assays revealed that tumor ECM-educated macrophages efficiently stimulated cancer cell invasion through a mechanism involving CCL18. Notably, the high expression of this chemokine at the invasive front of human colorectal tumors correlated with advanced tumor staging. Our approach evidences that normal and tumor decellularized matrices constitute excellent scaffolds when trying to recreate complex microenvironments to understand basic mechanisms of disease or therapeutic resistance.

Mesenteric lymph node granulomatous lesions in naturally infected wild boar (Sus scrofa) in Portugal--Histological, immunohistochemical and molecular aspects.

Several studies have demonstrated that wildlife reservoirs of mycobacteria are responsible for the maintenance and spreading of the infection to livestock and wildlife counterparts. Recent data report the role of wild boar (Sus scrofa) as a reservoir for Mycobacterium bovis. This study was conducted to evaluate the chronic inflammatory response in the mesenteric lymph nodes (MLN) of wild boar with granulomatous lymphadenitis (n=30). Morphological parameters of the lesions were recorded. The expression of CD3 and CD79α molecules was evaluated by immunohistochemistry. Molecular genotyping and culture to identify mycobacteria were performed. The lesions consisted mainly of stage III and stage IV granulomas. CD3 and CD79α positive cells were observed in 15 (50%) and in 11 (36.6%) MLN, respectively. In these lesions, higher percentages of T lymphocytes were found and a limited number of animals exhibited a tendency for an increased percentage of B lymphocytes. Our results suggest that there are similar percentages and distribution patterns of CD3 and CD79α in the lesions, regardless of the presence of Mycobacterium avium subsp. paratuberculosis (Map), M. bovis or Map-M. bovis co-infection, and confirm that wild boar is both susceptible and could be an important Map and M. bovis wild reservoir in the study area.

New Insights into Mycobacterium bovis Prevalence in Wild Mammals in Portugal.

A survey to determine the prevalence of Mycobacterium bovis in wild mammals in Portugal was conducted by testing samples from hunted animals and those found dead between 2009 and 2013. In this study, we investigated 2116 wild mammals. Post-mortem examinations were performed, and tissues were collected from wild mammals representing 8 families and 11 different species, with a total of 393 animals analysed. Cultures were performed, and acid-fast isolates were identified by PCR. Tissues were also screened for Mycobacterium bovis by directly extracting DNA and testing for the Mycobacterium bovis-specific sequences. Mycobacterium bovis prevalence was 26.9% (95% CI: 22.8-31.5%). Mycobacterium bovis was recorded in 106 of the 393 studied species: prevalence by species were 26.9% (95% CI: 16.8-40.2%) in red foxes, 20.0% (95% CI: 7.0-45.2%) in Egyptian mongooses, 21.4% (95% CI: 16.2-27.7%) in wild boar and 38.3% (95% CI: 29.9-47.4%) in red deer. Mycobacterium bovis infection was detected in six of eight taxonomic families. For some species, the small sample sizes obtained were a reflection of their restricted range and low abundance, making estimates of infection prevalence very difficult (1 beech marten of 4; 1 Eurasian otter of 3; 2 common genet of 3). Infection was not detected in European badgers, hedgehog, wild rabbits and hare. The results of this study confirm the presence of Mycobacterium bovis infection in wild carnivores in Portugal.

Macrophages stimulate gastric and colorectal cancer invasion through EGFR Y(1086), c-Src, Erk1/2 and Akt phosphorylation and smallGTPase activity.

