Inhibition of extracellular matrix assembly induces the expression of osteogenic markers in skeletal muscle cells by a BMP-2 independent mechanism

<p>Abstract</p> <p>Background</p> <p>The conversion of one cell type into another has been suggested to be, at the molecular level, the consequence of change(s) in the expression level of key developmental genes. Myoblasts have the ability to differentiate either to skeletal muscle or osteogenic lineage depending of external stimuli. Extracellular matrix (ECM) has been shown to be essential for skeletal muscle differentiation, through its direct interaction with myoblasts' cell receptors. We attempt to address if ECM also plays a role in the osteogenic differentiation of skeletal muscle cells.</p> <p>Results</p> <p>Inhibition of proteoglycan sulfation by sodium chlorate in myoblast cultures strongly affects ECM synthesis and deposition and induces the expression of the osteogenic lineage markers alkaline phosphatase (ALP) and osteocalcin in mononuclear cells. Induction of ALP by sodium chlorate does not affect the expression of specific muscle determination transcription factors, such as MyoD and Myf-5, in the same cells. The osteogenic transcription factor Cbfa-1 expression is also unaffected. Induction of ALP is not inhibited by a soluble form of BMP receptor IA. This suggests that the deviation of the myogenic pathway of C2C12 myoblasts into the osteogenic lineage by inhibitors of proteoglycan sulfation is BMP-2 independent. The increase of osteogenic markers expression can be totally prevented by an exogenous ECM. Interestingly, a similar BMP-2-independent ALP activity induction can be observed in myoblasts cultured on an ECM previously synthesized by BMP-2 treated myoblasts. Under <it>in vivo </it>conditions of increased ECM turn-over and deposition, as in the <it>mdx </it>dystrophic muscle and during skeletal muscle regeneration, an induction and relocalization of ALP is observed in a subpopulation of skeletal muscle fibers, whereas in normal skeletal muscle, ALP expression is restricted to blood vessels and some endomysial mononuclear cells.</p> <p>Conclusion</p> <p>These results suggest that signals arising from the ECM induce the expression of osteogenic markers in muscle cells by a mechanism independent of BMP-2 and without affecting the expression of key muscle or osteogenic determination genes. An induction and relocalization of ALP is also observed in <it>mdx </it>and regenerating skeletal muscles, <it>in vivo </it>conditions of increased muscle ECM deposition or turnover.</p

Evaluation of the Effect of Azospirillum brasilense and Mycorrhizal Fungi of the Soil in Yacon Grown in a Greenhouse

In order to evaluate the effect of Azospirillum brasilense and mycorrhizal fungi in the soil on the nutrition of the yacon crop (Smallanthus sonchifolius [(Poeppig & Endlicher) H. Robinson]), determinations of agronomic parameters and the health status of the plants were carried out, under greenhouse conditions. The tests were carried out at the time of the implantation of the culture: the propagules were inoculated with A. brasilense and with native mycorrhizal fungi, generating four treatments, including the control and the co-inoculation of the consortium of the microorganisms under study (T0: control or control without inoculation; T1: inoculation with native A. brasilense; T2: inoculation with native mycorrhizal fungi and T3: joint inoculation with A. brasilense and native mycorrhizal fungi. The results indicate that co-inoculation with A. brasilense and native mycorrhizal fungi increased plant growth in height, leaf area, biomass, dry matter and yields significantly in greenhouse production. It was determined that the application of the selected microorganisms has a plant growth promoting effect, increasing growth and productivity, greenhouse grown yacón.Fil: Di Barbaro, Maria Gabriela. Universidad Nacional de Catamarca; ArgentinaFil: Andrada, Horacio Enrique. Universidad Nacional de Catamarca; ArgentinaFil: del Valle, Eleodoro Eduardo. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Ciencias Agropecuarias del Litoral. - Universidad Nacional del Litoral. Instituto de Ciencias Agropecuarias del Litoral.; Argentina. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; ArgentinaFil: Brandan, Celia Ines. Universidad Nacional de Tucumán; Argentin

Actividad biológica de suelos cultivados con yacón (Smallanthus sonchifolius Poep. & Endl) H. Robinson, inoculados con Azospirillum brasilense y hongos micorrícicos nativos

