Fabrication of Functional Cell Sheets with Human Thyrocytes from Non-Tumorous Thyroid Tissue

BACKGROUND: Engineered cell sheet transplantation has been considered an alternative physiological therapy for endocrine disorders. In this study, we attempted to fabricate functional human thyroid cell sheets using the engineering technology by culturing primary thyrocytes in free-feeder monolayers and assessed their proliferation and function in two different media. METHODS: The non-tumorous tissues (approximately 2 g) were dissected during surgery. Primary human thyroid cells were isolated by mechanical dispersion and treatment with isolation solution. The cells were cultured on tissue culture dishes or temperature-responsive culture dishes to induce the formation of detached cell sheets. RESULTS: Primary thyroid cells isolated from nine patients were positive for thyroid transcription factor 1, thyroglobulin (TG) and cytokeratin 7. Cell sheets with follicles were fabricated by cells incubated in both Dulbecco's Modified Eagle Medium (DMEM) and hepatocyte-defined medium (HDM) culture medium. The diameter and thickness of sheets fabricated in HDM were larger and thicker than those fabricated from DMEM. Furthermore, the cells incubated in HDM secreted higher levels of fT3 and fT4 than those incubated in DMEM. The thyroid peroxidase and TG mRNA of cells maintained in HDM were higher than those in cells maintained in DMEM. CONCLUSION: HDM appears suitable as a culture medium for maintaining primary thyrocytes and fabricating functional cell sheets. These in vitro findings may contribute to the development of appropriate culture conditions for human thyrocytes as well as engineered functional cell sheets.

Optimization of Microenvironments Inducing Differentiation of Tonsil-Derived Mesenchymal Stem Cells into Endothelial Cell-Like Cells

BACKGROUND: Stem cell engineering is appealing consideration for regenerating damaged endothelial cells (ECs) because stem cells can differentiate into EC-like cells. In this study, we demonstrate that tonsil-derived mesenchymal stem cells (TMSCs) can differentiate into EC-like cells under optimal physiochemical microenvironments.METHODS: TMSCs were preconditioned with Dulbecco's Modified Eagle Medium (DMEM) or EC growth medium (EGM) for 4 days and then replating them on Matrigel to observe the formation of a capillary-like network under light microscope. Microarray, quantitative real time polymerase chain reaction, Western blotting and immunofluorescence analyses were used to evaluate the expression of gene and protein of EC-related markers.RESULTS: Preconditioning TMSCs in EGM for 4 days and then replating them on Matrigel induced the formation of a capillary-like network in 3 h, but TMSCs preconditioned with DMEM did not form such a network. Genome analyses confirmed that EGM preconditioning significantly affected the expression of genes related to angiogenesis, blood vessel morphogenesis and development, and vascular development. Western blot analyses revealed that EGM preconditioning with gelatin coating induced the expression of endothelial nitric oxide synthase (eNOS), a mature EC-specific marker, as well as phosphorylated Akt at serine 473, a signaling molecule related to eNOS activation. Gelatin-coating during EGM preconditioning further enhanced the stability of the capillary-like network, and also resulted in the network more closely resembled to those observed in human umbilical vein endothelial cells.CONCLUSION: This study suggests that under specific conditions, i.e., EGM preconditioning with gelatin coating for 4 days followed by Matrigel, TMSCs could be a source of generating endothelial cells for treating vascular dysfunction.

Influence of voxel size on cone-beam computed tomography-based detection of vertical root fractures in the presence of intracanal metallic posts

PURPOSE: This study was performed to evaluate the influence of voxel size and the accuracy of 2 cone-beam computed tomography (CBCT) systems in the detection of vertical root fracture (VRF) in the presence of intracanal metallic posts. MATERIALS AND METHODS: Thirty uniradicular extracted human teeth were selected and randomly divided into 2 groups (VRF group, n=15; and control group, n=15). The VRFs were induced by an Instron machine, and metallic posts were placed in both groups. The scans were acquired by CBCT with 4 different voxel sizes: 0.1 mm and 0.16 mm (for the Eagle 3D V-Beam system) and 0.125 mm and 0.2 mm (for the i-CAT system) (protocols 1, 2, 3, and 4, respectively). Interobserver and intraobserver agreement was assessed using the Cohen kappa test. Sensitivity and specificity were evaluated and receiver operating characteristic analysis was performed. RESULTS: The intraobserver coefficients indicated good (0.71) to very good (0.83) agreement, and the interobserver coefficients indicated moderate (0.57) to very good (0.80) agreement. In respect to the relationship between sensitivity and specificity, a statistically significant difference was found between protocols 1 (positive predictive value: 0.710, negative predictive value: 0.724) and 3 (positive predictive value: 0.727, negative predictive value: 0.632) (P < .05). The least interference due to artifact formation was observed using protocol 2. CONCLUSION: Protocols with a smaller voxel size and field of view seemed to favor the detection of VRF in teeth with intracanal metallic posts.

