Extracción de proteínas secretadas por el fitopatógeno Macrophomina phaseolina: Selección de un método eficiente que incluye estímulo con tejido de su hospedero

El hongo necrotrófico, mundialmente distribuido, Macrophomina phaseolina es el agente causal de enfermedades de cultivos de importancia económica como la podredumbre carbonosa de la soja. Este hongo secreta una amplia variedad de proteínas y metabolitos que le permiten invadir la planta e iniciar el proceso de infección. La contribución de las proteínas con actividad hidrolítica secretadas por este hongo en el proceso de infección ha sido ampliamente estudiada, las proteínas sin actividad enzimática también podrían poseer un papel importante en este proceso. El análisis de proteínas totales permitiría ampliar el conocimiento sobre este patógeno, y establecer estrategias más eficientes para su control. El objetivo del presente trabajo fue evaluar tres métodos de extracción de proteínas secretadas por M. phaseolina. El hongo fue crecido en medio líquido PDB y Czapek-Dox (CZP) con y sin suplemento de hojas de soja. Las proteínas fueron extraídas del filtrado liofilizado del medio PDB utilizando tres métodos de extracción, y analizadas mediante SDS-PAGE. Se seleccionó el método de precipitación de proteínas con ácido tricloroacético en acetona ya que mostró una mejor resolución del perfil proteico. El filtrado de M. phaseolina crecido en PDB suplementado con hojas de soja (MpPDBs) presentó un mayor rendimiento de extracción de proteínas secretadas entre todas las condiciones evaluadas. Los perfiles proteicos del medio PDB con y sin suplemento, presentaron siete bandas diferenciales, una de ellas específica, detectada en MpPDBs. Estos resultados constituyen una base para estudios sobre el papel de proteínas secretadas por el hongo en el proceso de infección

Chemoselective and stereoselective lithium carbenoid mediated cyclopropanation of acyclic allylic alcohols

The reaction of geraniol with different lithium carbenoids generated from n-BuLi and the corresponding dihaloalkane has been evaluated. The reaction occurs in a chemo and stereoselective manner, which is consistent with a directing effect from the oxygen of the allylic moiety. Furthermore, a set of polyenes containing allylic hydroxyl or ether groups were chemoselectively and stereoselectively converted into the corresponding gem-dimethylcyclopropanes in one single step in moderate to good yields mediated by a lithium carbenoid generated in situ by the reaction of n-BuLi and 2,2-dibromopropane

A novel PKC activating molecule promotes neuroblast differentiation and delivery of newborn neurons in brain injuries

Neural stem cells are activated within neurogenic niches in response to brain injuries. This results in the production of neuroblasts, which unsuccessfully attempt to migrate toward the damaged tissue. Injuries constitute a gliogenic/non-neurogenic niche generated by the presence of anti-neurogenic signals, which impair neuronal differentiation and migration. Kinases of the protein kinase C (PKC) family mediate the release of growth factors that participate in different steps of the neurogenic process, particularly, novel PKC isozymes facilitate the release of the neurogenic growth factor neuregulin. We have demonstrated herein that a plant derived diterpene, (EOF2; CAS number 2230806-06-9), with the capacity to activate PKC facilitates the release of neuregulin 1, and promotes neuroblasts differentiation and survival in cultures of subventricular zone (SVZ) isolated cells in a novel PKC dependent manner. Local infusion of this compound in mechanical cortical injuries induces neuroblast enrichment within the perilesional area, and noninvasive intranasal administration of EOF2 promotes migration of neuroblasts from the SVZ towards the injury, allowing their survival and differentiation into mature neurons, being some of them cholinergic and GABAergic. Our results elucidate the mechanism of EOF2 promoting neurogenesis in injuries and highlight the role of novel PKC isozymes as targets in brain injury regeneration

Antibacterial activity of the alkaloid extract and isolated compounds from Croton bonplandianum Baill. (Euphorbiaceae)

ABSTRACT The antibacterial activity of the alkaloid extract from the leaves of Croton bonplandianum Baill. and its main compounds, sparsiflorine and crotsparine, was tested against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa by the resazurin microtitre-plate method. Pure compounds were identified by spectroscopic techniques, mainly 1D and 2D NMR. The alkaloid extract showed activity particularly against the S. aureus and P. aeruginosa. Regarding the pure compounds, the crotsparine was inactive against the microorganisms assayed, whereas the sparsiflorine indicated a moderate activity similar to the alkaloid extract. The Pseudomonas aeruginosa was the most sensitive of the tested microorganisms with MIC of 0.141 mg/mL. The results suggest that the activity of the extract may be credited mainly to the presence of the sparsiflorine. Although the activity of the sparsiflorine does not get close to the antimicrobial drugs in clinical use, it still could be a lead compound for the development of new antibacterial substances

