Development of a Rainbow Trout (Oncorhynchus mykiss) Intestinal In Vitro Platform for Profiling Amino Acid Digestion and Absorption of a Complete Diet.

The ever-increasing number and variation of raw materials utilized to provide alternative feed formulations continues to allow for a more sustainable and flexible approach. Testing all these options in vivo is still the most robust and reliable manner to pick the best raw material candidates, but it requires the use of large numbers of animals and is time-consuming and expensive. Therefore, we are developing an in vitro platform that can provide a reliable evaluation of new ingredients. The main aim of this work was to combine an in vitro digestion protocol of extruded, commercially relevant aquafeeds with the exposure of intestinal epithelial cells to the extracted bio-available fraction (BAF). The results show that 250,000 cells/cm2 represents the optimal seeding density and that up to 50% BAF concentration for up to 24 h had no negative effects on the epithelial barrier morphology and function. It is possible to determine amino acid digestibility and bioavailability in all the experimental conditions (with and without BSA, at 25% and 50% dilution) and at all time points (0, 6, and 24 h). However, BAF concentration, the medium used for its dilution, and the length of exposure to the different epithelial cell lines can all influence the results and, therefore, must be selected according to the final aim of the experiment.

Preclinical Detection of Prions in Blood of Nonhuman Primates Infected with Variant Creutzfeldt-Jakob Disease.

Variant Creutzfeldt-Jakob disease (vCJD) is caused by prion infection with bovine spongiform encephalopathy and can be transmitted by blood transfusion. Protein misfolding cyclic amplification (PMCA) can detect prions in blood from vCJD patients with 100% sensitivity and specificity. To determine whether PMCA enables prion detection in blood during the preclinical stage of infection, we performed a blind study using blood samples longitudinally collected from 28 control macaques and 3 macaques peripherally infected with vCJD. Our results demonstrate that PMCA consistently detected prions in blood during the entire preclinical stage in all infected macaques, without false positives from noninfected animals, when using the optimized conditions for amplification of macaque prions. Strikingly, prions were detected as early as 2 months postinoculation (>750 days before disease onset). These findings suggest that PMCA has the potential to detect vCJD prions in blood from asymptomatic carriers during the preclinical phase of the disease.

Dentoskeletal and aesthetic effects of mandibular protraction appliance (MPA) using Ricketts analysis / Efeitos dentoesqueléticos e tegumentares do aparelho de protração mandibular (APM) usando a análise cefalométrica de Ricketts

Objective: This study was designed to evaluate the skeletal, dental and soft tissue effects of mandibular deficiency treatment with the mandibular protraction appliance (MPA) using 12 factors of the Ricketts analysis. Material and method: This cross-sectional retrospective study sample consisted of a group (n = 27), with Class II malocclusion, convex facial profile, increased horizontal trespass and mandibular deficiency, with initial mean age of 12.27 and final of 15.18 years, treated with fixed appliance combined with the MPA, in an average time of 2.9 years. Initial and final radiographs were investigated using Ricketts analysis. The dependent t-test was used to compare the initial and final phases of the MPA group, with a significance level of 5%. Result: Statistically significant differences were observed for dental changes such as retrusion (p=0.000) and palatal inclination of the maxillary incisors (p=0.000); protrusion (p=0.000) and buccal inclination of the mandibular incisors (p=0.000); increased interincisal angle (p=0.002) and improved molar ratio (p=0.003). There was also a restriction of the anterior displacement of the maxilla (p=0.000) and a decrease in the mandibular plane angle (p=0.024). The variable inferior labial protrusion with significance (p=0.000), reiterated the improvement in the profile. Conclusion: The effects of MPA on correction of malocclusion Class II, verified by Ricketts analysis occurred predominantly by dentoalveolar changes, decrease in the Mandibular Plane Angle, and restriction of anterior displacement of the maxilla, which contributed to the improvement in the patient's profile.