Fractured styloid process masquerading as neck pain: Cone-beam computed tomography investigation and review of the literature

Historically, Eagle syndrome is a term that has been used to describe radiating pain in the orofacial region, foreign body sensation, and/or dysphagia due to a unilateral or bilateral elongated styloid process impinging upon the tonsillar region. Because elongated styloid processes–with or without associated Eagle syndrome–can present with various symptoms and radiographic findings, it can be challenging for healthcare practitioners to formulate an accurate diagnosis. Abnormal styloid anatomy can lead to a multitude of symptoms, including chronic orofacial/neck pain, thus masquerading as more commonly diagnosed conditions. In this report, we describe a patient who presented to our department with styloid process elongation and fracture. A careful history, physical examination, and a conebeam computed tomography (CBCT) investigation led to the diagnosis. The patient was then referred for appropriate care. This case report demonstrates the utilization of CBCT in differentiating a fracture site from a pseudo-joint that might mimic a fracture.

β-arrestin2 Affects Cardiac Progenitor Cell Survival through Cell Mobility and Tube Formation in Severe Hypoxia

BACKGROUND AND OBJECTIVES: β-arrestin2 (β-arr2) basically regulates multiple signaling pathways in mammalian cells by desensitization and internalization of G-protein coupled receptors (GPCRs). We investigated impacts of β-arr2 on survival, mobility, and tube formation of cardiac progenitor cells (CPCs) obtained from wild-type (WT) mouse (CPC-WT), and β-arr2 knock-out (KO) mouse (CPC-KO) cultured in presence or absence of serum and oxygen as non-canonical roles in GPCR system. METHODS: CPCs were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 -based media containing fetal bovine serum and growth factors. Survival of 2 types of CPCs in hypoxia and/or serum deprivation was measured by fluorescence-activated cell sorting. Wound healing ability, and tube formation ability on Matrigel of 2 kinds of CPCs were compared in normoxic and hypoxic cultures. Protein expression related to survival and mobility were measured with the Western blot for each culture conditions. RESULT: CPC-KO showed significantly worse mobility in the wound healing assay and in tube formation on Matrigel especially in hypoxic culture than did the CPC-WT. Also, CPC-KO showed significantly higher apoptosis fraction in both normoxic and hypoxic cultures than did the CPC-WT. Expression of proteins associated with cell survival and mobility, e.g., protein kinase B (Akt), β-catenin, and glycogen synthase kinase-3β (GSK-3β) was significantly worse in CPC-KO. CONCLUSIONS: The CPC-KO had significantly worse cell mobility, tube formation ability, and survival than the CPC-WT, especially in the hypoxic cultures. Apparently, β-arr2 is important on CPC survival by means of mobility and tube formation in myocardial ischemia.

Feeder Cells Free Rabbit Oral Mucosa Epithelial Cell Sheet Engineering

The optimal cell culture method of autologous oral mucosal epithelial cell sheet is not well established for a safe transplantation on to the patients' ocular surface. Animal serum and 3T3 mouse feeder cells are currently being used to stimulate the growth of the epithelial cells. However, the use of animal compounds can have potential side effects for the patient after transplantation of the engineered cell sheet. In the present study, we focused on engineering a rabbit oral mucosal epithelial cell sheet without 3T3 mouse feeder cells using a mix of Dulbecco's Modified Eagle Medium/Bronchial Epithelial Cell Growth Medium culture media (DMEM/BEGM). Autologous oral mucosal epithelial cell sheets, engineered with DMEM/BEGM feeder cell free culture media, were compared to those cultured in presence of serum and feeder cells. Using a DMEM/BEGM mix culture media, feeder cell free culture condition, autologous oral mucosal epithelial cells reached confluence and formed a multilayered sheet. The phenotype of engineered cell sheets cultured with DMEM/BEGM were characterized and compared to those cultured with serum and feeder. Hematoxylin and eosin staining showed the formation of a similar stratified multilayer cell sheets, in both culture conditions. The expression of deltaN-p63, ABCG2, PCNA, E-cadherin, Beta-catenin, CK3, CK4, CK13, Muc5AC, was similar in both culture conditions. We demonstrated that rabbit autologous oral mucosal epithelial cell sheet can be engineered, in feeder cell free conditions. The use of the DMEM/BEGM culture media to engineer culture autologous oral mucosa epithelial cell sheet will help to identify key factors involved in the growth and differentiation of oral mucosal epithelial cells.

