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Chinese Journal of Biotechnology ; (12): 1376-1384, 2021.
Article in Chinese | WPRIM | ID: wpr-878639


To rapidly and accurately manipulate genome such as gene deletion, insertion and site mutation, the whole genome of a very virulent strain Md5 of Marek's disease virus (MDV) was inserted into bacterial artificial chromosome (BAC) through homogeneous recombination. The recombinant DNA was electroporated into DH10B competent cells and identified by PCR and restriction fragment length polymorphism analysis. An infectious clone of Md5BAC was obtained following transfection into chicken embryo fibroblast (CEF) cells. Furthermore, a lorf10 deletion mutant was constructed by two step Red-mediated homologous recombination. To confirm the specific role of gene deletion, the lorf10 was reinserted into the original site of MDV genome to make a revertant strain. All the constructs were rescued by transfection into CEF cells, respectively. The successful packaging of recombinant viruses was confirmed by indirect immunofluorescence assay. The results of growth kinetics assay and plaques area measurement showed that the lorf10 is dispensable for MDV propagation in vitro. Overall, this study successfully constructed an infectious BAC clone of MDV and demonstrated its application in genome manipulation; the knowledge gained from our study could be further applied to other hepesviruses.

Animals , Chick Embryo , Chickens , Chromosomes, Artificial, Bacterial , DNA, Recombinant , Herpesvirus 2, Gallid/genetics , Marek Disease
Braz. j. med. biol. res ; 54(3): e10291, 2021. tab, graf
Article in English | LILACS | ID: biblio-1153518


The vascular network expansion and functioning are important factors affecting normal intra-uterine fetal development. This study addressed the previously reported antiangiogenic potential of beta-2-glycoprotein I (β2GPI) in vivo in the chick embryo model of angiogenesis. The effects of two naturally occurring β2GPI forms on the development of the chorioallantoic membrane (CAM) vessels and the chicken embryo were investigated. β2GPI monomers and dimers were obtained by fractioned purification and characterized using SDS-PAGE, immunoblot, and ELISA. The egg exposure was performed by injection of small volumes of 2.5 µg/mL solutions of the β2GPI subfractions. Angiogenesis was evaluated through quantitative measurements of vascular architecture parameters in the captured CAM images, using computational analysis of texture contrasts and computer vision techniques. Quantitative information was assigned to the CAM vasculature modifications. In vivo, the β2GPI dimer completely halted the formation of CAM vessels and led to embryo death after 48 h of exposure. The β2GPI monomer allowed the embryo to develop up to the 10th day, despite early changes of CAM vessels. The impaired normal vessel growth proceeded as a self-limited effect. The β2GPI monomer-exposed eggs showed reduced vascularization on the 6th day of incubation, but embryos were viable on the 10th day of incubation, with ingurgitated CAM vessels implying sequelae of the angiogenesis inhibition. Both subfractions impaired CAM vasculature development. The β2GPI dimer proved to be largely more harmful than the β2GPI monomer. β2GPI modification by cleavage or dimerization may play a role in angiogenesis control in vivo.

Chickens , Chorioallantoic Membrane , Chick Embryo , Neovascularization, Physiologic , Angiogenesis Inhibitors/pharmacology , beta 2-Glycoprotein I
Braz. J. Vet. Res. Anim. Sci. (Online) ; 57(2): e166086, mai. 2020. ilus, tab, graf
Article in English | ID: biblio-1122174


Avian coronavirus (AvCoV) infects a range of tissues in chickens and several other avian species. Although the virus can be isolated in chicken embryos, only a few strains of the 6 genotypes/33 lineages can grow in cell lines, with the Beaudette strain (GI-1 lineage) being the most used for in vitro studies. Considering the differences between cell lines and chicken embryos as habitats for AvCoV, this study aimed to assess the diversity of the genes coding for the nonstructural protein 3 (nsp3) and spike envelope protein (S) after serial passages in BHK-21 and Vero cells. After 14 passages of an embryo-adapted Beaudette strain, the virus loads fluctuated in both cell lines, with the highest loads being 8.72 log genome copies/µL for Vero and 6.36 log genome copies/µL for BHK-21 cells. No polymorphisms were found for nsp3; regarding S, not only aa substitutions (Vero: 8th passage A150S, and 14th S150A; BHK-21: 4th S53F, 8th F53Y, and 8th S95R), but also minor variants could be detected on chromatograms with fluctuating intensities. As the regions of these aa substitutions are within the receptor-binding domain of S, it can be speculated that differences in cell receptors between Vero and BHK-21 cells and the speed of cell death led to the selection of different dominant strains, while the stability of nsp3 supports its function as a protease involved in AvCoV replication. In conclusion, AvCoV quasispecies evolution is influenced by the biological model under consideration, and a gradual transition is seen for minor and major variants.(AU)

O Coronavírus aviário AvCoV infecta uma variedade de tecidos de galinhas e de outras espécies aviárias. Apesar de este vírus poder ser isolado em ovos embrionados de galinha, apenas alguns dos 6 genótipos / 33 linhagens podem crescer em cultivo celular, sendo a cepa Beuadette (linhagem GI-11) a mais utilizada para estudos in vitro. Considerando as diferentes linhagens celulares e ovos embrionados como habitats para o AvCoV, este estudo teve por objetivo estudar a diversidade de genes que codificam para a proteína não-estrutural 3 (nsp3) e espícula (S) após passagens seriadas em células BHK-21 e VERO. Após 14 passagens, de uma amostra Beuadette adaptada a ovos embrionados, os títulos virais variaram em ambas as células, com os maiores títulos sendo de 8,72 log cópias genômicas/µL para Vero e 6,36 cópias genômicas/µL para BHK-21. Nenhum polimorfismo foi encontrando para nsp3. Considerando a proteína S, não somente foram encontradas substituições de aminoácidos (Vero: 8a passagem A150S e 14a passagem S150A; BHK-21: 4a passagem S53F, 8a passagem F53Y e S95R), mas também, variantes subconsensuais foram detectadas pelos cromatogramas com intensidades flutuantes. Uma vez que as regiões destes aa se encontram no domínio de ligação de receptor de S, pode-se especular que diferenças em receptores celulares entre Vero e BHK-21, além da velocidade da morte celular, levaram à seleção de diferentes cepas dominantes, enquanto que a estabilidade de nsp3 concorda com sua função como protease com papel na replicação de AvCoV. Como conclusão, a evolução de quase-espécies de AvCoV é influenciada pelo modelo biológico sob consideração e uma transição gradual é vista para variantes dominantes e subdominantes.(AU)