Objetivo: Este estudo analisou as alterações dentárias, esqueléticas e tegumentares promovidas pelo Aparelho de Protração Mandibular (APM) por meio da análise de Ricketts. Material e método: A amostra contou com 27 pacientes (14 meninas e 13 meninos) com má oclusão de Classe II, perfil facial convexo, trespasse horizontal aumentado e deficiência mandibular, com idade média inicial de 12,27 e final de 15,18 anos, tratados com aparelho fixo combinado com o APM. A comparação das telerradiografias iniciais (T1) e finais (T2) foi realizada pelo teste t dependente, com nível de significância de 5%. Resultado: Observou-se diferença estatisticamente significante para a retrusão (p=0.000) e lingualização dos incisivos superiores (p=0.000), protrusão (p=0.000) e vestibularização dos incisivos inferiores (p=0.000), aumento do ângulo interincisivos (p=0.002), melhora da relação molar (p=0.003), restrição do deslocamento anterior da maxila (p=0.000), diminuição do ângulo do plano mandibular (p=0.024) e melhora do perfil facial (p=0.000). Conclusão: O APM promoveu alterações dentoalveolares, observadas principalmente pela diminuição do ângulo do plano mandibular e restrição do deslocamento para anterior da maxila que contribuíram para a melhora do perfil do paciente.

Rescue of homeostatic regulation of striatal excitability and locomotor activity in a mouse model of Huntington's disease.

We describe a fast activity-dependent homeostatic regulation of intrinsic excitability of identified neurons in mouse dorsal striatum, the striatal output neurons. It can be induced by brief bursts of activity, is expressed on a time scale of seconds, limits repetitive firing, and can convert regular firing patterns to irregular ones. We show it is due to progressive recruitment of the KCNQ2/3 channels that generate the M current. This homeostatic mechanism is significantly reduced in striatal output neurons of the R6/2 transgenic mouse model of Huntington's disease, at an age when the neurons are hyperactive in vivo and the mice begin to exhibit locomotor impairment. Furthermore, it can be rescued by bath perfusion with retigabine, a KCNQ channel activator, and chronic treatment improves locomotor performance. Thus, M-current dysfunction may contribute to the hyperactivity and network dysregulation characteristic of this neurodegenerative disease, and KCNQ2/3 channel regulation may be a target for therapeutic intervention.

Tratamento da má oclusão de Classe II, divisão 1, com Ortopedia Funcional removível e Ortodontia fixa: relato de caso e controle de cinco anos / Treatment of Class II division 1 maloclusion with removable functional orthopedics and fixes orthodontics: a case report with a 5-years follow-up

Recursos ortopédicos e ortodônticos, utilizados de forma associada, têm-se mostrado uma opção terapêutica eficiente no tratamento da má oclusão de Classe II, divisão 1, desde que o paciente ainda apresente um potencial de crescimento. Entretanto, as alterações nas relações esqueléticas, dentárias e tegumentares só podem ser consideradas satisfatórias, caso permaneçam estáveis. No referido artigo, é apresentado um tratamento combinado ortopédico/ortodôntico, numa má oclusão de Classe II, divisão 1, em paciente em fase de crescimento e desenvolvimento craniofacial, onde a má oclusão foi tratada em duas fases. Na primeira fase utilizou-se um aparelho ortopédico funcional removível, tipo Ativador por um ano, seguido de terapia ortodôntica fixa concomitantemente à contenção ativa com o ativador. Na fase final, o tratamento foi complementado com elásticos de classe II, terminando essa fase fixa em 02 anos e 05 meses. Com isso contabilizou-se um tempo total de tratamento combinado ortopédico/ortodôntico da Classe II, divisão 1, com 3 anos e 05 meses de tratamento. Os resultados foram satisfatórios, com adequação do trepasse horizontal e vertical, ajuste da relação molar, adequado selamento labial e obtenção de um bom perfil, tendendo ao reto. Após 5 anos de remoção do aparelho fixo, foi realizado um controle, onde se verificou manutenção dos resultados ortodônticos e faciais, com manutenção do perfil reto, bom selamento labial passivo, trespasse vertical e horizontal adequados e relação de Classe I de Angle mantidos.

Extração atípica de incisivos centrais superiores no tratamento da má oclusão de Classe II / Atypical extraction of maxillary central incisors for Class II malocclusion treatment

O presente trabalho tem como objetivo relatar um caso de Classe II, divisão 1, tratado na fase adulta, com extrações de incisivos centrais superiores. A paciente apresentava má oclusão de Classe II completa com apinhamento anterossuperior. Os incisivos centrais superiores apresentavam restaurações de resina extensas e uma lesão gengival na região do dente 11. Observou-se, nas radiografias periapicais, que os dentes 11 e 21 possuíam tratamento endodôntico e pouca estrutura coronária remanescente. Devido a tais condições, optou-se por extraí-los e por fechar o espaço ortodonticamente. A mecânica foi realizada por meio da retração anterossuperior associada a elásticos de Classe III, e, no lugar dos incisivos, colocou-se uma prótese parcial removível. Ao final do tratamento e após a reanatomização dos incisivos laterais e caninos, obteve-se resultados estéticos e funcionais bastante satisfatórios. A estabilidade foi avaliada três anos após o tratamento, onde foi observada pequena reabertura do espaço das extrações (0,5mm); no entanto, outros aspectos, como a relação anteroposterior, se mantiveram estáveis.