Repeated Glucose Deprivation/Reperfusion Induced PC-12 Cell Death through the Involvement of FOXO Transcription Factor

BACKGROUND: Cognitive impairment and brain damage in diabetes is suggested to be associated with hypoglycemia. The mechanisms of hypoglycemia-induced neural death and apoptosis are not clear and reperfusion injury may be involved. Recent studies show that glucose deprivation/reperfusion induced more neuronal cell death than glucose deprivation itself. The forkhead box O (FOXO) transcription factors are implicated in the regulation of cell apoptosis and survival, but their role in neuronal cells remains unclear. We examined the role of FOXO transcription factors and the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt and apoptosis-related signaling pathways in PC-12 cells exposed to repeated glucose deprivation/reperfusion. METHODS: PC-12 cells were exposed to control (Dulbecco's Modified Eagle Medium [DMEM] containing 25 mM glucose) or glucose deprivation/reperfusion (DMEM with 0 mM glucose for 6 hours and then DMEM with 25 mM glucose for 18 hours) for 5 days. MTT assay and Western blot analysis were performed for cell viability, apoptosis, and the expression of survival signaling pathways. FOXO3/4',6-diamidino-2-phenylindole staining was done to ascertain the involvement of FOXO transcription factors in glucose deprivation/reperfusion conditions. RESULTS: Compared to PC-12 cells not exposed to hypoglycemia, cells exposed to glucose deprivation/reperfusion showed a reduction of cell viability, decreased expression of phosphorylated Akt and Bcl-2, and an increase of cleaved caspase-3 expression. Of note, FOXO3 protein was localized in the nuclei of glucose deprivation/reperfusion cells but not in the control cells. CONCLUSION: Repeated glucose deprivation/reperfusion caused the neuronal cell death. Activated FOXO3 via the PI3K/Akt pathway in repeated glucose deprivation/reperfusion was involved in genes related to apoptosis.

Resource availability and diet in Harpy Eagle breeding territories on the Xingu River, Brazilian Amazon / Disponibilidade e consumo de presas por harpia no entorno de territórios de nidificação no rio Xingu, Amazônia Oriental Brasileira

Abstract In the Tapajos-Xingu interfluve, one of the largest birds of prey, the Harpy Eagle, is under intense anthropogenic pressure due to historical and recent reductions in forest cover. We studied prey availability and use by Harpy Eagle on six breeding territories on the low- and mid-Xingu River, between 2013 and 2015. We evaluated food resource availability using the environmental-surveys database from two methods: terrestrial surveys (RAPELD method) and fauna rescue/flushing before vegetation suppression for the Belo Monte Hydroelectric Complex construction. Harpy Eagle diet was identified by prey remains sampled around six nest trees. Eighteen species of mammals, birds and reptiles comprised the prey items. Most prey species were sloths, primates and porcupines, which have arboreal habits and are found in forested areas, but two species, hoatzin and iguana, are usually associated with riverine habitats. The proportion of prey from each species predated on the nest best studied was different from estimated availability (χ2 = 54.23; df = 16; p < 0.001), however there was a positive correlation (rs = 0.7; p < 0.01) between prey species consumed and abundance available, where the predation was more on species more abundant. Continuous monitoring of the Harpy Eagle diet at these nests could evidence changes in the assemblage of prey species available for Harpy Eagles, due to changes in the seasonal flood pulse of the Xingu River to be caused by the operation of the hydroelectric dam, and changes in habitat features by forest reduction around breeding territories. We believe that it is important to consider the protection of remnants of forested areas in the landscape matrix surrounding the breeding territories to maintain the food resource availability and allow all pairs to successfully reproduce.

