Role of TGF-beta beta and BMP7 in the pathogenesis of oral submucous fibrosis

To understand the molecular pathogenesis of oral submucous fibrosis (OSF), which is a chronic inflammatory disease, gene expression profiling was performed in 10 OSF tissues against 8 pooled normal tissues using oligonucleotide arrays. Microarray results revealed differential expression of 5288 genes (P < a parts per thousand currency sign 0.05 and fold change >= a parts per thousand yen 1.5). Among these, 2884 are upregulated and 2404 are downregulated. Validation employing quantitative real-time PCR and immunohistochemistry confirmed upregulation of transforming growth factor-beta beta 1 (TGF-beta beta 1), TGFBIp, THBS1, SPP1, and TIG1 and downregulation of bone morphogenic protein 7 (BMP7) in OSF tissues. Furthermore, activation of TGF-beta beta pathway was evident in OSF as demonstrated by pSMAD2 strong immunoreactivity. Treatment of keratinocytes and oral fibroblasts by TGF-beta beta confirmed the regulation of few genes identified in microarray including upregulation of connective tissue growth factor, TGM2, THBS1, and downregulation of BMP7, which is a known negative modulator of fibrosis. Taken together, these data suggest activation of TGF-beta beta signaling and suppression of BMP7 expression in the manifestation of OSF.

Activation of TGF-beta Pathway by Areca Nut Constituents: A Possible Cause of Oral Submucous Fibrosis

Oral submucous fibrosis (OSF) is a chronic inflammatory disease characterized by the accumulation of excess collagen, and areca nut chewing has been proposed as an important etiological factor for disease manifestation. Activation of transforming growth factor-beta signaling has been postulated as the main causative event for increased collagen production in OSF. Oral epithelium plays important roles in OSF, and arecoline has been shown to induce TGF-beta in epithelial cells. In an attempt to understand the role of areca nut constituents in the manifestation of OSF, we studied the global gene expression profile in epithelial cells (HaCaT) following treatment with areca nut water extract or TGF-beta. Interestingly, 64% of the differentially regulated genes by areca nut water extract matches with the TGF-beta induced gene expression profile. Out of these, expression of 57% of genes was compromised in the presence of ALK5 (T beta RI) inhibitor and 7% were independently induced by areca nut, highlighting the importance of TGF-beta in areca nut actions. Areca nut water extract treatment induced p-SMAD2 and TGF-beta downstream targets in HaCaT cells but not in human gingival fibroblast cells (hGF), suggesting epithelial cells could be the source of TGF-beta in promoting OSF. Water extract of areca nut consists of polyphenols and alkaloids. Both polyphenol and alkaloid fractions of areca nut were able to induce TGF-beta signaling and its downstream targets. Also, SMAD-2 was phosphorylated following treatment of HaCaT cells by Catechin, Tannin and alkaloids namely Arecoline, Arecaidine and Guvacine. Moreover, both polyphenols and alkaloids induced TGF-beta 2 and THBS1 (activator of latent TGF-beta) in HaCaT cells suggesting areca nut mediated activation of p-SMAD2 involves up-regulation and activation of TGF-beta. These data suggest a major causative role for TGF-beta that is induced by areca nut in OSF progression.

Oncogenic MicroRNA-155 Down-regulates Tumor Suppressor CDC73 and Promotes Oral Squamous Cell Carcinoma Cell Proliferation IMPLICATIONS FOR CANCER THERAPEUTICS

The CDC73 gene is mutationally inactivated in hereditary and sporadic parathyroid tumors. It negatively regulates beta-catenin, cyclin D1, and c-MYC. Down-regulation of CDC73 has been reported in breast, renal, and gastric carcinomas. However, the reports regarding the role of CDC73 in oral squamous cell carcinoma (OSCC) are lacking. In this study we show that CDC73 is down-regulated in a majority of OSCC samples. We further show that oncogenic microRNA-155 (miR-155) negatively regulates CDC73 expression. Our experiments show that the dramatic up-regulation of miR-155 is an exclusive mechanism for down-regulation of CDC73 in a panel of human cell lines and a subset of OSCC patient samples in the absence of loss of heterozygosity, mutations, and promoter methylation. Ectopic expression of miR-155 in HEK293 cells dramatically reduced CDC73 levels, enhanced cell viability, and decreased apoptosis. Conversely, the delivery of a miR-155 antagonist (antagomir-155) to KB cells overexpressing miR-155 resulted in increased CDC73 levels, decreased cell viability, increased apoptosis, and marked regression of xenografts in nude mice. Cotransfection of miR-155 with CDC73 in HEK293 cells abrogated its pro-oncogenic effect. Reduced cell proliferation and increased apoptosis of KB cells were dependent on the presence or absence of the 3'-UTR in CDC73. In summary, knockdown of CDC73 expression due to overexpression of miR-155 not only adds a novelty to the list of mechanisms responsible for its down-regulation in different tumors, but the restoration of CDC73 levels by the use of antagomir-155 may also have an important role in therapeutic intervention of cancers, including OSCC.