MHC class I protein is expressed by neurons and neural progenitors in mid-gestation mouse brain.

Proteins of the major histocompatibility complex class I (MHCI) are known for their role in the vertebrate adaptive immune response, and are required for normal postnatal brain development and plasticity. However, it remains unknown if MHCI proteins are present in the mammalian brain before birth. Here, we show that MHCI proteins are widely expressed in the developing mouse central nervous system at mid-gestation (E9.5-10.5). MHCI is strongly expressed in several regions of the prenatal brain, including the neuroepithelium and olfactory placode. MHCI is expressed by neural progenitors at these ages, as identified by co-expression in cells positive for neuron-specific class III ß-tubulin (Tuj1) or for Pax6, a marker of neural progenitors in the dorsal neuroepithelium. MHCI is also co-expressed with nestin, a marker of neural stem/progenitor cells, in olfactory placode, but the co-localization is less extensive in other regions. MHCI is detected in the small population of post-mitotic neurons that are present at this early stage of brain development, as identified by co-expression in cells positive for neuronal microtubule-associated protein-2 (MAP2). Thus MHCI protein is expressed during the earliest stages of neuronal differentiation in the mammalian brain. MHCI expression in neurons and neural progenitors at mid-gestation, prior to the maturation of the adaptive immune system, is consistent with MHCI performing non-immune functions in prenatal brain development. These results raise the possibility that disruption of the levels and/or patterns of MHCI expression in the prenatal brain could contribute to the pathogenesis of neurodevelopmental disorders.

Frizzled-1 is involved in the neuroprotective effect of Wnt3a against Abeta oligomers.

The activation of the canonical Wnt signaling pathway protects hippocampal neurons against the toxicity of Alzheimer's amyloid-beta-peptide (Abeta), however, the role played by the Wnt receptors Frizzleds, has not been studied. We report here that Frizzled-1 mediates the activation of the canonical Wnt/beta-catenin pathway by Wnt3a in PC12 cells. In addition, the protective effect of Wnt3a against the toxicity of Abeta oligomers was modulated by Frizzled-1 expression levels in both PC12 cells and hippocampal neurons. Over-expression of Frizzled-1 significantly increased cell survival induced by Wnt3a and diminished caspase-3 activation, while knocking-down Frizzled-1 expression by antisense oligonucleotides decreased the Wnt3a protection. Over-expression of wild-type beta-catenin, but not a transcriptionally inactive mutated version, prevented the toxicity of Abeta suggesting that the transcription of Wnt target genes may be involved in these events. This was confirmed by co-transfecting both Frizzled-1 and the inactive form of beta-catenin, which does not elicited protection levels similar to those showed with endogenous beta-catenin. Our results indicate that Wnt3a protects from Abeta-oligomers toxicity by activating the canonical Wnt signaling pathway through the Frizzled-1 receptor, suggesting a therapeutic potential for this signaling pathway in the treatment of Alzheimer's disease.

The functional links between prion protein and copper.

Prion diseases are fatal neurodegenerative disorders associated with the conversion of the cellular prion protein (PrPC) into a pathologic isoform. Although the physiological function of PrPC remains unknown, evidence relates PrPC to copper metabolism and oxidative stress as suggested by its copper-binding properties in the N-terminal octapeptide repeat region. This region also reduces copper ions in vitro, and this reduction ability is associated with the neuroprotection exerted by the octarepeat region against copper in vivo. In addition, the promoter region of the PrPC gene contains putative metal response elements suggesting it may be regulated by heavy metals. Here we address some of the evidence that support a physiological link between PrPC and copper. Also, in vivo experiments suggesting the physiological relevance of PrPC interaction with heparan sulfate proteoglycans are discussed.