Resumo O interflúvio Tapajós-Xingu, um dos centros de endemismo da Amazônia, onde habita uma das mais poderosas aves de rapina, a harpia, é também alvo de intensa pressão antrópica com reduções históricas da cobertura florestal. Analisamos a disponibilidade de vertebrados e o consumo de presas por harpia em seis territórios de reprodução no Rio Xingu, entre 2013 e 2015. A disponibilidade de recursos alimentares foi baseada nos dados de amostragens por dois métodos: levantamentos terrestres pelo método RAPELD e resgate/afugentamento da fauna antes da supressão da vegetação para construção de infra-estrutura para o Complexo Hidrelétrico Belo Monte. As espécies de presas consumidas pela harpia foram identificadas a partir de vestígios coletados em seis ninhos. Dezoito espécies de mamíferos, aves e répteis compuseram a dieta no baixo e médio Rio Xingu, com maior proporção de preguiças, macacos e porcos-espinhos, incluindo duas espécies (Opisthocomus hoazin e Iguana iguana) associadas aos hábitats criados por rios. A proporção de indivíduos das espécies predadas no ninho com maior número de coletas foi diferente da disponibilidade (χ2 = 54,23; gl = 16; p < 0,001), no entanto, existe correlação positiva (rs = 0,7; p < 0,01) entre o consumo de presas e a abundância disponível, com a predação maior sobre espécies mais abundantes. A riqueza de presas indica que as florestas remanescentes na região foram fonte eficiente de recursos alimentares para a reprodução e manutenção da harpia. O monitoramento da dieta da harpia nos ninhos pode apresentar alterações relacionadas às mudanças no pulso sazonal de inundação do Xingu, provocadas pelo futuro funcionamento da usina hidrelétrica e à redução da floresta no entorno dos ninhos localizados no Reservatório Xingu e Intermediário. É importante considerar a proteção dos remanescentes florestais no entorno dos territórios de nidificação para garantir a disponibilidade de presas e a reprodução efetiva dos casais de harpia na região.

Abundance of Harpy and Crested Eagles from a reservoir-impact area in the Low- and Mid-Xingu River / Abundância de Gavião-real e Gavião-real falso numa área sob impacto de reservatório no Baixo e Médio rio Xingu

Abstract In the Brazilian Amazon, two monospecific genera, the Harpy Eagle and Crested Eagle have low densities and are classified by IUCN as Near Threatened due to habitat loss, deforestation, habitat degradation and hunting. In this study, we evaluate occurrence of these large raptors using the environmental surveys database from Belo Monte Hydroelectric Power Plant. Integrating the dataset from two methods, we plotted a distribution map along the Xingu River, including records over a 276-km stretch of river. Terrestrial surveys (RAPELD method) were more efficient for detecting large raptors than standardized aquatic surveys, although the latter were complementary in areas without modules. About 53% of the records were obtained during activities of wildlife rescue/flushing, vegetation suppression or in transit. Between 2012 and 2014, four Harpy Eagles were removed from the wild; two shooting victims, one injured by collision with power lines and one hit by a vehicle. Also, seven nests were mapped. The mean distance between Harpy Eagle records was 15 km along the river channel, with a mean of 20 km between nests near the channel, which allowed us to estimate 20 possible pairs using the alluvial forest, riverine forest and forest fragments. Territories of another ten pairs will probably be affected by inundation of the Volta Grande channel, which is far from the main river. The average distance between Crested Eagle records was 16 km along the river channel. The only nest found was 1.3 km away from a Harpy Eagle nest. The remnant forests are under threat of being replaced by cattle pastures, so we recommend that permanently protected riparian vegetation borders (APP) be guaranteed, and that forest fragments within 5 km of the river be conserved to maintain eagle populations.

Resumo Na Amazônia brasileira dois gêneros mono-específicos, Harpia e Morphnus, caracterizam-se por baixa densidade e estão classificados pelo IUCN como Quase Ameaçados, porém ocorrem sobre grande parte do território nacional, suas principais ameaças são a fragmentação florestal, a degradação de hábitat e a caça. Neste estudo avaliamos a abundância destas duas grandes aves de rapina utilizando a base de dados dos programas ambientais da UHE Belo Monte, integrando-se dois métodos para construir um mapa de distribuição ao longo de 245 km do rio Xingu. Os levantamentos terrestres pelo método RAPELD mostraram-se mais eficientes para os registros de grandes águias quando comparado aos esforços padronizados aquáticos, entretanto estes foram complementares na ausência de módulos. Cinquenta e tres por cento foram registros ocasionais durante a supressão da vegetação, afugentamento ou deslocamento. Entre 2012-2014 quatro harpias foram removidas da natureza, dois indivíduos alvo de disparos, uma por colisão com rede elétrica, e outra por atropelamento. Sete ninhos mapeados, a distância média entre os registros de harpia na calha e margens do rio foi de 15 km, 20 km distância média entre ninhos, o que permitiu estimar um total de 20 casais utilizando as florestas aluviais em uma distancia de 270 km, incluindo matas ciliares e os fragmentos fora da margem do rio. Estimamos que territórios de outros 10 casais usando a Volta Grande longe do rio principal também serão afetados pela inundação. A distância média entre os registros de Morphnus foi 16 km ao longo do rio, o único ninho mapeado estava distante 1.3 km do ninho de Harpia. Estes fragmentos florestais estão sendo substituídos por pastagens, ressaltando-se a importância da manutenção das áreas de preservação permanentes (APP) e a proteção destes fragmentos em diversos formatos de áreas de proteção, para diminuir a degradação dos mesmos e garantir a manutenção das populações destes grandes predadores na região do rio Xingu.