Extracción de proteínas secretadas por el fitopatógeno Macrophomina phaseolina: Selección de un método eficiente que incluye estímulo con tejido de su hospedero

The globally distributed necrotrophic fungus Macrophomina phaseolina is the causal agent of economically important crop diseases such as soybean charcoal rot. This fungus secretes a wide variety of proteins and metabolites that allow it to invade the plant and initiate the infection process. The role of fungi secreted proteins with hydrolytic activity in the infection process has been extensively studied; proteins without enzymatic activity could also play an important role in this process. The analysis of total proteins would allow to broaden the knowledge about this pathogen and establish more efficient strategies for its control. The objective of the present work was to evaluate three methods for the extraction of proteins secreted by M. phaseolina. The fungus was grown in potato dextrose broth (PDB) and Czapek-Dox (CZP) with and without soybean leaf supplementation. Proteins were extracted from the lyophilized filtrate of PDB medium using three extraction methods and analyzed by SDS-PAGE. The protein precipitation with trichloroacetic acid in acetone was selected because it showed a better resolution of the protein profile. The filtrate of M. phaseolina grown in PDB supplemented with soybean (MpPDBs) presented the highest yield of protein extraction of secreted proteins among all conditions evaluated. The protein profiles of PDB medium with and without supplementation showed seven differential bands, one of the specific, detected in MpPDBs. These results constitute a basis for studies on the implication of proteins secreted by the fungus in the infection process.El hongo necrotrófico, mundialmente distribuido, Macrophomina phaseolina es el agente causal de enfermedades de cultivos de importancia económica como la podredumbre carbonosa de la soja. Este hongo secreta una amplia variedad de proteínas y metabolitos que le permiten invadir la planta e iniciar el proceso de infección. La contribución de las proteínas con actividad hidrolítica secretadas por este hongo en el proceso de infección ha sido ampliamente estudiada, las proteínas sin actividad enzimática también podrían poseer un papel importante en este proceso. El análisis de proteínas totales permitiría ampliar el conocimiento sobre este patógeno, y establecer estrategias más eficientes para su control. El objetivo del presente trabajo fue evaluar tres métodos de extracción de proteínas secretadas por M. phaseolina. El hongo fue crecido en medio líquido PDB y Czapek-Dox (CZP) con y sin suplemento de hojas de soja. Las proteínas fueron extraídas del filtrado liofilizado del medio PDB utilizando tres métodos de extracción, y analizadas mediante SDS-PAGE. Se seleccionó el método de precipitación de proteínas con ácido tricloroacético en acetona ya que mostró una mejor resolución del perfil proteico. El filtrado de M. phaseolina crecido en PDB suplementado con hojas de soja (MpPDBs) presentó un mayor rendimiento de extracción de proteínas secretadas entre todas las condiciones evaluadas. Los perfiles proteicos del medio PDB con y sin suplemento, presentaron siete bandas diferenciales, una de ellas específica, detectada en MpPDBs. Estos resultados constituyen una base para estudios sobre el papel de proteínas secretadas por el hongo en el proceso de infección

Screening of Natural Products Inhibitors of SARS-CoV-2 Entry

The COVID-19 pandemic has led to the search for new molecules with antiviral activity against SARS-CoV-2. The entry of the virus into the cell is one of the main targets for inhibiting SARS-CoV-2 infection. Natural products are an important source of new therapeutic alternatives against diseases. Pseudotyped viruses allow the study of SARS-CoV-2 viral entry inhibitors, and due to their simplicity, they allow the screening of a large number of antiviral candidates in Biosafety Level 2 facilities. We used pseudotyped HIV-1 with the D614G SARS-CoV-2 spike glycoprotein to test its ability to infect ACE2-expressing HEK 293T cells in the presence of diverse natural products, including 21 plant extracts, 7 essential oils, and 13 compounds from plants and fungi. The 50% cytotoxic concentration (CC50) was evaluated using the resazurin method. From these analyses, we determined the inhibitory activity of the extract of Stachytarpheta cayennensis, which had a half-maximal inhibitory concentration (IC50) of 91.65 µg/mL, a CC50 of 693.5 µg/mL, and a selectivity index (SI) of 7.57, indicating its potential use as an inhibitor of SARS-CoV-2 entry. Moreover, our work indicates the usefulness of the pseudotyped-virus system in the screening of SARS-CoV-2 entry inhibitors