El suelo está influenciado por las raíces de las plantas, ya que los exudados radicales afectan los procesos físicos, químicos y biológicos del suelo. El objetivo de la investigación fue evaluar la actividad microbiana en suelos donde se ha cultivado yacón (Smallanthus sonchifolius), inoculado con microorganismos promotores del crecimiento de las plantas. El diseño experimental empleado fue bloques al azar con tres repeticiones por tratamiento. Cada repetición correspondió a una parcela de 3 x 3m con 25 plantas, en 5 líneas de cultivo distanciadas a 70 cm. Se aplicaron cuatro tratamientos en el momento de la plantación y consistieron en la inoculación de propágulos de yacón con Azospirillum brasilense (T1), hongos micorrícicos nativos (T2), con el consorcio microbiano (T3) y en los tratamientos testigos los propágulos sin inocular con estos microorganismos (T0). Muestras de suelo fueron colectadas en lotes cultivados con yacón situados en la Provincia de Catamarca, al noroeste de Argentina, al momento de implantación y de cosecha durante tres ciclos agrícolas. Se determinó la Actividad Biológica Total (ABT) por Hidrólisis del Diacetato de Fluoresceína a las muestras colectadas. La ABT del suelo fue afectada por los diferentes tratamientos del cultivo, la cual se elevó en los suelos estudiados, difiriendo estadísticamente con los testigos. También se observaron diferencias significativas entre la ABT de los suelos al inicio de los cultivos con los valores registrados a la cosecha de este. La inoculación de propágulos de yacón con A. brasilense y hongos micorrícicos nativos afecta la ABT de los suelos cultivados, aumentando significativamente.The soil is influenced by the roots of plants because the exudate radicals affect its physical, chemical, and biological processes. The aim of this research was to evaluate the microbial activity of soils cultivated with yacón (Smallanthus sonchifolius Poep. & Endl) H. Robinson and inoculated with microorganisms which promote plant growth. The experimental design used was randomized blocks with three repetitions per treatment. Each repetition corresponded to a 3 x 3m plot with 25 plants, in 5 cultivation lines with a distance of 70 cm apart. Four treatments were applied at the time of plantation. These consisted of yacón propagules inoculated with Azospirillum brasilense (T1) and native mycorrhizal fungi (T2) inoculated with the microbial consortium (T3). In the control treatments, the propagules were not inoculated with these microorganisms (T0). Soil samples were collected in lots cultivated with yacón located in the Province of Catamarca, in the northwest of Argentina. The samples were collected at the time of implantation and harvest during three agricultural cycles. The total Biological Activity (TBA) of the collected samples was determined by Fluorescein Diacetate Hydrolysis. The TBA of the soil was affected by the different crop treatments, which rose in the analyzed soils and was statistically different from the control treatments. Significant differences were also observed between the TBA of the soils at the beginning of the crops in comparison with the recorded values of the crop at the time of harvest. The inoculation of yacón propagules with A. brasilense and native mycorrhizal fungi affects the TBA of the cultivated soils marking a significant increase.Fil: Di Barbaro, Maria Gabriela. Universidad Nacional de Catamarca; ArgentinaFil: Andrada, Horacio Enrique. Universidad Nacional de Catamarca; ArgentinaFil: del Valle, Eleodoro Eduardo. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Ciencias Agropecuarias del Litoral. - Universidad Nacional del Litoral. Instituto de Ciencias Agropecuarias del Litoral.; Argentina. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; ArgentinaFil: Brandan, Celia Ines. Universidad Nacional de Tucumán; Argentin

Biofertilization of topinambur with Azospirillum brasilense and native mycorrhical fungi, cultivated in the Central Valley of Catamarca, Argentina

To evaluate the effect of Azospirillum and mycorrhizal soil fungi on the nutrition of the Jerusalem artichoke crop (Helianthus tuberosus L.), determinations of agronomic parameters and the health status of the plants were carried out under field conditions. The tests were carried out, at the time of the implantation of the culture: the ?seeds? were inoculated with A. brasilense and with native mycorrhizal fungi, generating four treatments including the control and the co-inoculation of the consortium of the microorganisms under study (T0: control or uninoculated control; T1: inoculation with native A. brasilense; T2: inoculation with native mycorrhizal fungi and T3: joint inoculation with A. brasilense and native mycorrhizal fungi). The results indicate that co-inoculation with A. brasilense and native mycorrhizal fungi, significantly increased plant growth in height, leaf area, biomass, dry matter, and yields. It was determined that the application of the selected microorganisms has a promoting effect of plant growth, increasing the growth and productivity of the topinambur crop.Fil: Di Barbaro, Maria Gabriela. Universidad Nacional de Catamarca; ArgentinaFil: Andrada, Horacio Enrique. Universidad Nacional de Catamarca; ArgentinaFil: Batallan Morales, Silvana Rosa. Universidad Nacional de Catamarca; ArgentinaFil: Espeche Acosta, Eliana. Universidad Nacional de Catamarca; ArgentinaFil: Rizo, Melisa. Universidad Nacional de Catamarca; ArgentinaFil: Gonzalez Basso, Maria Valeria. Universidad Nacional de Catamarca; ArgentinaFil: del Valle, Eleodoro Eduardo. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Ciencias Agropecuarias del Litoral. - Universidad Nacional del Litoral. Instituto de Ciencias Agropecuarias del Litoral.; Argentina. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; ArgentinaFil: Brandan, Celia Ines. Universidad Nacional de Tucumán; Argentin