Abundance of Harpy and Crested Eagles from a reservoir-impact area in the Low- and Mid-Xingu River / Abundância de Gavião-real e Gavião-real falso numa área sob impacto de reservatório no Baixo e Médio rio Xingu

In the Brazilian Amazon, two monospecific genera, the Harpy Eagle and Crested Eagle have low densities and are classified by IUCN as Near Threatened due to habitat loss, deforestation, habitat degradation and hunting. In this study, we evaluate occurrence of these large raptors using the environmental surveys database from Belo Monte Hydroelectric Power Plant. Integrating the dataset from two methods, we plotted a distribution map along the Xingu River, including records over a 276-km stretch of river. Terrestrial surveys (RAPELD method) were more efficient for detecting large raptors than standardized aquatic surveys, although the latter were complementary in areas without modules. About 53% of the records were obtained during activities of wildlife rescue/flushing, vegetation suppression or in transit. Between 2012 and 2014, four Harpy Eagles were removed from the wild; two shooting victims, one injured by collision with power lines and one hit by a vehicle. Also, seven nests were mapped. The mean distance between Harpy Eagle records was 15 km along the river channel, with a mean of 20 km between nests near the channel, which allowed us to estimate 20 possible pairs using the alluvial forest, riverine forest and forest fragments. Territories of another ten pairs will probably be affected by inundation of the Volta Grande channel, which is far from the main river. The average distance between Crested Eagle records was 16 km along the river channel. The only nest found was 1.3 km away from a Harpy Eagle nest. The remnant forests are under threat of being replaced by cattle pastures, so we recommend that permanently protected riparian vegetation borders (APP) be guaranteed, and that forest fragments within 5 km of the river be conserved to maintain eagle populations.

Na Amazônia brasileira dois gêneros mono-específicos, Harpia e Morphnus, caracterizam-se por baixa densidade e estão classificados pelo IUCN como Quase Ameaçados, porém ocorrem sobre grande parte do território nacional, suas principais ameaças são a fragmentação florestal, a degradação de hábitat e a caça. Neste estudo avaliamos a abundância destas duas grandes aves de rapina utilizando a base de dados dos programas ambientais da UHE Belo Monte, integrando-se dois métodos para construir um mapa de distribuição ao longo de 245 km do rio Xingu. Os levantamentos terrestres pelo método RAPELD mostraram-se mais eficientes para os registros de grandes águias quando comparado aos esforços padronizados aquáticos, entretanto estes foram complementares na ausência de módulos. Cinquenta e tres por cento foram registros ocasionais durante a supressão da vegetação, afugentamento ou deslocamento. Entre 2012-2014 quatro harpias foram removidas da natureza, dois indivíduos alvo de disparos, uma por colisão com rede elétrica, e outra por atropelamento. Sete ninhos mapeados, a distância média entre os registros de harpia na calha e margens do rio foi de 15 km, 20 km distância média entre ninhos, o que permitiu estimar um total de 20 casais utilizando as florestas aluviais em uma distancia de 270 km, incluindo matas ciliares e os fragmentos fora da margem do rio. Estimamos que territórios de outros 10 casais usando a Volta Grande longe do rio principal também serão afetados pela inundação. A distância média entre os registros de Morphnus foi 16 km ao longo do rio, o único ninho mapeado estava distante 1.3 km do ninho de Harpia. Estes fragmentos florestais estão sendo substituídos por pastagens, ressaltando-se a importância da manutenção das áreas de preservação permanentes (APP) e a proteção destes fragmentos em diversos formatos de áreas de proteção, para diminuir a degradação dos mesmos e garantir a manutenção das populações destes grandes predadores na região do rio Xingu.

Resource availability and diet in Harpy Eagle breeding territories on the Xingu River, Brazilian Amazon / Disponibilidade e consumo de presas por harpia no entorno de territórios de nidificação no rio Xingu, Amazônia Oriental Brasileira

