Filter Retardation Assay for Detecting and Quantifying Polyglutamine Aggregates Using Caenorhabditis elegans Lysates

Protein aggregation is a hallmark of several neurodegenerative diseases and is associated with impaired protein homeostasis. This imbalance is caused by the loss of the protein's native conformation, which ultimately results in its aggregation or abnormal localization within the cell. Using a C. elegans model of polyglutamine diseases, we describe in detail the filter retardation assay, a method that captures protein aggregates in a cellulose acetate membrane and allows its detection and quantification by immunoblotting

Nuclear/Cytoplasmic Fractionation of Proteins from Caenorhabditis elegans

C. elegans is widely used to investigate biological processes related to health and disease. To study protein localization, fluorescently-tagged proteins can be used in vivo or immunohistochemistry can be performed in whole worms. Here, we describe a technique to localize a protein of interest at a subcellular level in C. elegans lysates, which can give insight into the location, function and/or toxicity of proteinsNational Institutes of Health National Centre for Research Resources (NIH)European Research Council (ERC)USANIH National Center for Research Resources (NCRR)Japan National BioResource Projec

Regulation of protein homeostasis in neurodegenerative diseases:the role of coding and non-coding genes

Protein homeostasis is fundamental for cell function and survival, because proteins are involved in all aspects of cellular function, ranging from cell metabolism and cell division to the cell's response to environmental challenges. Protein homeostasis is tightly regulated by the synthesis, folding, trafficking and clearance of proteins, all of which act in an orchestrated manner to ensure proteome stability. The protein quality control system is enhanced by stress response pathways, which take action whenever the proteome is challenged by environmental or physiological stress. Aging, however, damages the proteome, and such proteome damage is thought to be associated with aging-related diseases. In this review, we discuss the different cellular processes that define the protein quality control system and focus on their role in protein conformational diseases. We highlight the power of using small organisms to model neurodegenerative diseases and how these models can be exploited to discover genetic modulators of protein aggregation and toxicity. We also link findings from small model organisms to the situation in higher organisms and describe how some of the genetic modifiers discovered in organisms such as worms are functionally conserved throughout evolution. Finally, we demonstrate that the non-coding genome also plays a role in maintaining protein homeostasis. In all, this review highlights the importance of protein and RNA homeostasis in neurodegenerative diseases

Aggregatibacter (Actinobacillus) Actinomycetemcomitans y Enfermedad Periodontal

La enfermedad periodontal ha sido asociada siempre al biofilm o placa dental. El mismo está compuesto por microorganismos taxonómicamente diversos y se localiza principalmente, en el surco gingival. Su composición es tan compleja, que la mayoría de las bacterias comensales de la cavidad bucal están presentes. En el biofilm que produce enfermedades gingivoperiodontales, las bacterias Gram negativas representan la biota dominante y entre ellos Aggregatibacter (Actinobacillus) actinomycetemcomitans reviste particular importancia. Este microorganismo posee elementos estructurales y genera distintos productos que se comportan como antígenos frente al huésped, quien responde de maneras diferentes, siendo éste un factor esencial en la progresión de la enfermedad. Es importante conocer la patogenicidad de la población constituyente del biofilm y su acción sobre los tejidos periodontales, así como, destacar que el buen diagnóstico tanto clínico como radiográfico sumado al estudio microbiológico permitirá instaurar el tratamiento adecuado para el mejor pronóstico evitando eventuales complicaciones sistémicas que pudieran comprometer el estado general del paciente

Reactivación de Lactobacillus spp. aislados de saliva y alimentos

Objetivo: El objetivo del presente estudio fue evaluar la recuperación in vitro del género Lactobacillus de origen bucal y alimentario en medios referenciados como selectivos tales como Rogosa y MRS. Material y Método: se seleccionaron pacientes con caries activas y se recolectaron muestras de saliva que se cultivaron en medio Rogosa; a partir del aislamiento primario las cepas se conservaron en caldo MRS. Se procesaron muestras de leche y agente coagulante artesanal utilizado en la producción de quesos artesanales, los aislamientos primarios se efectuaron en medio Rogosa. Resultados: se aislaron, purificaron y conservaron cepas de Lactobacillus spp. a partir de muestras de saliva y alimentos (leche y agente coagulante artesanal). Conclusión: se demostró que la conservacióna corto y largo plazo del microorganismo en caldo MRS con 15% de glicerol (crioprotector) a -20ºC, pudieron, posteriormente ser reactivadas