Primary Microcephaly Gene MCPH1 Shows Signatures of Tumor Suppressors and Is Regulated by miR-27a in Oral Squamous Cell Carcinoma

Mutations in the MCPH1 (microcephalin 1) gene, located at chromosome 8p23.1, result in two autosomal recessive disorders: primary microcephaly and premature chromosome condensation syndrome. MCPH1 has also been shown to be downregulated in breast, prostate and ovarian cancers, and mutated in 1/10 breast and 5/41 endometrial tumors, suggesting that it could also function as a tumor suppressor (TS) gene. To test the possibility of MCPH1 as a TS gene, we first performed LOH study in a panel of 81 matched normal oral tissues and oral squamous cell carcinoma (OSCC) samples, and observed that 14/71 (19.72%) informative samples showed LOH, a hallmark of TS genes. Three protein truncating mutations were identified in 1/15 OSCC samples and 2/5 cancer cell lines. MCPH1 was downregulated at both the transcript and protein levels in 21/41 (51.22%) and 19/25 (76%) OSCC samples respectively. A low level of MCPH1 promoter methylation was also observed in 4/40 (10%) tumor samples. We further observed that overexpression of MCPH1 decreased cellular proliferation, anchorage-independent growth in soft agar, cell invasion and tumor size in nude mice, indicating its tumor suppressive function. Using bioinformatic approaches and luciferase assay, we showed that the 3'-UTR of MCPH1 harbors two non-overlapping functional seed regions for miR-27a which negatively regulated its level. The expression level of miR-27a negatively correlated with the MCPH1 protein level in OSCC. Our study indicates for the first time that, in addition to its role in brain development, MCPH1 also functions as a tumor suppressor gene and is regulated by miR-27a.

MicroRNA-125a Reduces Proliferation and Invasion of Oral Squamous Cell Carcinoma Cells by Targeting Estrogen-related Receptor alpha IMPLICATIONS FOR CANCER THERAPEUTICS

Estrogen-related receptor (ESRRA) functions as a transcription factor and regulates the expression of several genes, such as WNT11 and OPN. Up-regulation of ESRRA has been reported in several cancers. However, the mechanism underlying its up-regulation is unclear. Furthermore, the reports regarding the role and regulation of ESRRA in oral squamous cell carcinoma (OSCC) are completely lacking. Here, we show that tumor suppressor miR-125a directly binds to the 3UTR of ESRRA and represses its expression. Overexpression of miR-125a in OSCC cells drastically reduced the level of ESRRA, decreased cell proliferation, and increased apoptosis. Conversely, the delivery of an miR-125a inhibitor to these cells drastically increased the level of ESRRA, increased cell proliferation, and decreased apoptosis. miR-125a-mediated down-regulation of ESRRA impaired anchorage-independent colony formation and invasion of OSCC cells. Reduced cell proliferation and increased apoptosis of OSCC cells were dependent on the presence of the 3UTR in ESRRA. The delivery of an miR-125a mimic to OSCC cells resulted in marked regression of xenografts in nude mice, whereas the delivery of an miR-125a inhibitor to OSCC cells resulted in a significant increase of xenografts and abrogated the tumor suppressor function of miR-125a. We observed an inverse correlation between the expression levels of miR-125a and ESRRA in OSCC samples. In summary, up-regulation of ESRRA due to down-regulation of miR-125a is not only a novel mechanism for its up-regulation in OSCC, but decreasing the level of ESRRA by using a synthetic miR-125a mimic may have an important role in therapeutic intervention of OSCC and other cancers.