Chick Embryo , Viral Nonstructural Proteins , Coronavirus Infections/veterinary , Spike Glycoprotein, Coronavirus , Gammacoronavirus
Int. j. morphol ; 38(1): 135-139, Feb. 2020. graf
Article in Spanish | LILACS | ID: biblio-1056411


La angiogénesis es el proceso por el cual se forman nuevos vasos sanguíneos a partir de otros ya existentes. Para que esto se lleve a cabo de forma correcta debe existir un balance entre los factores proangiogénicos y los factores antiangiogénicos dentro del microambiente tisular. Por otra parte, la existencia de productos químicos naturales como los polifenoles, que son capaces de adquirirse en la dieta, inducen a estos factores a intervenir en el proceso de angiogénesis. Se administraron los polifenoles en filtros de metilcelulosa sobre la membrana alantocoriónica de huevos White Leghorn, manteniendo el posterior desarrollo normal del feto. Se utilizaron 15 fetos de pollo fijados en formalina tamponada, a los cuales se extrajo el corazón. El procesamiento de las muestras de corazón se realizó a través de técnicas histológicas, histoquímicas e inmunohistoquímica. Finalmente se evaluó la presencia del VEGF y la capacidad de formar vasos sanguíneos bajo el tratamiento con los polifenoles. La inmunorreactividad fue cuantificada mediante Image J®. Los resultados indican que Ácido cafeico y Pinocembrina disminuyen la densidad microvascular y la expresión de VEGF en corazones de fetos de pollo tratados con estos polifenoles. Tanto el Ácido Cafeico como la Pinocembrina cumplen un rol inhibitorio en el proceso de angiogénesis fisiológica en corazón de pollo, pudiendo modular las vías de señalización mediadas por los VEGFR o modulando la disponibilidad de VEGF. Estos polifenoles podrían utilizarse para el estudio de otros tejidos asociados a angiogénesis patológica.

Angiogenesis is the process by which new blood vessels are formed from other existing ones. A balance between proangiogenic factors and anti-angiogenic factors within the microenvironment must exist for the process to be carried out correctly. Similarly, the existence of natural chemicals such as polyphenols, which are capable of being acquired in the diet, induce these factors in the angiogenic process. Polyphenols were administered in the methylcellulose filters on the of chorioallantoic membrane of White Leghorn eggs, maintaining the normal posterior development of the fetus. 15 chicken fetuses were fixed in buffered formalin, obtaining the hearts to histological processing, performing histological, histochemical and immunohistochemical techniques. VEGF levels and the ability of the blood vessels growing under the stimulation of the polyphenols were evaluated. Immunoreactivity was quantified by Image J. The results indicate that caffeic acid and pinocembrin decreased microvascular density and VEGF expression in hearts stimulated with these polyphenols. Both the caffeic and pinocembrin acids play an inhibitory role in the physiological angiogenesis process in the chicken heart, which decrease the microvascular density and could act by modulating the signaling pathways mediated by the VEGFR or by modulating the availability of VEGF. The use of these polyphenols could be useful in studies of other tissues associated with pathological angiogenesis.

Animals , Caffeic Acids/pharmacology , Neovascularization, Physiologic/drug effects , Chick Embryo , Polyphenols/pharmacology
Medicina (B.Aires) ; 79(1,supl.1): 62-67, abr. 2019. tab
Article in Spanish | LILACS | ID: biblio-1002607


La exposición prenatal al alcohol es causa de alteraciones somáticas, cognitivas y conductuales que se agrupan bajo el término de trastorno del espectro alcohólico fetal (TEAF). La evolución a largo plazo de los sujetos afectados a menudo es desfavorable, especialmente a nivel académico y adaptativo social. En el perfil neuropsicológico es característica la disfunción ejecutiva a menudo asociada a trastornos de la conducta que evolucionan en muchos casos hacia la delincuencia a partir de la adolescencia y en la edad adulta. Se han descrito también déficits de las habilidades sociales y la empatía. La exposición prenatal al alcohol constituye la causa más frecuente de trastorno del neurodesarrollo adquirido y prevenible.

Prenatal exposure to alcohol is the cause of cognitive and behavioural disorders grouped under the term fetal alcohol spectrum disorders (FASD). The long-term evolution of subjects with FASD is often unfavourable, especially in social and academic fields. Executive dysfunction is a hallmark deficit for children with FASD with increased rates of externalizing behaviours, such as aggressiveness and frequently delinquency in adolescence and adulthood. Deficits in social skills, empathy and communication ability are frequent observed among FASD. Prenatal exposure to alcohol is the most frequent cause of acquired and preventable neurodevelopmental disorder.