Composición y abundancia de poblaciones fúngicas y bacterianas y sus actividades: respuesta al manejo agroecológico

La agroecología propone la autorregulación para lograr un equilibrio agrícola sustentable. Por lo tanto, considerando la rápida respuesta de las comunidades microbianas ante los pequeños cambios en el uso del suelo, el objetivo de este estudio fue evaluar su respuesta ante el manejo agroecológico versus convencional de la agricultura extensiva. El muestreo del suelo se llevó a cabo en 2016 y 2017 en tres tratamientos diferentes utilizando la secuencia soja/maíz (Glycine max L./Zea mays L.) como cultivo principal: Agroecológico (AE), convencional con cultivos de cobertura (CC) y convencional sin cultivos de cobertura (control). Las especies utilizadas como cultivos de cobertura fueron trigo (Triticum aestivum), vicia (Vicia sativa L.), avena (Avena sativa L.) y nabo forrajero (Raphanus sativus L.). El tratamiento CC mostró el mayor valor de hidrólisis de fluoresceína de diacetato, con valores 63,2% y 12,1% mayores que AE y el control, respectivamente. Sin embargo, el tratamiento AE registró la mayor relación hongo:bacteria (44,8) y el cociente metabólico más bajo (1,14), lo que indica una mejora en la eficiencia metabólica y la calidad del suelo. No se registraron diferencias significativas en la abundancia de las comunidades fúngicas y bacterianas entre los tratamientos. Los resultados sugieren que el manejo agroecológico se caracterizó por el predominio de los hongos del suelo y una mayor eficiencia metabólica microbiana en comparación con el manejo convencional. Se evidenció un uso más eficiente de los sustratos de carbono en los sistemas agroecológicos, lo que podría contrarrestar el efecto negativo de la falta de fertilización sintética y la labranza reducida en el largo plazo. Este trabajo demuestra que las herramientas agrícolas sustentables con un manejo adecuado se pueden utilizar efectivamente para preservar la calidad del suelo.EEA PergaminoFil: Chavarria, Diego N. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina.Fil: Pérez Brandan, Carolina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Salta; Argentina.Fil: Serri, Dannae Lilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina.Fil: Meriles, José M. Universidad Nacional de Córdoba. Instituto Multidisciplinario de Biología Vegetal (IMBIV- CONICET); Argentina.Fil: Restovich, Silvina Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Pergamino. Laboratorio Suelo; Argentina.Fil: Andriulo, Adrian Enrique. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Pergamino. Laboratorio Suelo; Argentina.Fil: Jacquelin, Luis. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Pergamino. Laboratorio Suelo; Argentina.Fil: Vargas Gil, Silvina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina

Use of an orthovoltage X-ray treatment unit as a radiation research system in a small-animal cancer model