Lipid coated mesoporous silica nanoparticles as an oral delivery system for targeting and treatment of intravacuolar Salmonella infections

Lipid coated mesoporous silica nanoparticle (L-MSN) were synthesized for oral delivery of ciprofloxacin for intracellular elimination of Salmonella pathogen. The particle size was found to be between 50-100 nm with a lipid coat of approximately 5 nm thickness. The lipid coating was achieved by sonication of liposomes with the MSN particles and evaluated by CLSMand FTIR studies. The L-MSN particles exhibited lower cytotoxicity compared to bare MSN particles. Ciprofloxacin, a fluoroquinolone antibiotic, loaded into the L-MSN particles showed enhanced antibacterial activity against free drug in in vitro assays. The lipid coat was found to aid in intravacuolar targeting of the drug cargo as observed by confocal microscopy studies. We also observed that a lower dose of antibiotic was sufficient to clear the pathogen from mice and increase their survivability using the L-MSN oral delivery system.

Combined inhalation and oral supplementation of Vitamin A and Vitamin D: A possible prevention and therapy for tuberculosis

Tuberculosis is continuing as a problem of mankind. With evolution, MDR and XDR forms of tuberculosis have emerged from drug sensitive strain. MDR and XDR strains are resistant to most of the antibiotics, making the management more difficult. BCG vaccine is not providing complete protection against tuberculosis. Therefore new infections are spreading at a tremendous rate. At the present moment there is experimental evidence to believe that Vitamin A and Vitamin D has anti-mycobacterial property. It is in this context, we have hypothesized a host based approach using the above vitamins that can cause possible prevention and cure of tuberculosis with minimal chance of resistance or toxicity. (C) 2015 Elsevier Ltd. All rights reserved.

Role of Areca Nut Induced TGF-beta and Epithelial-Mesenchymal Interaction in the Pathogenesis of Oral Submucous Fibrosis

Areca nut consumption has been implicated in the progression of Oral Submucous fibrosis (OSF); an inflammatory precancerous fibrotic condition. Our previous studies have demonstrated the activation of TGF-beta signaling in epithelial cells by areca nut components and also propose a role for epithelial expressed TGF-beta in the pathogenesis of OSF. Although the importance of epithelial cells in the manifestation of OSF has been proposed, the actual effectors are fibroblast cells. However, the role of areca nut and TGF-beta in the context of fibroblast response has not been elucidated. Therefore, to understand their role in the context of fibroblast response in OSF pathogenesis, human gingival fibroblasts (hGF) were treated with areca nut and/or TGF-beta followed by transcriptome profiling. The gene expression profile obtained was compared with the previously published transcriptome profiles of OSF tissues and areca nut treated epithelial cells. The analysis revealed regulation of 4666 and 1214 genes by areca nut and TGF-beta treatment respectively. The expression of 413 genes in hGF cells was potentiated by areca nut and TGF-beta together. Further, the differentially expressed genes of OSF tissues compared to normal tissues overlapped significantly with areca nut and TGF-beta induced genes in epithelial and hGF cells. Several positively enriched pathways were found to be common between OSF tissues and areca nut + TGF-beta treated hGF cells. In concordance, areca nut along with TGF-beta enhanced fibroblast activation as demonstrated by potentiation of alpha SMA, gamma SMA and collagen gel contraction by hGF cells. Furthermore, TGF-beta secreted by areca nut treated epithelial cells influenced fibroblast activation and other genes implicated in fibrosis. These data establish a role for areca nut influenced epithelial cells in OSF progression by activation of fibroblasts and emphasizes the importance of epithelial-mesenchymal interaction in OSF.