The interactions between cancer cells and their microenvironment are crucial for malignant progression, as they modulate invasion-related activities. Tumor-associated macrophages are generally considered allies in the process of tumor progression in several types of cancer, although their role on gastric and colorectal carcinomas is still poorly understood. In this report, we studied the influence of primary human macrophages on gastric and colorectal cancer cells, considering invasion, motility/migration, proteolysis and activated intracellular signaling pathways. We demonstrated that macrophages stimulate cancer cell invasion, motility and migration, and that these effects depend on matrix metalloproteinase (MMP) activity and on the activation of epidermal growth factor receptor (EGFR) (at the residue Y(1086)), PLC-γ (phospholipase C-gamma) and Gab1 (GRB2-associated binding protein-1), as evidenced by siRNA (small interference RNA) experiments. Epidermal growth factor (EGF)-immunodepletion impaired macrophage-mediated cancer cell invasion and motility, suggesting that EGF is the pro-invasive and pro-motile factor produced by macrophages. Macrophages also induced gastric and colorectal cancer cell phosphorylation of Akt, c-Src and ERK1/2, and led to an increase of RhoA and Cdc42 activity. Interestingly, whereas macrophage-mediated cancer cell c-Src and ERK1/2 phosphorylation occurred downstream EGFR activation, Akt phosphorylation seems to be a parallel event, taking place in an EGFR-independent manner. The involvement of EGF, EGFR-downstream signaling partners and MMPs in macrophage-mediated invasion provides novel insights into the molecular crosstalk established between cancer cells and macrophages, opening new perspectives for the design of new and more efficient therapeutic strategies to counteract cancer cell invasion.

Maximal respiratory pressures among adolescent swimmers.

Maximal inspiratory pressures (MIP) and maximal expiratory pressures (MEP) are useful indices of respiratory muscle strength in athletes. The aims of this study were: to describe the strength of the respiratory muscles of Olympic junior swim team, at baseline and after a standard physical training; and to determine if there is a differential inspiratory and expiratory pressure response to the physical training. A cross-sectional study evaluated 28 international-level swimmers with ages ranging from 15 to 17 years, 19 (61 %) being males. At baseline, MIP was found to be lower in females (P = .001). The mean values reached by males and females were: MIP(cmH2O) = M: 100.4 (± 26.5)/F: 67.8 (± 23.2); MEP (cmH2O) = M: 87.4 (± 20.7)/F: 73.9 (± 17.3). After the physical training they reached: MIP (cmH2O) = M: 95.3 (± 30.3)/F: 71.8 (± 35.6); MEP (cmH2O) = M: 82.8 (± 26.2)/F: 70.4 (± 8.3). No differential pressure responses were observed in either males or females. These results suggest that swimmers can sustain the magnitude of the initial maximal pressures. Other studies should be developed to clarify if MIP and MEP could be used as a marker of an athlete's performance.

Development and application of stir bar sorptive extraction with polyurethane foams for the determination of testosterone and methenolone in urine matrices.

This work describes the development, validation, and application of a novel methodology for the determination of testosterone and methenolone in urine matrices by stir bar sorptive extraction using polyurethane foams [SBSE(PU)] followed by liquid desorption and high-performance liquid chromatography with diode array detection. The methodology was optimized in terms of extraction time, agitation speed, pH, ionic strength and organic modifier, as well as back-extraction solvent and desorption time. Under optimized experimental conditions, convenient accuracy were achieved with average recoveries of 49.7 8.6% for testosterone and 54.2 ± 4.7% for methenolone. Additionally, the methodology showed good precision (<9%), excellent linear dynamic ranges (>0.9963) and convenient detection limits (0.2-0.3 µg/L). When comparing the efficiency obtained by SBSE(PU) and with the conventional polydimethylsiloxane phase [SBSE(PDMS)], yields up to four-fold higher are attained for the former, under the same experimental conditions. The application of the proposed methodology for the analysis of testosterone and methenolone in urine matrices showed negligible matrix effects and good analytical performance.

Análise de custo-benefício da vacinação com Rev.1 da brucelose ovina e caprina no norte de Portugal entre 2000 e 2005 / Cost-benefit analysis of sheep and goat brucellosis vaccination with Rev.1 in the north of Portugal from 2000 to 2005