<p>Abstract</p> <p>Background</p> <p>We explore the use of a clinical orthovoltage X-ray treatment unit as a small-animal radiation therapy system in a tumoral model of cervical cancer.</p> <p>Methods</p> <p>Nude mice were subcutaneously inoculated with 5 × 10⁶HeLa cells in both lower limbs. When tumor volume approximated 200 mm³treatment was initiated. Animals received four 2 mg/kg intraperitoneal cycles (1/week) of cisplatin and/or 6.25 mg/kg of gemcitabine, concomitant with radiotherapy. Tumors were exposed to 2.5 Gy/day nominal surface doses (20 days) of 150 kV X-rays. Lead collimators with circular apertures (0.5 to 1.5 cm diameter) were manufactured and mounted on the applicator cone to restrict the X-ray beam onto tumors. X-ray penetration and conformality were evaluated by measuring dose at the surface and behind the tumor lobe by using HS GafChromic film. Relative changes in tumor volume (RTV) and a clonogenic assay were used to evaluate the therapeutic response of the tumor, and relative weight loss was used to assess toxicity of the treatments.</p> <p>Results</p> <p>No measurable dose was delivered outside of the collimator apertures. The analysis suggests that dose inhomogeneities in the tumor reach up to ± 11.5% around the mean tumor dose value, which was estimated as 2.2 Gy/day. Evaluation of the RTV showed a significant reduction of the tumor volume as consequence of the chemoradiotherapy treatment; results also show that toxicity was well tolerated by the animals.</p> <p>Conclusion</p> <p>Results and procedures described in the present work have shown the usefulness and convenience of the orthovoltage X-ray system for animal model radiotherapy protocols.</p

Purification of rat liver N-heparan-sulfate sulfotransferase

N-Heparan-sulfate sulfotransferase catalyzes the transfer of sulfate from 3\u27-phosphoadenilyl sulfate to the nitrogen of glucosamine in heparan sulfate. This reaction is an obligatory step for subsequent epimerization of D-glucuronic to L-iduronic acid and of O-sulfation of the sugar chains. We have purified this sulfotransferase from rat liver membranes to apparent homogeneity using a combination of conventional and affinity chromatography on DEAE-Sephacel, heparin-agarose, 3\u27,5\u27-ADP-agarose, wheat germ-Sepharose, and finally a glycerol gradient. The pure enzyme is a glycoprotein with an apparent molecular weight of 97,000. It was enriched in specific activity 65,000-fold over the homogenate. The recovery of activity was 4% of that of the homogenate. Preliminary enzymatic characterization of the purified sulfotransferase indicates a high degree of substrate specificity. Transfer of sulfate occurs to heparan sulfate, N-heparan sulfate, and N-desulfated heparin, but not to N-acetylated heparan sulfate, N-acetylated heparin, chondroitin, chondroitin sulfate, and tyrosine-containing tripeptides

Hypoxia and HIF-1α induce CTGF expression in fibroblast and myoblasts, and require TGF-β Smad pathway signaling

status: publishe

Nilotinib impairs skeletal myogenesis by increasing myoblast proliferation

Abstract Background Tyrosine kinase inhibitors (TKIs) are effective therapies with demonstrated antineoplastic activity. Nilotinib is a second-generation FDA-approved TKI designed to overcome Imatinib resistance and intolerance in patients with chronic myelogenous leukemia (CML). Interestingly, TKIs have also been shown to be an efficient treatment for several non-malignant disorders such fibrotic diseases, including those affecting skeletal muscles. Methods We investigated the role of Nilotinib on skeletal myogenesis using the well-established C2C12 myoblast cell line. We evaluated the impact of Nilotinib during the time course of skeletal myogenesis. We compared the effect of Nilotinib with the well-known p38 MAPK inhibitor SB203580. MEK1/2 UO126 and PI3K/AKT LY294002 inhibitors were used to identify the signaling pathways involved in Nilotinib-related effects on myoblast. Adult primary myoblasts were also used to corroborate the inhibition of myoblasts fusion and myotube-nuclei positioning by Nilotinib. Results We found that Nilotinib inhibited myogenic differentiation, reducing the number of myogenin-positive myoblasts and decreasing myogenin and MyoD expression. Furthermore, Nilotinib-mediated anti-myogenic effects impair myotube formation, myosin heavy chain expression, and compromise myotube-nuclei positioning. In addition, we found that p38 MAPK is a new off-target protein of Nilotinib, which causes inhibition of p38 phosphorylation in a similar manner as the well-characterized p38 inhibitor SB203580. Nilotinib induces the activation of ERK1/2 and AKT on myoblasts but not in myotubes. We also found that Nilotinib stimulates myoblast proliferation, a process dependent on ERK1/2 and AKT activation. Conclusions Our findings suggest that Nilotinib may have important negative effects on muscle homeostasis, inhibiting myogenic differentiation but stimulating myoblasts proliferation. Additionally, we found that Nilotinib stimulates the activation of ERK1/2 and AKT. On the other hand, we suggest that p38 MAPK is a new off-target of Nilotinib. Thus, there is a necessity for future studies to investigate the long-term effects of TKIs on skeletal muscle homeostasis, along with potential detrimental effects in cell differentiation and proliferation in patients receiving TKI therapies