Epithelial atrophy in oral submucous fibrosis is mediated by copper (II) and arecoline of areca nut

Exposure of oral cavity to areca nut is associated with several pathological conditions including oral submucous fibrosis (OSF). Histopathologically OSF is characterized by epithelial atrophy, chronic inflammation, juxtaepithelial hyalinization, leading to fibrosis of submucosal tissue and affects 0.5% of the population in the Indian subcontinent. As the molecular mechanisms leading to atrophied epithelium and fibrosis are poorly understood, we studied areca nut actions on human keratinocyte and gingival fibroblast cells. Areca nut water extract (ANW) was cytotoxic to epithelial cells and had a pro-proliferative effect on fibroblasts. This opposite effect of ANW on epithelial and fibroblast cells was intriguing but reflects the OSF histopathology such as epithelial atrophy and proliferation of fibroblasts. We demonstrate that the pro-proliferative effects of ANW on fibroblasts are dependent on insulin-like growth factor signalling while the cytotoxic effects on keratinocytes are dependent on the generation of reactive oxygen species. Treatment of keratinocytes with arecoline which is a component of ANW along with copper resulted in enhanced cytotoxicity which becomes comparable to IC50 of ANW. Furthermore, studies using cyclic voltammetry, mass spectrometry and plasmid cleavage assay suggested that the presence of arecoline increases oxidation reduction potential of copper leading to enhanced cleavage of DNA which could generate an apoptotic response. Terminal deoxynucleotidyl transferase dUTP Nick End Labeling assay and Ki-67 index of OSF tissue sections suggested epithelial apoptosis, which could be responsible for the atrophy of OSF epithelium.

Genomic amplification upregulates estrogen-related receptor alpha and its depletion inhibits oral squamous cell carcinoma tumors in vivo

The ESRRA gene encodes a transcription factor and regulates several genes, such as WNT11 and OPN, involved in tumorigenesis. It is upregulated in several cancers, including OSCC. We have previously shown that the tumor suppressor miR-125a targets ESRRA, and its downregulation causes upregulation of ESRRA in OSCC. Upregulation of ESRRA in the absence of downregulation of miR-125a in a subset of OSCC samples suggests the involvement of an alternative mechanism. Using TaqMan (R) copy number assay, here we report for the first time that the genomic amplification of ESRRA causes its upregulation in a subset of OSCC samples. Ectopic overexpression of ESRRA led to accelerated cell proliferation, anchorage-independent cell growth and invasion, and inhibited apoptosis. Whereas, knockdown of ESRRA expression by siRNA led to reduced cell proliferation, anchorage-independent cell growth and invasion, and accelerated apoptosis. Furthermore, the delivery of a synthetic biostable ESRRA siRNA to OSCC cells resulted in regression of xenografts in nude mice. Thus, the genomic amplification of ESRRA is another novel mechanism for its upregulation in OSCC. Based on our in vitro and in vivo experiments, we suggest that targeting ESRRA by siRNA could be a novel therapeutic strategy for OSCC and other cancers.

Determinación de la implementación de buenas prácticas pecuarias, para el proceso de rastreabilidad de carne en fincas ganaderas de "El Coral", Chontales

Este trabajo se llevó a cabo en el Municipio de “El Coral”, localizado al sureste de la ciudad de Managua. Los objetivos del presente estudio fueron: a) Elaborar el Protocolo de Buenas Prácticas Pecuarias; b) Determinar el grado de cumplimento de BPP en fincas ganaderas del municipio de “El Coral”; y c) Elaborar una estrategia para la implementación del programa de rastreabilidad de carne bovina. La metodología utilizada para el primer objetivo consistió en una revisión de fuentes secundarias y una encuesta a personal clave en tres aspectos de los ítems del Manual de BPP y se abordó tres aspectos: si están de acuerdo con cada ítem, si es exigible y si tienen otro ítem que agregar al protocolo; en el caso del segundo objetivo consistió en un diagnóstico modificado de PROGANIC, con preguntas cerradas aplicado a 70 fincas y su procesamiento en un programa de cómputo Excel; y en el caso del tercer objetivo se aplico una matriz de estrategia para la determinación del rol y proceso de implementación. Se obtuvo como resultado de 29 personas claves que fueron encuestadas, 55 (98%) de los 56 (100%) ítems en total del protocolo propuesto obtuvieron un consenso positivo de parte de los encuestados, para que estos ítem fueran aplicados en el diagnóstico de finca y se resulto un “Protocolo de BPP” de 50 ítem en total, de un total de 72 ítem que tenia el “Manual de BPP de la producción primaria” de la resolución 117-2004 de la Asamblea Nacional. El diagnóstico practicado a 70 fincas ganaderas que representan el 21% del municipio, mostró que el 100% de las fincas ganaderas no cumplen con las BPP definidas en el protocolo de BPP, siendo 9 ítems sobre manejo los que más afectan el no cumplimiento de las BPP. El resultado de la matriz de estrategia muestra que se debe organizar, definir el rol y establecer un mecanismo para la implementación del sistema de rastreabilidad de carne bovina en “El Coral”. Se concluye: a) El “Protocolo de Buenas Prácticas Pecuarias” aplicado en este estudio es apropiado y valido para fincas ganaderas; b) Ninguna de las fincas ganaderas cumple con las BPP para fincas ganaderas; d) Las principales causas de la no aplicación de las BPP son: el desconocimiento de las mismas; la falta de asistencia técnica directa y el bajo incentivo de la producción y e) la principal estrategia para la implementación del programa de rastreabilidad en el municipio, es la sensibilización a todos los actores locales que participaran en el programa.