Humans , Animals , Female , Pregnancy , Developmental Disabilities/diagnosis , Fetal Alcohol Spectrum Disorders/diagnosis , Prognosis , Social Behavior Disorders/etiology , Chick Embryo , Developmental Disabilities/complications , Developmental Disabilities/physiopathology , Uncertainty , Diagnostic Errors , Fetal Alcohol Spectrum Disorders/physiopathology
Int. j. morphol ; 37(1): 48-53, 2019. graf
Article in Spanish | LILACS | ID: biblio-990003


RESUMEN: Los niveles de VEGF y su unión a sus receptores son etapas claves en la regulación de la angiogénesis. El ácido acetilsalicílico (AAS), ampliamente utilizado en tratamiento post infarto al miocardio ha mostrado poseer un efecto antiangiogénico en modelos tumorales. Este efecto potencialmente contraproducente requiere ser estudiado en miocardio. El objetivo del presente trabajo es cuantificar el efecto de AAS y de ácido salicílico (AS) sobre la vascularización en membrana alantocoriónica (MAC) y sobre los niveles de VEGF-A y VEGFR2 en miocardio de embriones de pollo. Para ello, treinta fetos de pollo White Leghorn fueron instilados a los 10 días de gestación con 60 µL de DMSO 0,1 % (control) o conteniendo además 0,3 µmol de AAS o AS. A las 48 horas se realizó procesamiento histológico de MAC para recuento de vasos sanguíneos y de tejido cardíaco para cuantificar VEGF-A y VEGFR2 por inmunohistoquímica. La inmunorreactividad fue cuantificada mediante Image J. Tanto AAS como AS disminuyeron la densidad microvascular de MAC. En miocardio, AAS aunque no AS, disminuyó la concentración de VEGFR2. No hubo efecto sobre VEGF-A. En nuestro modelo experimental, fetos de pollo a los 10 días de gestación también se observó el efecto inhibidor de AAS sobre la angiogénesis en MAC. La disminución de VEGFR2 en cardiomiocitos sugiere que AAS también afecta la angiogénesis en miocardio sano, modificando la disponibilidad del receptor a VEGF. Estos hallazgos nos permiten postular que AAS podría interferir con la regeneración de tejido, en situaciones como post infarto al miocardio.

SUMMARY: The VEGF levels and its binding to its receptors are key stages in the regulation of angiogenesis. Acetylsalicylic acid (ASA), widely used in post-myocardial infarction treatment, has been shown to have an anti-angiogenic effect in tumor models. This potentially counterproductive effect requires to be studied in myocardium. The aim of this study is to quantify the effect of ASA and salicylic acid (SA) on the vascularization in chick allantochorionic membrane (CAM) and on the levels of VEGF-A and VEGFR2 in myocardium of chicken embryos. Thirty White Leghorn chicken fetuses were instilled at 10 days of gestation with 60 mL of 0.1 % DMSO (control) or also containing 0.3 mmol of ASA or SA. After 48 hours, CAM histological processing was performed to count blood vessels and heart tissue to quantify VEGFA and VEGFR2 by immunohistochemistry. Immunoreactivity was quantified by Image J. Both ASA and SA decreased CAM microvascular density. In myocardium, AAS, although not SA, decreased the concentration of VEGFR2. There was no effect on VEGF-A. In our experimental model, chicken fetuses at 10 days of gestation, the inhibitory effect of ASA on angiogenesis in CAM were also observed. The decrease in VEGFR2 in cardiomyocytes suggests that ASA also affects angiogenesis in healthy myocardium, modifying the availability of the receptor to VEGF. These findings allow us to postulate that ASA could interfere with tissue regeneration, when it is required, as post myocardial infarction.

Animals , Chick Embryo , Aspirin/pharmacology , Salicylic Acid/pharmacology , Vascular Endothelial Growth Factor Receptor-2/drug effects , Vascular Endothelial Growth Factor A/drug effects , Heart/drug effects , Immunohistochemistry , Neovascularization, Physiologic/drug effects , Vascular Endothelial Growth Factor Receptor-2/analysis , Vascular Endothelial Growth Factor Receptor-2/metabolism , Vascular Endothelial Growth Factor A/analysis , Vascular Endothelial Growth Factor A/metabolism
Int. j. morphol ; 37(1): 344-348, 2019. tab, graf
Article in Spanish | LILACS | ID: biblio-990049


RESUMEN: El proceso angiogénico se define como el proceso en el que los vasos sanguíneos generan brotes dando como resultado neovascularidad. Un desbalance en el proceso angiogénico contribuye a numerosos desórdenes inflamatorios, infecciosos, isquémicos, inmunológicos y malignos. En el territorio maxilofacial se pueden encontrar patologías neoplásicas benignas de desarrollo local con un marcado componente angiogénico que determinan su crecimiento y agresividad. Sin embargo, existe escasa evidencia de cómo tratarlas en base al control de la angiogénesis. Terry & Jacoway (1994) desarrollaron un protocolo de tratamiento para lesiones neoplásicas benignas con un importante componente vascular que se utiliza actualmente. Este protocolo consiste en la infiltración intralesional de una suspensión de triamcinolona 10 mg/ml más una solución de anestésico local de uso odontológico como la lidocaína al 2 % asociada a epinefrina en una concentración de 1:200.000. Sin embargo, el uso de epinefrina podría disminuir la acción antiangiogénica de la triamcinolona al ser un vasoconstrictor. El objetivo de este estudio es comparar el efecto antiangiogénico, en la membrana alantocoriónica de pollo (MAC), de esta suspensión versus el efecto de la triamcinolona sin asociar a anestésicos locales. Los resultados del efecto antiangiogénico en la MAC de pollo, obtenidos en la investigación concluyeron que la suspensión de triamcinolona asociada a lidocaína con epinefrina es similar al de la suspensión de triamcinolona sin asociar a anestésicos locales. Además, se logró determinar que las suspensiones de triamcinolona sin asociar a anestésicos locales y las asociadas a anestésicos locales con o sin vasoconstrictor poseen un marcado efecto antiangiogénico, en la MAC de pollo, en comparación al grupo control.