In the Tapajos-Xingu interfluve, one of the largest birds of prey, the Harpy Eagle, is under intense anthropogenic pressure due to historical and recent reductions in forest cover. We studied prey availability and use by Harpy Eagle on six breeding territories on the low- and mid-Xingu River, between 2013 and 2015. We evaluated food resource availability using the environmental-surveys database from two methods: terrestrial surveys (RAPELD method) and fauna rescue/flushing before vegetation suppression for the Belo Monte Hydroelectric Complex construction. Harpy Eagle diet was identified by prey remains sampled around six nest trees. Eighteen species of mammals, birds and reptiles comprised the prey items. Most prey species were sloths, primates and porcupines, which have arboreal habits and are found in forested areas, but two species, hoatzin and iguana, are usually associated with riverine habitats. The proportion of prey from each species predated on the nest best studied was different from estimated availability (χ2 = 54.23; df = 16; p < 0.001), however there was a positive correlation (rs = 0.7; p < 0.01) between prey species consumed and abundance available, where the predation was more on species more abundant. Continuous monitoring of the Harpy Eagle diet at these nests could evidence changes in the assemblage of prey species available for Harpy Eagles, due to changes in the seasonal flood pulse of the Xingu River to be caused by the operation of the hydroelectric dam, and changes in habitat features by forest reduction around breeding territories. We believe that it is important to consider the protection of remnants of forested areas in the landscape matrix surrounding the breeding territories to maintain the food resource availability and allow all pairs to successfully reproduce.

RO interflúvio Tapajós-Xingu, um dos centros de endemismo da Amazônia, onde habita uma das mais poderosas aves de rapina, a harpia, é também alvo de intensa pressão antrópica com reduções históricas da cobertura florestal. Analisamos a disponibilidade de vertebrados e o consumo de presas por harpia em seis territórios de reprodução no Rio Xingu, entre 2013 e 2015. A disponibilidade de recursos alimentares foi baseada nos dados de amostragens por dois métodos: levantamentos terrestres pelo método RAPELD e resgate/afugentamento da fauna antes da supressão da vegetação para construção de infra-estrutura para o Complexo Hidrelétrico Belo Monte. As espécies de presas consumidas pela harpia foram identificadas a partir de vestígios coletados em seis ninhos. Dezoito espécies de mamíferos, aves e répteis compuseram a dieta no baixo e médio Rio Xingu, com maior proporção de preguiças, macacos e porcos-espinhos, incluindo duas espécies (Opisthocomus hoazin e Iguana iguana) associadas aos hábitats criados por rios. A proporção de indivíduos das espécies predadas no ninho com maior número de coletas foi diferente da disponibilidade (χ2 = 54,23; gl = 16; p < 0,001), no entanto, existe correlação positiva (rs = 0,7; p < 0,01) entre o consumo de presas e a abundância disponível, com a predação maior sobre espécies mais abundantes. A riqueza de presas indica que as florestas remanescentes na região foram fonte eficiente de recursos alimentares para a reprodução e manutenção da harpia. O monitoramento da dieta da harpia nos ninhos pode apresentar alterações relacionadas às mudanças no pulso sazonal de inundação do Xingu, provocadas pelo futuro funcionamento da usina hidrelétrica e à redução da floresta no entorno dos ninhos localizados no Reservatório Xingu e Intermediário. É importante considerar a proteção dos remanescentes florestais no entorno dos territórios de nidificação para garantir a disponibilidade de presas e a reprodução efetiva dos casais de harpia na região.

Nonsurgical treatment of stylohyoid (Eagle) syndrome: a case report

Eagle syndrome is a rare condition caused by elongation of the styloid process or calcification of the stylohyoid ligament. Patients with Eagle syndrome typically present with dysphagia, dysphonia, cough, voice changes, otalgia, sore throat, facial pain, foreign body sensation, headache, vertigo, and neck pain. Here we report a case in which the patient initially presented with sore throat, left-sided facial pain, and cough. This case report provides a brief review of the diagnosis and nonsurgical management of this rare syndrome.

Eagle's syndrome: a case report

Eagle's syndrome is a disease caused by an elongated styloid process or calcified stylohyoid ligament. Eagle defined the disorder in 1937 by describing clinical findings related to an elongated styloid process, which is one of the numerous causes of pain in the craniofacial and cervical region. The prevalence of individuals with this anatomic abnormality in the adult population is estimated to be 4% with 0.16% of these individuals reported to be symptomatic. Eagle's syndrome is usually characterized by neck, throat, or ear pain; pharyngeal foreign body sensation; dysphagia; pain upon head movement; and headache. The diagnosis of Eagle's syndrome must be made in association with data from the clinical history, physical examination, and imaging studies. Patients with increased symptom severity require surgical excision of the styloid process, which can be performed through an intraoral or an extraoral approach. Here, we report a rare case of stylohyoid ligament bilaterally elongated to more than 60 mm in a 51-year-old female. We did a surgery by extraoral approach and patient's symptom was improved.