Evaluacion de los factores del proceso de incubacion que intervienen en la ventana de nacimiento de los pollitos, en la incubadora PIPASA-Nicaragua, en el periodo de Enero a Julio, 2009

Espinoza Gutiérrez, LF; Matey Lechado, MS. 2009. Evaluación de los factores del proceso de incubación que intervienen en la ventana de nacimiento de los pollitos en la Incubadora PIPASA-Nicaragua, en el periodo de Enero a Julio ,2009. Tesis MV. En el grado de licenciatura.Managua, NI Facultad de Ciencia Animal de la Universidad Nacional Agraria (UNA).76p. Fueron evaluados los factores de exposición del huevo: temperatura,humedad relativa, volteo,tiempo de almacenamiento del huevo y edad de la reproductora, el efecto de las variaciones de estos factores sobre la calidad de los pollitos en el proceso de incubación, sobre la ventana de nacimiento de los pollitos, también se identificaron las principales muertes embrionarias (embriopatías) en la planta de Incubación PIPASA Nicaragua. Se realizaron diecisiete experimentos durante: la recepción, selección, y encharolado, almacenamiento en frío, atemperado o precalentamiento, carga a la maquina incubadora, ovocoscopia o miraje, toma de peso, trasferencia a la maquina nacedora y selección de los pollitos por su calidad. Se identificaron tres zonas (Z1, Z2 y Z3) dentro de la máquina; se tomaron tres charolas (pruebas) con huevos en las posiciones de: superior,media e inferior; después de ser transferidas se elaboro un horario de inspección de nacimiento durante las 48 horas (43, 38, 33, 23, y 13) de incubación en la nacedora. Se midieron los factores ambientales (temperatura, humedad) con huevos sensores (datalogger) para monitorear las fluctuaciones de los mismos cada cinco minutos. Una vez sacado el pollito se clasifico según su calidad (A, B) y se realizo la necropsia a huevos no picados para identificar las muertes embrionarias. Mediante ANDEVA se determino el efecto ocasionado por la ubicación (posición y zona) en el proceso de incubación sobre las variables dependientes (pollos nacidos), observando que el inicio del nacimiento (33 h antes del saque) fue de 3.76 % para las bandejas superiores, 1,2% para las bandejas medias y 0.25% para las bandejas inferiores durante la IV inspección, posteriormente (23 hantes del saque) fue 55.54%, 36.62% y 18.67%, respectivamente; para la V inspección, a las 13 horas,fue de un 86.35%, 82.10% y por 74.31% posición de bandejas (VI inspección).Durante la VII inspección, momento del saque, proyectaron un 88.82% para la posición superior,87.91% posición media,81.76% posición inferior.En lo referente al efecto de la zona se encontró que la zona 3 tuvo un valor significativo(P< 0.001)sobre el porcentaje de pollos nacidos de un 58% en comparación con la zona1 y en la zona 2 que ambas obtuvieron 54 %.Al evaluar la edad de la reproductora se observó que en reproductoras menores de 40 semanas de edad el tiempo de incubación fue mayor (497.5 horas); para lotes de 41 a 49 semanas de edad fue de 493 horas promedios y de 494 horas para los lotes mayores de 50 a 54 semanas. El volteo del huevo fértil no influyó sobre la ventana de nacimiento, puesto que este se realizaba de forma adecuada tanto en frecuencia, ángulo, eje y plano de rotación. Existe una relación directa entre la posición de la charola y la calidad del pollo, encontrando mayor calidad en las bandejas superiores con un 28.65% de pollos clase A con relación a las medias e inferiores de 24. 40% y 23.18%, respectivamente. Las principales embriopatías encontradas en la necropsia corresponden a malas posiciones (38%), un 27% de huevos infértiles, 17% de embriones vivos , 5% de embriones con deformidades y 5% de huevos contaminados (huevo bomba)y la edad de mortalidad mas alta fue 18-21 días conun 30%.