SUMMARY: Angiogenesis is defined as the process through which new blood vessels form from previously existing vessels. Several inflammatory, infectious, ischemic, immunological and malignant disorders are caused by the lack of adequate angiogenesis balance. In the maxillofacial area, there are invasive benign neoplastic pathologies with a strong angiogenic component, which determines aggressive behavior and growth. Studies in the literature are scarce regarding treatment of these conditions based on angiogenesis control. Currently, the protocol used to treat these maxillofacial benign neoplastic lesions, was developed in 1994 by Terry & Jacoway and has a strong angiogenic component. Consequently lesions are treated via intra-lesion administration of triamcinolone 10 mg / mL, a solution used in dental local anesthetic, such as lidocaine 2 %, in conjunction with epinephrine at a concentration of 1:200,000. The objective of this study was to compare the antiangiogenic effect of this protocol in chicken chorioallantoic membrane (CAM) without the use of local anesthetic. The results of the antiangiogenic effect in the CAM obtained in this study concluded that the effect of the suspension of triamcinolone associated to lidocaine with epinephrine, is similar to the suspension of triamcinolone without associating local anesthetics. Furthermore, it was determined that suspensions of triamcinolone without local anesthetic, and those associated to local anesthetic with, and without vasoconstrictor have a strong antiangiogenic effect in CAM compared to the control group.

Animals , Chick Embryo , Triamcinolone/administration & dosage , Epinephrine/administration & dosage , Angiogenesis Inhibitors/administration & dosage , Chorioallantoic Membrane/drug effects , Lidocaine/administration & dosage , Anesthetics, Local/administration & dosage , Neovascularization, Pathologic
Int. j. morphol ; 36(2): 569-575, jun. 2018. graf
Article in English | LILACS | ID: biblio-954156


In order to compare Wnt/ beta-catenin expression in mouse and chick facial primordia during development, we performed an immunohistochemical analysis of both protein expressions in E12 to E17 mouse embryos and in E3 and E10 chick embryos. During odontogenesis, from bud to bell stage, both proteins exhibit similar fixation patterns, with epithelial and mesenchymal immunoreactivity, consistant with literature data. Double labelling demonstrates that the same cells express both antigens, even in undifferentiated mesenchyme. The enamel knot, and the ameloblastic and odontoblastic layers are stained at the same manner. In the chick, Wnt and beta-catenin are diffusely present on craniofacial mesenchyme. In both species, premuscular blastemata express Wnt and b-catenin, but Wnt is specifically expressed on the perichondrium and ossification centers, suggesting a role independent from beta-catenin pathway.

Animals , Chick Embryo , Mice , Tooth/embryology , Embryo, Nonmammalian/metabolism , Wnt Proteins/metabolism , beta Catenin/metabolism , Immunohistochemistry , Fluorescent Antibody Technique
Rev. bras. geriatr. gerontol. (Online) ; 21(2): 205-214, Apr.-Mar. 2018. tab
Article in English, Portuguese | LILACS | ID: biblio-958911


Objective: To analyze the association between sociodemographic profile, health profile and the quality of life of caregivers of elderly people diagnosed with dementia. Method: A cross-sectional study was carried out in a geriatric outpatient unit with 35 elderly caregivers diagnosed with dementia. A socio-demographic and health characterization form was used as well as the WHOQOL-bref scale. To verify the association of the variables, the Spearman or Pearson correlation tests were applied, according to the normality of the data. Results: A moderate association between caregiver age and quality of life was found, as well as between total time of care and quality of life, which reveals that the care provided to the elderly with dementia has repercussions on the quality of life. The domains of the WHOQOL-bref with the highest means were Social and Physical Relations, and those with lower mean values ​​were Psychological and Environmental. Conclusion: The identification of conditions that influence the quality of life of caregivers of elderly people with dementia allows actions to promote, protect and recover the health and well-being of those who provide care to be established, so that this care is both for themselves and for the elderly.

Objetivo: Analisar associação entre o perfil sociodemográfico, características do cuidado e a qualidade de vida de cuidadores de idosos diagnosticados com demência. Método: Estudo transversal, realizado em uma unidade ambulatorial geriátrica com 35 cuidadores de idosos diagnosticados com demência. Foi utilizado uma ficha de caracterização sociodemográfica e de saúde assim como a escala WHOQOL-bref. Para verificar a associação das variáveis idade, qualidade de vida e tempo total de cuidado foram aplicados os testes de "correlação de Spearman" ou "Pearson", conforme a normalidade dos dados. Resultados: Constatou-se associação moderada entre idade do cuidador e sua qualidade de vida, assim como tempo total de cuidado e qualidade de vida, o que mostra que o cuidado prestado ao idoso demenciado gera repercussão na qualidade de vida. Os domínios do WHOQOL-bref que tiveram melhores médias foram Relações Sociais e Físico, e os que apresentaram menores médias foram Psicológico e Meio Ambiente. Conclusão: A identificação de condições que influenciam a qualidade de vida dos cuidadores de idosos com demência permite o estabelecimento de ações de promoção, proteção e recuperação da saúde e do bem-estar de quem cuida, de forma que este cuidado transcenda para si e para o idoso.

Humans , Male , Female , Middle Aged , Aged , Chick Embryo , Quality of Life , Health of the Elderly , Nursing , Caregivers , Dementia
Arq. bras. med. vet. zootec. (Online) ; 69(5): 1259-1266, set.-out. 2017. tab
Article in Portuguese | ID: biblio-879203


Este estudo objetivou avaliar os efeitos da inoculação de proteína isolada de soja (P.I.S.) em ovos embrionados de matrizes semipesadas com diferentes idades. Foram utilizados 320 ovos embrionados de matrizes semipesadas da linhagem Rhode Island Red com 35 e 70 semanas de idade. Ao 17º dia de incubação, os ovos foram inoculados com 0,5mL de solução diretamente na cavidade alantoide. O delineamento experimental utilizado foi o inteiramente ao acaso, em esquema fatorial (2 x 4), constituído por dois fatores: idades das matrizes (matrizes jovens e matrizes velhas) e soluções inoculadas (ovo íntegro; 0,5% de solução salina; 1% de P.I.S. + 0,5% de solução salina; e 2% de P.I.S. + 0,5% de solução salina), totalizando oito tratamentos contendo 40 ovos cada. Os dados coletados foram analisados pelo teste de Tukey a 5% de significância. A inoculação de proteína isolada de soja apresentou efeito positivo sobre a relação pinto-ovo, porém sem proporcionar melhora nos rendimentos de incubação, no desenvolvimento dos órgãos e no desempenho de pintos na fase pré-inicial. A idade da matriz influenciou diretamente as variáveis avaliadas, em que ovos oriundos de aves velhas apresentaram maior peso, maior mortalidade embrionária na fase tardia, pintos mais pesados e pior conversão alimentar.(AU)