The influence of serum substituents on serum-free Vero cell conditioned culture media manufactured from Dulbecco's modified Eagle medium in mouse embryo culture

OBJECTIVE: This study was conducted to examine the influences of supplementation of the serum substituents and available period of serum-free Vero cell conditioned media (SF-VCM) manufactured from Dulbecco's modified Eagle medium cultured with Vero cells for in vitro development of mouse preimplantation embryos. METHODS: A total of 1,099 two-cell embryos collected from imprinting control region mice were cultured in SF-VCM with 10% and 20% human follicular fluid (hFF), serum substitute supplement (SSS), and serum protein substitute (SPS). Development of embryos was observed every 24 hours. Results between different groups were analyzed by chi-square test, and considered statistically significant when P-value was less than 0.05. RESULTS: The rates of embryonic development cultured in SF-VCM supplemented with serum substituents were significantly higher compare with serum-free group (P < 0.05). The rates of embryonic development after 48 hours (morula< or =) and 96 hours (blastocyst< or =) were significantly higher in 20% SSS and 10% SPS than in 20% hFF supplementation (P < 0.05). And the rates of embryonic development after 96 hours (hatching blastocyst< or =) were significantly higher in 10% SPS (94.5%) than in 20% SSS (82.6%) and 20% hFF supplementation (68.5%). The rates of embryonic development according to storage period of the SF-VCM supplemented with 10% SPS showed no significant difference between control, 2 weeks and 4 weeks group. However developmental rate in 6 weeks storage group was significantly lower than other groups. CONCLUSION: The rate of embryonic development after 96 hours (hatching blastocyst< or =) was significantly higher in SF-VCM supplemented with 10% SPS. And storage period of media up to 4 weeks did not affect on embryonic development.

A unique case of Turner syndrome accompanying prolactinoma and unexpected elongated styloid process: Clinical and cone-beam computed tomographic features

Turner syndrome (TS) is one of the most common chromosomal abnormalities, with an estimated frequency among female live births of 1/2,000-3,000. The syndrome is characterized by the partial or complete absence of one X chromosome (45,X karyotype). We reported a unique case of a 40-year-old woman with TS accompanying unexpected elongated styloid process specific to Eagle syndrome (ES) and followed up-prolactinoma. The present article is the first report to define the cone-beam computed tomographic (CBCT) features of TS accompanying ES. Patients with TS carry various risks that make treatment more complicated; thus advanced imaging techniques for proper treatment and follow-up are extremely important. In the light of CBCT examination, craniofacial abnormalities specific to TS and accompanying syndromes such as the crowding of teeth especially in the maxillary anterior region caused by maxillary narrowness, micrognatic maxilla and mandible, relative mandibular retrusion, malocclusion, open-bite, and an elongated styloid process (length of 32.7 mm) on the right side were illustrated in detail.

Clinicoradiologic evaluation of styloid process calcification

PURPOSE: This study was performed to investigate the prevalence, morphology, and calcification pattern of the elongated styloid process in the Mathura population and its relation to gender, age, and mandibular movements. MATERIALS AND METHODS: The study analyzed digital panoramic radiographs of 2,706 adults. The elongated styloid process was classified with the radiographic appearance based on the morphology and calcification pattern. The limits of mandibular protrusion were evaluated for each subject. The data were analyzed by using a Student's t-test and chi-squared test with significance set at p=0.05. RESULTS: Bilateral elongation having an "elongated" type styloid process with a "partially mineralized" pattern was the most frequent type of styloid process. No correlation was found between styloid process type and calcification pattern on the one hand and gender on the other, although elongated styloid was more prevalent in older and male populations (p0.05). CONCLUSION: Dentists should recognize the existence of morphological variation in elongated styloid process or Eagle syndrome apparent on panoramic radiographs. We found higher prevalence of elongated styloid process in the population of the Mathura region when compared with other Indian populations. The calcification of the styloid process was more common in the older age group with no correlation to gender, mandibular movement and site. "Type I" with a "partially calcified" styloid process was observed more frequently in the population studied.

Isolation of human mesenchymal stem cells from the skin and their neurogenic differentiation in vitro