Estudio del proceso de implementación y certificación del sistema HACCP en las Empresas Callejas, S.A., Exotic Food, Apronot y Tropifrutas

El presente trabajo fue realizado en las empresas procesadoras de frutas tropicales de Nicaragua que han venido desarrollando actividades encaminadas a la certificación HACCP según datos suministrados por el Ministerio Agropecuario y Forestal (MAG-FOR), las empresas visitadas fueron: APRONOT, ubicada en el Municipio de San Marcos Departamento de Carazo, La empresa Callejas Sequeira, S.A. ubicada en la ciudad de Granada, La empresa Exotic Food ubicada en el Km 107 carretera a Rivas y la empresa TROPIFRUTAS ubicada en Nueva Guinea, Región Autónoma del Atlántico Sur. Se consultaron fuentes secundarías existentes en el país sobre el Análisis de Riesgos y Críticos Puntos de Control (HACCP), así como información de Internet. Para conocer la situación actual del proceso de certificación y adopción del Análisis de Riesgos y Críticos Puntos de Control (HACCP) en la Industria Agroalimentaria se procedió a entrevistar a funcionarios del Ministerio Agropecuario y Forestal (MAGFOR), y del Ministerio de Fomento, Industria y Comercio (MIFIC). La industria agroalimentaria de Nicaragua ha adoptado el sistema de Análisis de Riesgos y Críticos Puntos de Control (HACCP) y se encuentra en un proceso de verificación y Auditoria para obtener la certificación, todas las empresas entrevistadas mostraron toda la voluntad de certificar su producto ya que les abre las puertas en la búsqueda de mejores oportunidades de mercados para sus productos. Las mayores dificultades para lograr la certificación radican en la falta de recursos financieros para ejecutar las recomendaciones de los inspectores, así como la dificultad de garantizar la trazabilidad del origen de la materia prima.

Estudio del proceso de floracion inducido por el acido giberelico (AG3) en nueve cultivares de quequisque (Xanthosoma spp) en Nicaragua

Se evaluó la floración inducida por tres aplicaciones (semanal y quincenal) de 0,500 y 1000 ppm de acido giberélico (AG3) en los cultivares de quequisque San Ramón, El Tuma, San Antonio, La Rampla, Malaquías,San Lucas, Casitas, Masaya y Apalí. Se utilizó el diseño de bloque completo al azar, con tres bloques, cuatro plantas/bloque y 12 plantas/cultivar. Se evaluó la altura de planta, grosor del pseudotallo, número de hojas y área foliar; momento de floración, número de flores, estructuras relacionadas a la floración, germinación del polen y receptividad del ovario. Previo a la aplicación los cultivares no diferían en desarrollo morfológico. El AG3 retrasó drásticamente el crecimiento de las plantas. Los cultivares iniciaron la floración 88-114 días después de la primera aplicación (ddpa). San Ramón, San Antonio y Malaquías iniciaron la floración 88 ddpa. La Rampla y San Lucas la iniciaron 114 ddpa. La floración duró dos meses. Las plantas produjeron 8 flores promedio. El cultivar San Antonio aplicado con 500 ppm de AG3 semanalmente produjo 102 flores. Aplicaciones quincenales de 1000 ppm indujeron 25 flores en San Ramón y El Tuma, 8 en San Lucas y Casitas. Además se produjeron: brácteas, brácteas múltiples, bráctea hoja de bandera y brácteas cubriendo hoja de bandera. La germinación del polen fue 90-100%. Altas temperaturas al inicio de la floracion afectaron la ántesis y la producción del polen. La receptividad del ovario fue 100%. Este es el primer estudio de inducción de floración en quequisque cuyos resultados son la base de futuros trabajos de mejora genética en el cultivo.