This study aimed to evaluate the influence of inoculation of isolated soy protein (P.I.S.) in embryonated eggs of semi-heavy with different ages. 320 embryonated eggs were used from semi-heavy breeders of Rhode Island Red lineage with 35 and 70 weeks of age. In the 17th day of incubation, the eggs were inoculated with 0.5 ml of solution in the allantoic cavity. The experimental method was completely randomized in factorial scheme (2 x 4), consisting of two factors: matrices ages (young and old matrices) and inoculation solutions (non-inoculated eggs; 0.5% of saline solution; 1% of P.I.S. + 0.5% of saline solution; and 2% of P.I.S. + 0.5% of saline solution, totalling eight treatments containing 40 eggs each. The inoculation of isolated soy protein showed positive effect on the chick-egg relation, albeit without improving incubation yields, organ development and chicks's performance in the pre-initial phase. The age of the breeder directly influenced the variables evaluated, with eggs from older breeder showing better weight, higher embryonic mortality in late phase, heavier chicks and worse feed conversion.(AU)

Animals , Chick Embryo/growth & development , Chickens , Eggs/analysis , Embryonic Development , Proteins/analysis , Soybeans
Braz. j. microbiol ; 48(3): 403-409, July-Sept. 2017. tab, graf
Article in English | LILACS | ID: biblio-889142


Abstract Very high gravity (VHG) technology was employed on industrial scale to produce ethanol from molasses (fermented) as well as by-products formation estimation. The effect of different Brix° (32, 36 and 40) air-flow rates (0.00, 0.20, 0.40, and 0.60 vvm) was studied on ethanol production. The maximum ethanol production was recorded to be 12.2% (v/v) at 40 Brix° with 0.2 vvm air-flow rate. At optimum level aeration and 40 Brix° VHG, the residual sugar level was recorded in the range of 12.5-18.5 g/L, whereas the viable cell count remained constant up to 50 h of fermentation and dry matter production increased with fermentation time. Both water and steam consumption reduced significantly under optimum conditions of Brix° and aeration rate with compromising the ethanol production. Results revealed VHG with continuous air flow is viable technique to reduce the ethanol production cost form molasses at commercial scale.

Chick Embryo , Ethanol/metabolism , Industrial Microbiology/methods , Molasses/microbiology , Saccharomyces cerevisiae/metabolism , Fermentation , Hypergravity , Industrial Microbiology/instrumentation , Molasses/analysis , Molasses/economics , Saccharomyces cerevisiae/genetics
Int. j. morphol ; 35(1): 208-211, Mar. 2017. ilus
Article in English | LILACS | ID: biblio-840956


The aim of this study was to observe morphological changes of the cultured otocysts isolated from various stages of the chick embryo. Isolated otocysts were dissected from embryonic day, E2.5-4.5 of incubation (HH stage 16-26) according to stages of developing inner ear. Morphology of the chick otocyst exhibited an ovoid shape. The width and height of the otocyst were 0.2 mm and 0.3 mm, respectively. Elongation of a tube-like structure, the endolymphatic duct, was found at the dorsal aspect of the otocyst. The cultured otocyst is lined by the otic epithelium and surrounding periotic mesenchymal cells started to migrate outwards the lateral aspect of such epithelium. Notably, the acoustic-vestibular ganglion (AVG) was observed at the ventrolateral aspect of the otocyst. Appearance of AVG in vitro can be applied for studying chemical-induced ototoxicity and sensorineural hearing loss. It was concluded that the organ-cultured otocyst of the chick embryo could be used as a model to study sensory organ development of avian inner ear.

El objetivo de este estudio fue observar los cambios morfológicos de otocistos cultivados aislados en las diversas etapas del desarrollo del embrión de pollo. Otocistos aislados fueron obtenidos de embriones día, E2.5-4.5 de incubación (HH etapa 16-26) de acuerdo a las etapas de desarrollo del oído interno. El otocisto de pollo presentó una morfología ovoide. El ancho y la altura del otocisto fue de 0,2 mm y 0,3 mm, respectivamente. En la cara dorsal del otocisto se visualizó el alargamiento de una estructura similar a un tubo, el conducto endolinfático. El otocisto cultivado está revestido por epitelio ótico y células mesenquimatosas perióticas que comienzan a migrar hacia el exterior de la cara lateral en búsqueda del epitelio. En particular, el ganglio acústico-vestibular (GAV) fue observado en la parte ventrolateral del otocisto. La aparición de GAV in vitro puede ser aplicado para el estudio de la ototoxicidad inducida por productos químicos y la pérdida de audición neurosensorial. Se concluyó que el otocisto cultivado de embrión de pollo podría ser utilizado como un modelo para estudiar el desarrollo de órganos sensoriales del oído interno aviar.