The functional links between prion protein and copper

Prion diseases are fatal neurodegenerative disorders associated with the conversion of the cellular prion protein (PrPC) into a pathologic isoform. Although the physiological function of PrPC remains unknown, evidence relates PrPC to copper metabolism and oxidative stress as suggested by its copper-binding properties in the N-terminal octapeptide repeat region. This region also reduces copper ions in vitro, and this reduction ability is associated with the neuroprotection exerted by the octarepeat region against copper in vivo. In addition, the promoter region of the PrPC gene contains putative metal response elements suggesting it may be regulated by heavy metals. Here we address some of the evidence that support a physiological link between PrPC and copper. Also, in vivo experiments suggesting the physiological relevance of PrPC interaction with heparan sulfate proteoglycans are discussed.

Human-like rodent amyloid-beta-peptide determines Alzheimer pathology in aged wild-type Octodon degu.

It is generally accepted that human Alzheimer's disease (AD) neuropathology markers are completely absent in rodent brains. We report here that an aged wild-type South American rodent, Octodon degu, expresses neuronal beta-amyloid precursor protein (beta-APP695) displaying both intracellular and extracellular deposits of amyloid-beta-peptide (Abeta), intracellular accumulations of tau-protein and ubiquitin, a strong astrocytic response and acetylcholinesterase (AChE)-rich pyramidal neurons. The high amino acid homology (97.5%) between deguAbeta and humanAbeta sequences is probably a major factor in the appearance of AD markers in this aged rodent. Our results indicate that aged O. degu constitutes the first wild-type rodent model for neurodegenerative processes associated to AD.

The N-terminal copper-binding domain of the amyloid precursor protein protects against Cu2+ neurotoxicity in vivo.

The amyloid precursor protein (APP) contains a Cu binding domain (CuBD) localized between amino acids 135 and 156 (APP135-156), which can reduce Cu2+ to Cu1+ in vitro. The physiological function of this APP domain has not yet being established; nevertheless several studies support the notion that the CuBD of APP is involved in Cu homeostasis. We used APP synthetic peptides to evaluate their protective properties against Cu2+ neurotoxicity in a bilateral intra-hippocampal injection model. We found that human APP135-156 protects against Cu2+-induced neurotoxic effects, such as, impairment of spatial memory, neuronal cell loss, and astrogliosis. APP135-156 lacking two histidine residues showed protection against Cu2+; however, APP135-156 mutated in cysteine 144, a key residue in the reduction of Cu2+ to Cu1+, did not protect against Cu2+ neurotoxicity. In accordance with recent reports, the CuBD of the Caenorhabditis elegans, APL-1, protected against Cu2+ neurotoxicity in vivo. We also found that Cu2+ neurotoxicity is associated with an increase in nitrotyrosine immunofluorescence as well as with a decrease in Cu2+ uptake. The CuBD of APP therefore may play a role in the detoxification of brain Cu.

Acetylcholinesterase-Abeta complexes are more toxic than Abeta fibrils in rat hippocampus: effect on rat beta-amyloid aggregation, laminin expression, reactive astrocytosis, and neuronal cell loss.

Neuropathological changes generated by human amyloid-beta peptide (Abeta) fibrils and Abeta-acetylcholinesterase (Abeta-AChE) complexes were compared in rat hippocampus in vivo. Results showed that Abeta-AChE complexes trigger a more dramatic response in situ than Abeta fibrils alone as characterized by the following features observed 8 weeks after treatment: 1). amyloid deposits were larger than those produced in the absence of AChE. In fact, AChE strongly stimulates rat Abeta aggregation in vitro as shown by turbidity measurements, Congo Red binding, as well as electron microscopy, suggesting that Abeta-AChE deposits observed in vivo probably recruited endogenous Abeta peptide; 2). the appearance of laminin expressing neurons surrounding Abeta-AChE deposits (such deposits are resistant to disaggregation by laminin in vitro); 3). an extensive astrocytosis revealed by both glial fibrillary acidic protein immunoreactivity and number counting of reactive hypertrophic astrocytes; and 4). a stronger neuronal cell loss in comparison with Abeta-injected animals. We conclude that the hippocampal injection of Abeta-AChE complexes results in the appearance of some features reminiscent of Alzheimer-like lesions in rat brain. Our studies are consistent with the notion that Abeta-AChE complexes are more toxic than Abeta fibrils and that AChE triggered some of the neurodegenerative changes observed in Alzheimer's disease brains.