In the North of Portugal, a mass vaccination programme of small ruminants was conducted from 2001 to 2004. A study of cost-benefit was carried out for the 2000/2005 period to ascertain the economic benefits of this strategy. In order to estimate the cost of the zoonosis, the compensation costs paid to farmers for culled animals in the Brucellosis Eradication Campaign, data from vaccine Rev. 1 costs, and costs of people internment due to brucellosis were studied. An increase in the cost was observed from 2000 to 2001 (aproximately US$ 110,000), essentially due to compensation for culled animals, but also from vaccination and human internment costs. However, a progressive decrease was observed from 2001 to 2004 (from more than US$1,200,000 in 2001 to US$180,000 in 2004), roughly US$ 1,020,000 less. As the main conclusion, the annual cost of this zoonosis decreased by an average of almost US$600,000 per year, and a total of more than three million dollars were saved from 2000 to 2005. The results of this study suggest that mass vaccination with Rev. 1 reduced overall costs and was effective in reducing animal and human brucellosis costs.

No norte de Portugal, procedeu-se à vacinação massiva contra a brucelose em pequenos ruminantes entre 2001 e 2004. Neste estudo, efetuou-se uma análise de custo benefício entre 2000 e 2005 para apurar os benefícios econômicos dessa estratégia. Para estimar o custo da zoonose, estudaram-se três variáveis: a indenização paga aos proprietários dos animais eliminados durante a campanha de erradicação da brucelose, o custo da vacina Rev. 1 e os custos de internamento das pessoas infectadas por brucelose nas unidades de Serviços de Saúde. Durante os anos em estudo, observou-se um aumento de custo de aproximadamente US$110.000, entre 2000 e 2001, principalmente, devido à indenização dos animais eliminados e à aplicação da vacina e os custos por internamento de pessoas infectadas, seguindo-se uma redução acentuada entre 2001 e 2004 (de US$1.200.000 para US$180.000 em 2004), o que perfez US$1.020.000 poupados. O custo anual da zoonose, em relação às variáveis estudadas, diminuiu em média US$600.000 por ano, sendo poupados mais de US$ 3.000.000 entre 2000 e 2005. Estes resultados sugerem que a vacinação com Rev. 1 reduziu os custos totais da infecção humana e animal relativamente às variáveis estudadas.

Comparative evaluation of PCR in Ziehl-Neelsen stained smears and PCR in tissues for diagnosis of Mycobacterium avium subsp. paratuberculosis.

Thirty six tissues from sheep, previously diagnosed with paratuberculosis, were tested by PCR in positive Ziehl-Neelsen staining smears of tissues, and PCR in tissues targeting IS900 specific for Mycobacterium avium subsp. paratuberculosis. DNA amplification was achieved in 33.3% Ziehl-Neelsen smears, and in 61.1% tissue samples. Combination of both techniques found 66.7% samples as positive. Combination of techniques would, therefore, increase the sensitivity of diagnosis.

Coloração de Ziehl-Neelsen como método rápido de diagnóstico de paratuberculose ovina / Ziehl-Neelsen staining as a fast method in the diagnosis of ovine paratuberculosis

Estudou-se a presença de bacilos álcool-ácido resistentes compatíveis com Mycobacterium avium subsp. paratuberculosis em esfregaços de fezes e tecidos de ovinos. Vinte e seis esfregaços de fezes e 104 de tecidos, pertencentes a 26 animais diagnosticados como paratuberculosos, foram analisados pelo método de Ziehl-Neelsen. Dezesseis (61,5 por cento) esfregaços fecais apresentaram bacilos álcool-ácido resistentes compatíveis no exame microscópico. Vinte animais (76,9 por cento) foram diagnosticados pelo método nos esfregaços de tecidos. Vinte e um animais apresentaram esfregaços positivos nas fezes e nos tecidos, simultaneamente. A sensibilidade de Ziehl-Neelsen para os esfregaços fecais, esfregaços de tecidos e para a combinação de ambos foi de 61,5 por cento, 76,9 por cento e 80,8 por cento, respectivamente.