Animals , Chick Embryo/anatomy & histology , Ear, Inner/embryology , Morphogenesis
Pakistan Journal of Pharmaceutical Sciences. 2017; 30 (4): 1341-1344
in English | IMEMR | ID: emr-189703


In the present study, anti-Avian influenza virus H9N2 activity of aqueous extracts [5, 10, 15, 20, 25%] of Zingiber officinalis and Allium sativum was evaluated. Embryo-toxicity was evaluated by histopathological scoring of Chorio-allantoic membrane of chick embryos. Cytotoxicity of extracts was determined by MTT assay on Vero cells. Aqueous extract of ginger had antiviral activity at 10, 15, 20 and 25% while garlic had activity at 15, 20 and 25%. Histopathological scoring of chorio-allantoic membrane for aqueous extracts [5, 10, 15, 20, 25%] of ginger [0.66+/-0.57, 1.33+/-0.57, 1.66+/-0.57, 2.66+/-0.57, 3.66+/-0.57, respectively] and garlic [1.00+/-0.00, 1.33+/-0.57, 2.00+/-0.00, 2.33+/-0.57, 3.66+/-0.57, respectively] was concentration dependant. MTT assay revealed cytotoxicity of both plants was also concentration dependent. Extracts of ginger [5, 10, 15, 20, 25%] had lower cytotoxicity [71, 59, 28, 22, 0 % cell survival, respectively] as compared to garlic [61, 36. 20, 11, 3% cell survival, respectively]. Overall results revealed that concentration of aqueous extract of ginger [10%], showing antiviral activity against H[9]N2, was less toxic to vero cells [> 50% cell survival]. It is insinuated that ginger may have anti- Avian influenza virus H9N2 potential and its active compounds needs further investigations

Influenza in Birds , Influenza A Virus, H9N2 Subtype , Plant Extracts , Garlic , Plant Structures , Chick Embryo
Biol. Res ; 50: 41, 2017. tab, graf
Article in English | LILACS | ID: biblio-950889


BACKGROUND: The marine environment is a rich source of bioactive natural products. Many of the marine bioactive compounds have been derived successfully from molluscs. Euchelus asper is a marine mollusc which is commonly found in the intertidal rocky regions of the Mumbai coast. The present study was focused on evaluating the anti-angiogenic and anti- proliferative activities of methanolic extract of Euchelus asper (EAME). METHODS: The anti-angiogenic activity of EAME (50-800 µg/mL) was assessed by chick chorio-allantoic membrane (CAM) model wherein multiple parameters in the CAM blood vessels were analysed through morphometric and histo-logical investigations. In vitro testing of EAME (5-20 µg/mL) included its cytotoxicity against three different cancer cell lines, its effect on cell proliferation by wound healing assay as well as their relevant molecular mechanisms. Statistical analysis was carried out by two-tailed student's t test for two unpaired groups. RESULTS: Analysis of CAM revealed that the extract is effective in reducing the branching points of the 1st order blood vessels or capillaries of CAM. Histological analysis of CAM showed significant decrease in capillary plexus and compartmentalization along with increase in mesodermal blood vessels, thus establishing its anti-angiogenicity. Further, EAME exhibited moderate but significant cytotoxicity against A549 non-small cell lung carcinoma cell line. We also demonstrated that the cytotoxicity of EAME in A549 was associated with its apoptotic activity by subG1 phase arrest. Lastly, EAME significantly reduced A549 proliferation by reducing the expression of Matrix metalloproteinase-2 (MMP-2) and Matrix metalloproteinase-9 (MMP-9). CONCLUSION: Overall, our study suggested that EAME has potential to inhibit tumour angiogenic and proliferative activity and may be a potential source for development of new anti-cancer pharmaceuticals.

Animals , Chick Embryo , Biological Products/pharmacology , Angiogenesis Inhibitors/pharmacology , Cell Proliferation/drug effects , Gastropoda/chemistry , Biological Products/isolation & purification , Angiogenesis Inhibitors/isolation & purification , Cell Line, Tumor/drug effects
Int. j. morphol ; 34(4): 1191-1196, Dec. 2016. ilus
Article in Spanish | LILACS | ID: biblio-840865


La acción antiangiogénica de los inhibidores del receptor de angiotensina II (ARA II), ha sido documentada previamente, sin embargo, no ha sido descrita la relación entre angiogénesis e inhibidores directos de la renina (DRIs), los cuales participan regulando el sistema renina-angiotensina-aldosterona (SRAA). El objetivo fue demostrar el efecto antiangiogénico de aliskireno, un DRI, en membranas alantocoriónicas (MAC) de pollo, para lo cual fueron instilados aliskireno y enalapril sobre MAC en distintas concentraciones para realizar su comparación posterior. En secciones histológicas seriadas se registró el número de vasos sanguíneos presentes en 9000 µm2 bajo microscopio de luz a máximo aumento, y se realizó análisis estadístico utilizando ANOVA y el test de Tukey para demostrar posibles diferencias. Los receptores tratados con aliskireno, en ambas concentraciones utilizadas, presentaron menor densidad vascular, en comparación con los controles, siendo ésta estadísticamente significativa a mayor concentración. Aliskireno en concentraciones altas tiene un efecto antiangiogénico en un modelo experimental de MAC. Este hallazgo plantea la necesidad de estudios posteriores, dada la proyección que podría tener el uso inhibidores directos de la renina. A partir de estos resultados, se podría pensar en la factibilidad del uso de aliskireno para la modulación de la angiogénesis en diversas enfermedades crónicas no transmisibles.

Angiogenesis is the formation of new blood vessels from pre-existing ones. Antiangiogenic effect of angiotensin receptor blockers has been reported, however, the relationship between direct renin inhibitors and angiogenesis has not been well described. To assess the antiangiogenic effect of aliskiren, a direct renin inhibitor, on chick embryo chorioallantoic membrane (CAM) assay. Aliskiren and enalapril were instilled in different concentrations and compared. In serial histological sections, the number of blood vessels per 9000 µm2 area under observation through optical microscope using maximum zoom, was registered. Statistical analysis using Anova and Tukey test in order to show possible differences, was performed. Receptors treated with aliskiren presented lower vascular density, which was statistically significant when a higher concentration was administered. High concentrations of aliskiren have an antiangiogenic effect on CAM assay. This finding means further studies are needed, because of the usefulness direct renin inhibitors could have. These results, also, might enhance the possibility of using aliskiren for regulating angiogenesis in the context of non-transmissible chronic diseases.