OBJECTIVES: This aim of this study was to effectively isolate mesenchymal stem cells (hSMSCs) from human submandibular skin tissues (termed hSMSCs) and evaluate their characteristics. These hSMSCs were then chemically induced to the neuronal lineage and analyzed for their neurogenic characteristics in vitro. MATERIALS AND METHODS: Submandibular skin tissues were harvested from four adult patients and cultured in stem cell media. Isolated hSMSCs were evaluated for their multipotency and other stem cell characteristics. These cells were differentiated into neuronal cells with a chemical induction protocol. During the neuronal induction of hSMSCs, morphological changes and the expression of neuron-specific proteins (by fluorescence-activated cell sorting [FACS]) were evaluated. RESULTS: The hSMSCs showed plate-adherence, fibroblast-like growth, expression of the stem-cell transcription factors Oct 4 and Nanog, and positive staining for mesenchymal stem cell (MSC) marker proteins (CD29, CD44, CD90, CD105, and vimentin) and a neural precursor marker (nestin). Moreover, the hSMSCs in this study were successfully differentiated into multiple mesenchymal lineages, including osteocytes, adipocytes, and chondrocytes. Neuron-like cell morphology and various neural markers were highly visible six hours after the neuronal induction of hSMSCs, but their neuron-like characteristics disappeared over time (24-48 hrs). Interestingly, when the chemical induction medium was changed to Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS), the differentiated cells returned to their hSMSC morphology, and their cell number increased. These results indicate that chemically induced neuron-like cells should not be considered true nerve cells. CONCLUSION: Isolated hSMSCs have MSC characteristics and express a neural precursor marker, suggesting that human skin is a source of stem cells. However, the in vitro chemical neuronal induction of hSMSC does not produce long-lasting nerve cells and more studies are required before their use in nerve-tissue transplants.

Genomic resources for the conservation and management of the harpy eagle (Harpia harpyja, Falconiformes, Accipitridae)

We report the characterization and optimization of 45 heterologous microsatellite loci, and the development of a new set of molecular sex markers for the conservation and management of the Neotropical harpy eagle (Harpia harpyja L. 1758). Of the 45 microsatellites tested, 24 were polymorphic, six monomorphic, 10 uncharacterizable due to multiple bands and five did not amplify. The observed gene diversity of the analyzed sample of H. harpyja was low and similar to that of other threatened Falconiformes. While a high proportion of the microsatellite markers were highly variable, individuals of H. harpyja could be differentiated by a joint analysis of just three (p = 2.79 x 10-4) or four markers (p = 2.89 x 10-5). Paternity could be rejected with 95.23 percent and 97.83 percent probabilities using the same three and four markers, respectively. The sex determination markers easily and consistently differentiated males from females even with highly degraded DNA extracted from naturally shed feathers. The markers reported in this study potentially provide an excellent set of molecular tools for the conservation and management of wild and captive H. harpyja and they may also prove useful for the enigmatic Neotropical crested eagle (Morphnus guianensis Daudin 1800).

Changes of Coregulators, MAP Kinase Activity and p27/kip1 with Estrogen or Antiestrogen Treatment in Breast Cancer Cell Line / 한국유방암학회지

PURPOSE: Estrogen, various polypeptide hormones and growth factors are associated with the development and progression of breast cancer. Coregulatory proteins are also associated with estrogen receptor (ER) transcriptional activity and tamoxifen resistance. Therefore, it is necessary to investigate the change of coregulator mRNAs and various cell proliferation proteins and cell cycle-related proteins after treatment with estrogen or antiestrogen. METHODS: MCF-7 cells were maintained in dextran-coated charcoal stripped 10% Dulbecco's Modified Eagle Medium (DMEM). To measure the change of the coactivators' (src-1, P/CAF, CBP, AIB1) mRNAs and corepressors' (SMRT, N-coR) mRNAs, multiple PCR was carried out using specific primers. In addition, intracellular proteins related to cell proliferation and cell cycle regulation were measured by performing Western blotting after treatment with estrogen or tamoxifen. The change of mitogen activated protein kinases was also measured by performing Western after tamoxifen treatment for 4 weeks. RESULTS: Coactivator mRNAs expression rapidly decreased in 15 min after estrogen treatment but this recovered to the initial level in 3 hr. The pattern was similar for the case of tamoxifen treatment. Corepressor mRNAs expression rapidly decreased in 15 min after estrogen treatment and it remained at a lower level until 24 hr after estrogen treatment. With tamoxifen treatment, the initial response was similar to the cases of estrogen treatment, but the xpression gradually increased 3 hr after tamoxifen treatment. Treatment of estrogen induced intracellular concentrations of c-myc and Ki-67 and it increased nuclear translocation of NF-kappaB and phosphor-ERK and it decreased the intracellular cell cycle suppressor p27/kip1. Tamoxifen treatment increased nuclear p27/kip1 but it decreased c-myc, NF-kappaB and phosphor-ERK. Long-term (4 weeks) treatment of tamoxifen was associated with decrease of activated ERK and p38 but there was no change in phospho-Akt level. CONCLUSION: Estrogen induced cell proliferation and the survival pathway-related factors, but it decreased the cell cycle suppressor p27/kip1. Long-term treatment with antiestrogen tamoxifen might decrease the MAPK activities in ERalpha-expressing tumor cells.