The presence of acid-fast bacilli compatible with Mycobacterium avium subsp. paratuberculosis in fecal and tissues smears was investigated using the Ziehl-Neelsen staining. A total of 26 fecal smears and 104 tissues smears collected from 26 sheep with confirmed paratuberculosis were analyzed. Sixteen (61.5 percent) fecal smears showed compatible with acid-fast bacilli on microscopic examination after staining. Twenty animals (76.9 percent) were diagnosed based on the positivity of tissues smears. The Ziehl-Neelsen sensitivities to faecal smears, tissues smears, and a combination of both were 61.5 percent, 76.9 percent, and 80.8 percent, respectively.

Projections from the anterior interposed nucleus to the red nucleus diminish with age in the mouse.

To analyse the effect of ageing on the projection of the anterior interposed nucleus to the red nucleus, we injected the retrograde tracer fluorogold in the red nucleus of 3-, 6- and 12-month-old mice. The number of labelled neurones in the anterior interposed nucleus fell by 9% between 3 and 6 months and by another 9% between 6 and 12 months (all P < 0.001). This suggests that loss of neurones from the cerebellar nuclei starts well before old age.

Development, optimisation and application of polyurethane foams as new polymeric phases for stir bar sorptive extraction.

In this contribution, polyurethane foams are proposed as new polymeric phases for stir bar sorptive extraction (SBSE). Assays performed for polyurethane synthesis demonstrated that four series of formulations (P(1), P(2), P(3) and P(4)) present remarkable stability and excellent mechanical resistance to organic solvents. For polymer clean-up treatment, acetonitrile proved to be the best solvent under sonification, ensuring the reduction of the contamination and interferences. SBSE assays performed on these polyurethane polymers followed by liquid desorption and high-performance liquid chromatography-diode array detection (LD-HPLC-DAD) or large volume injection-capillary gas chromatography-mass spectrometry (LD-LVI-GC-MS), showed that P(2) presents the best recovery yields for atrazine, 2,3,4,5-tetrachlorophenol and fluorene, used as model compounds in water samples at a trace level. SBSE(P(2)) assays performed on this polymer mixed up with several adsorbent materials, i.e. activated carbon, a mesoporous material and a calixarene, did not bring any advantages in relation with the polymeric matrix alone. The comparison between assays performed by SBSE(P(2)) and by the conventional SBSE(PDMS) showed much better performance for the former phase on aqueous samples spiked with atrazine, 2,3,4,5-tetrachlorophenol and fluorene, in which the foremost two analytes present recovery values 3- and 10-fold higher, respectively. The polyurethanes proposed as new polymeric phases for SBSE provided powerful capabilities for the enrichment of organic compounds from aqueous matrices, showing to be indicated mainly in the case of the more polar analytes.

Evaluation of the role of Ace1 and Yap1 in cadmium absorption using the eukaryotic cell model Saccharomyces cerevisiae.

In a previous paper, we demonstrated that the cytoplasmic level of glutathione-cadmium complex affects cadmium absorption by Saccharomyces cerevisiae, a usual eukaryotic cell model for studies of stress response. Furthermore, it was also observed that the absorption of this non-essential metal seems to be achieved by Zrt1, a zinc transporter of high affinity. Looking a little further into the control mechanism, we have verified that the deficiency in Ace1 impaired cadmium transport significantly. Ace1 is a transcription factor that activates the expression of CUP1, which encodes the S. cerevisiae metallothionein. On the other hand, the deficiency in the transcription factor Yap1 produced a two-fold increase in cadmium uptake. Cells lacking Yap1 showed low levels of glutathione, which could explain their higher capacity of absorbing cadmium. However, the mutant strain Ace1 deficient exhibited considerable amounts of glutathione. By using RT-PCR analysis, we observed that the lack of Yap1 activates the expression of both CUP1 and ZRT1, while the lack of Ace1 inhibited significantly the expression of these genes. Thus, metallothionein seems also to participate in the regulation of cadmium transport by controlling the expression of ZRT1. We propose that, at low levels of Cup1, the cytoplasmic concentration of essential metals, such as zinc, in free form (not complexated), increases, inhibiting ZRT1 expression. In contrast, at high levels of Cup1, the concentration of these metals falls, inducing ZRT1 expression and favoring cadmium absorption. These results confirm the involvement of zinc transport system with cadmium transport.