Animals , Amides/pharmacology , Chorioallantoic Membrane/drug effects , Fumarates/pharmacology , Neovascularization, Physiologic/drug effects , Analysis of Variance , Chick Embryo , Enalapril/pharmacology , Models, Animal , Renin/antagonists & inhibitors
Int. j. morphol ; 34(1): 57-62, Mar. 2016. ilus
Article in Spanish | LILACS | ID: lil-780475


Se evaluó el efecto del incremento de la temperatura de incubación en huevos de gallina Araucana sobre la tasa de eclosión, la mortalidad embrionaria, el peso del polluelo, del saco vitelino y de los órganos internos. Los huevos (n= 426) fueron distribuidos al azar en: a) grupo Control (n=215), incubados a 37,5 °C por 21 días y b) grupo Experimental (n= 211), incubados a 39,5 °C, por 18 días, disminuyéndose a 37,5 °C los 3 días restantes. En ambos grupos la incubación se realizó con una humedad relativa constante del 55 %. La tasa de mortalidad embrionaria total fue significativamente menor (P 0,05) para el grupo Control (14,9 vs 44,1 %) y consecuentemente la tasa de eclosión fue significativamente mayor (P 0,05) en este mismo grupo (97,8 vs. 80,5 %). El peso promedio de los polluelos y del saco vitelino a las 36 h post eclosión fue significativamente diferente (P 0,05) entre ambos grupos (37,1 vs. 36,3 y 2,5 vs 6,2 g para el grupo Control y Experimental respectivamente). Por otra parte, el peso de todos los órganos internos fue menor (P 0,05) en polluelos del grupo Experimental. Se concluye que el uso de una temperatura de incubación de 39,5 C por 18 días en huevos de gallina araucana influye negativamente, aumentando la mortalidad embrionaria, disminuyendo la tasa de eclosión, el peso del polluelo y de los órganos internos, y aumentando el peso del saco vitelino.

The aim of this study was to evaluate the effect of increasing incubation temperature of Araucana hen eggs on embryo mortality, hatchability,chick, yolk sac and internal organs weight. Eggs (n= 426) were randomly distributed into: a) Control group (n= 215), which were incubated at 37.5 °C for 21 days or b) Experimental group (n= 211), which were incubated at 39.5 °C, for 18 days and then at 37.5 °C for the remaining 3 days. In both groups eggs were incubated with a relative humidity of 55 %. The embryo mortality rate was greater (P 0.05) for the experimental than the control group (44.1 vs. 14.9 % respectively). On the contrary, hatching rate was greater (P 0.05) for the control than the experimental group (97.8 vs. 80.5 % respectively). The average weight of chicks, at 36 h of age, was lower (P 0.05) for the experimental than control group (36.3 vs. 37.01 g respectively). Also, increasing incubation temperature reduced (P 0.05) internal organs weight of chicks in the Experimental compared to Control group. On the other hand, yolk sac weight was reduced (P <0.05; 2.5 vs. 6.2 g for Control and Experimental group respectively). We conclude that increasing the incubation temperature of Araucana hen eggs up to 39.5 °C for 18 days has a negative effect on embryo survival, hatchability, chick and internal organs weight and absorption of yolk.

Animals , Chick Embryo , Chickens/growth & development , Temperature , Body Weight , Chickens/anatomy & histology , Eggs , Mortality , Organ Size
Chinese Journal of Virology ; (6): 39-45, 2016.
Article in Chinese | WPRIM | ID: wpr-296219


Small interfering ribonucleic acid (siRNA)-induced RNA degradation can inhibit viral infection, and has been investigated extensively for its efficacy as antiviral therapy. The potential therapeutic role of lentiviral-mediated short hairpin ribonucleic acid (shRNA) to Newcastle disease virus (NDV) replication in vivo has been explored less often. We constructed two recombinant lentiviral vectors containing shRNA against the phosphoprotein (P) of the NDV, RNAi-341 and RNAi-671. Recombinant shRNA lentivirus vectors were co-transfected into 293T cells, along with helper plasmids, to package the recombinant shRNA lentivirus. Lentivirus-based shRNAs were titrated and transduced into NDV-susceptible chicken embryo fibroblasts (CEFs) and chick embryos. Antiviral activity against the NDV strain was evaluated by virus titration and real-time reverse transcription-polymerase chain reaction. RNAi-341 and RNAi-671 strongly suppressed transient expression of a FLAG-tagged P fusion protein in 293T cells. RNAi-341 and RNAi-671 NDV reduced virus titers by 66.6-fold and 30.6-fold, respectively, in CEFs 16 h after infection. RNAi-341 and RNAi-671 reduced virus titers in specific pathogen-free chick embryos by 99% and 98%, respectively, 48 h after infection. Both shRNAs inhibited accumulation of not only P-gene mRNA, but also nucleocapsid, M-, F-, HN-, and L-gene mRNA. RNAi-341 silenced P-gene mRNA more potently than RNAi-671. These results suggest that shRNAs silencing the P gene had substantial antiviral properties and inhibited NDV replication in CEFs and chick embryos.

Animals , Chick Embryo , Chickens , Down-Regulation , Fibroblasts , Virology , Gene Targeting , Lentivirus , Genetics , Metabolism , Newcastle Disease , Virology , Newcastle disease virus , Genetics , Physiology , Phosphoproteins , Genetics , Metabolism , Poultry Diseases , Virology , RNA Interference , RNA, Small Interfering , Genetics , Metabolism , Viral Proteins , Genetics , Metabolism , Virus Replication
Chinese Journal of Virology ; (6): 70-76, 2016.
Article in Chinese | WPRIM | ID: wpr-296215


A quail-origin subtype of the influenza virus was isolated from a human-infecting H7N9 subtype of the avian influenza virus found in a live poultry market and was given the name A/Quail/Hangzhou/1/ 2013 (H9N2). We analyzed the whole genome of this virus and its biologic characteristics. Sequence analyses suggested that the: HA and NS genes belonged to a CK/BJ/1/94-like lineage; NA, NP, PA and PB1 genes belonged to a SH/F/98-like lineage; M and PB2 genes belonged to a G1-like lineage. Analyses of key amino acids showed that the cleavage site in HA protein was PSRSSR ↓ GL, and that the HA protein had a human receptor-binding site with Leu226. Deletion of amino acids 69 - 73 was detected in the stalk of NA protein, the M2 protein had an Asn31 mutation, and the NS1 protein had two mutations at Ser42, Ala149. The intravenous pathogenicity of this virus was 0.36. A study in chickens suggested that all inoculated birds shed the virus from the trachea and cloaca on the third day post-infection (p. i. ) until 11 days. All chickens that had direct contact shed the virus on the second day p. i. until 8 days. Results of virus reisolation suggested that lung and tracheal tissues could shed the virus in 5 days, whereas the other organs could shed the virus in 3 days. These results suggest that this virus strain is H9N2 subtype LPAIV, whose lineage is prevalent in mainland China. This research provides evidence on how to monitor and prevent the H9N2 subtype of the avian influenza virus.