Acetylcholinesterase induces neuronal cell loss, astrocyte hypertrophy and behavioral deficits in mammalian hippocampus.

Previous studies have demonstrated that acetylcholinesterase (AChE) promotes the assembly of amyloid-beta-peptides into neurotoxic amyloid fibrils and is toxic for chick retina neuronal cultures and neuroblastoma cells. Moreover, AChE is present in senile plaques in Alzheimer's disease (AD) brains. Here we have studied the effect of AChE on astrocytes and hippocampal neurons in vivo. Morphological as well as behavioral disturbances were analyzed after intrahippocampal injection of AChE. Rats were trained in the Morris water maze and assayed for behavioral parameters. Neuronal cell loss was found in the upper leaf of the dentate gyrus in rats injected with AChE in comparison with control animals. Glial fibrillary acidic protein immunoreactivity showed astrocytic hypertrophy and the magnitude of the response was associated with neuronal cell loss. Behavioral results show that injection of AChE produces cognitive impairment demonstrated by an altered water maze performance including (i) a higher escape latency score, (ii) a decreased spatial acuity and (iii) a shorter time of swimming in the platform quadrant. These findings indicate that a local increment in neuronal AChE concentration at the mammalian hippocampus, such as those present in amyloid deposits, may play a role in triggering neuropathological and behavioral changes such as those observed in AD brains.

The human prion octarepeat fragment prevents and reverses the inhibitory action of copper in the P2X4 receptor without modifying the zinc action.

Human prion protein fragments (PrP60-67 or PrP59-91) prevented and reversed the inhibition elicited by 5 micro m copper on the P2X4 receptor expressed in Xenopus laevis oocytes. A 60-s pre-application of 5 micro m copper caused a 69.2 +/- 2.6% inhibition of the 10 micro m adenosine triphosphate (ATP)-evoked currents, an effect that was prevented by mixing 5 micro m copper with 0.01-10 micro m of the PrP fragments 1-min prior to application. This interaction was selective, as PrP59-91 did not alter the facilitatory action of zinc. The EC50 of PrP60-67 and PrP59-91 for the reduction of the copper inhibition were 4.6 +/- 1 and 1.3 +/- 0.4 micro m, respectively. A synthetic PrP59-91 variant in which all four His were replaced by Ala was inactive. However, the replacement of Trp in each of the four putative copper-binding domains by Ala slightly decreased its potency. Furthermore, the application of 10 micro m PrP59-91 reversed the copper-evoked inhibition, restoring the ATP concentration curve to the same level as the non-inhibited state. Fragment 139-157 of betaA4 amyloid precursor protein also prevented the action of copper; its EC50 was 1.6 +/- 0.1 micro m; the metal chelator penicillamine was equipotent with PrP60-67, but carnosine was significantly less potent. Our findings highlight the role of PrP in copper homeostasis and hint at its possible role as a modulator of synapses regulated by this trace metal.

Wnt signaling involvement in beta-amyloid-dependent neurodegeneration.

Alzheimer's disease (AD) is a progressive dementia paralleled by selective neuronal death, which is probably caused by the cytotoxic effects of the amyloid-beta peptide (Abeta). We have observed that Abeta-dependent neurotoxicity induces a loss of function of Wnt signaling components and that activation of this signaling cascade prevent such cytotoxic effects. Therefore we propose that compounds which mimic this signaling cascade may be candidates for therapeutic intervention in Alzheimer's patients.

Hidroxiapatita microgranular: comportamento biológico em tecido subcutâneo de ratos / Hitologic evaluation of tissue response to microparticles of hydroxyapatite subcutaneously implanted in rats

Micropartículas ultrafinas de hidroxiapatita sintética foram implantadas na regiäo subcutânea de ratos. Os animais foram sacrificados nos intervalos de tempo de 3 e 24 horas e 7, 21, 30 e 60 dias após o implante, sendo os fragmentos teciduais incluídos em parafina e analisados pela microscopia óptica. A resposta tecidual induzida pela HA foi caracterizada, a partir do 7§ dia, por uma inflamaçäo crônica granulomatosa exuberante, com as células inflamatórias envolvendo as partículas. Neste trabalho näo se observou nenhuma resposta imunológica exacerbada, o que reforça a biocompatibilidade do material