Animals , Chick Embryo , Chickens , China , Genotype , Influenza A Virus, H9N2 Subtype , Classification , Genetics , Influenza in Birds , Virology , Phylogeny , Quail , Virology
Chinese Journal of Virology ; (6): 203-209, 2016.
Article in Chinese | WPRIM | ID: wpr-296196


In July 2009, some farms of breeding Muscovy ducks on the peak of egg laying suffered the decrease of hatching rate and the quality of the eggs showing low mortality and no evident respiratory symptoms. The swelling and congestive ovary was visible after autopsy. This study was brought out for the diagnosis of these cases. The virus was isolated and identified by the methods of virus culture in chicken embryo, physical and chemical properties test, hemagglutinin test, NDV (Newcastle diseases Virus) interference test, electron microscope observation, pathogenicity test and the gene sequence analysis. The results indicated the virus showed the characters of inducing dwarf embryo after inocubation, the sensibility to lipid solvent and the hemagglutination capacity after pancreatic enzyme treatment, the typical morphology of coronavirus, the interference to NDV replication and the homology among 84.7% - 99% of the particial N gene sequences to the reference IBV (Avian infectious bronchitis virus) strains. The strain was identified as IBV isolate and this study confirmed the pathogenicity of IBV to Muscovy ducks.

Amino Acid Sequence , Animals , Chick Embryo , Coronavirus Infections , Virology , Ducks , Virology , Female , Infectious bronchitis virus , Classification , Genetics , Molecular Sequence Data , Phylogeny , Poultry Diseases , Virology , Sequence Alignment
Rev. chil. cardiol ; 35(2): 127-132, 2016. ilus, tab, graf
Article in Spanish | LILACS | ID: lil-796798


Introducción: El ácido acetilsalicílico (AAS) es ampliamente utilizado en el manejo de patología cardiovascular. En modelos "in vitro" el AAS restringe la angiogénesis, atribuyéndose este efecto al bloqueo de ciclooxigenasa-1, manteniendo íntegra la zona adhe-rente endotelial, citotoxicidad directa y otras vías de señalización. Hipótesis: El AAS en concentración terapéutica antiplaquetaria utilizada en humanos ejerce un efecto antiangiogénico en modelo de membrana alantocorió-nica de pollo (MAC). Objetivo: Comparar la capacidad antiangiogénica del AAS en distintas concentraciones en MAC utilizando como punto de comparación la angiogénesis fisiológica de la MAC. Método: Se incubaron 46 huevos fecundados de gallinas White Leghorn, en cámara temperada a 37°C, provenientes del Instituto de Salud Pública de Chile. Mediante procedimiento descrito por Ribatti (2006), se instiló sobre filtro de metilcelulosa 10uL de Dimetilsulfóxido al 0.1% + m199, sin fármaco al control, asociado a AAS y ácido salicílico (AS) a los grupos de estudio en concentraciones 2mM y 5 mM. Posteriormente se fijó y analizó la muestra en forma ciega. Resultados: El promedio de vasos del control fue 21.8. Para el grupo AAS 2mM y 5mM fue 11.3 y 10, siendo para el grupo AS 2mM y 5mM 15.6 y 12.4. El análisis estadístico mediante ANOVA y t-Student muestra que todos los grupos que recibieron fármacos tuvieron una disminución significativa en el numero de vasos sanguíneos en relación al grupo control. No hubo diferencias significativas entre ambo grupos de AAS. El AS demostró tener mayor potencia antiangiogénica dosis dependiente. Discusión: En este estudio se demuestra que el AAS ejerce un efecto antiangiogénico en concentración terapéutica en condiciones fisiológicas de un modelo "in vivo".

Background: Acetylsalicylic acid (ASA) is widely used in the treatment of various cardiovascular disorders. In vitro, AAS decreases angiogenesis, through cyclo-oxigenase-1 blockade while keeping active the adherent endothelial zone, direct toxicity and other signaling pathways. Hypothesis: AAS at therapeutic anti plaquetary doses exerts an anti-angiogenic effect in the alanto choronic chicken membrane (ACM) Method: 46 fertilized eggs form White Leghorn hens were incubated at 37oC. 10 uL of 0.1% Dimethyl sulfoxide +Ml 19 with no drug were used as control, while experimental groups received ASA and Salicylic acid (SA), 2mM. After fixation, samples were analyzed in a blind fashion Results: The mean number of vessels was 21.8 for controls, 11.3 and 10 for ASA 2mM and ASA 5mM, respectively. Corresponding values for SA 2 and SA 5mM were 15.6 and 12.4, respectively. Thus, a statistically significant (ANOVA and Student's t) decrease in the number of vessels was observed in both ASA groups. SA showed had a greater potential for anti-angiogenesis in a dose dependent way. Conclusion: This study shows that ASA in therapeutic concentrations has an anti-angiogenic effect in a physiologic model in vivo.

Animals , Chick Embryo , Aspirin/pharmacology , Neovascularization, Physiologic/drug effects , Angiogenesis Inhibitors/pharmacology , Chorioallantoic Membrane , Chickens