Diseño y estandarización de la técnica de PCR para Porphyromonas gingivalis

El objetivo del presente trabajo fue diseñar y estandarizar la técnica de PCR para detección en líquido gingival de Porphyromonas gingivalis, en pacientes con enfermedad periodontal. Material y métodos: Se utilizaron iniciadores específicos para el gen ARNr 16s de Porphyromonas gingivalis. La especificidad de los iniciadores se ensayó utilizando material genético extraído de la cepa de referencia Porphyromonas gingivalis ATCC 33277. Se ajustaron las condiciones de amplificación y concentraciones de la mezcla de reacción. Para validar la técnica se aplicó a diez muestras clínicas de líquido gingival de pacientes con enfermedad periodontal. Resultados: Se vizualizaron bandas nítidas a 197pb utilizando cebadores específicos en seis muestras clínicas, y se obtuvo sensibilidad hasta 15 ug/ml de ADN purificado de la cepa de referencia ATCC 33277.Conclusiones: Se validó y estandarizó una PCR sencilla para la detección de Porphyromonas gingivalis en líquido gingiva

Intercambio de experiencias productivas sobre quesos artesanales en San Luis del Palmar, Corrientes

Considerando la ancestral tradición de la manufactura quesera en la Provincia de Corrientes y el acercamiento de pequeños productores zonales a la Universidad ocurrido en 2014. El Grupo BiMIA realizó, en el año 2015, una Jornada Práctica de elaboración de quesos en San Luis del Palmar, enfatizando las Buenas Prácticas de Manufactura y el uso de insumos tradicionales a fin de mantener esta usanza, que genera un producto de características particulares y puede ocasionar una mayor demanda, contribuyendo al desarrollo sustentable y sostenibilidad productiva de la región. Se presenta la experiencia y algunos resultados de importancia.

Identification of an RNA Polymerase III Regulator Linked to Disease-Associated Protein Aggregation.

Protein aggregation is associated with age-related neurodegenerative disorders, such as Alzheimer's and polyglutamine diseases. As a causal relationship between protein aggregation and neurodegeneration remains elusive, understanding the cellular mechanisms regulating protein aggregation will help develop future treatments. To identify such mechanisms, we conducted a forward genetic screen in a C. elegans model of polyglutamine aggregation and identified the protein MOAG-2/LIR-3 as a driver of protein aggregation. In the absence of polyglutamine, MOAG-2/LIR-3 regulates the RNA polymerase III-associated transcription of small non-coding RNAs. This regulation is lost in the presence of polyglutamine, which mislocalizes MOAG-2/LIR-3 from the nucleus to the cytosol. We then show biochemically that MOAG-2/LIR-3 can also catalyze the aggregation of polyglutamine-expanded huntingtin. These results suggest that polyglutamine can induce an aggregation-promoting activity of MOAG-2/LIR-3 in the cytosol. The concept that certain aggregation-prone proteins can convert other endogenous proteins into drivers of aggregation and toxicity adds to the understanding of how cellular homeostasis can be deteriorated in protein misfolding diseases

Deletion of SERF2 in mice delays embryonic development and alters amyloid deposit structure in the brain

In age-related neurodegenerative diseases, like Alzheimer's and Parkinson's, disease-specific proteins become aggregation-prone and form amyloid-like deposits. Depletion of SERF proteins ameliorates this toxic process in worm and human cell models for diseases. Whether SERF modifies amyloid pathology in mammalian brain, however, has remained unknown. Here, we generated conditional Serf2 knockout mice and found that full-body deletion of Serf2 delayed embryonic development, causing premature birth and perinatal lethality. Brain-specific Serf2 knockout mice, on the other hand, were viable, and showed no major behavioral or cognitive abnormalities. In a mouse model for amyloid-β aggregation, brain depletion of Serf2 altered the binding of structure-specific amyloid dyes, previously used to distinguish amyloid polymorphisms in the human brain. These results suggest that Serf2 depletion changed the structure of amyloid deposits, which was further supported by scanning transmission electron microscopy, but further study will be required to confirm this observation. Altogether, our data reveal the pleiotropic functions of SERF2 in embryonic development and in the brain and support the existence of modifying factors of amyloid deposition in mammalian brain, which offer possibilities for polymorphism-based interventions. </p

Deletion of SERF2 in mice delays embryonic development and alters amyloid deposit structure in the brain

In age-related neurodegenerative diseases, like Alzheimer's and Parkinson's, disease-specific proteins become aggregation -prone and form amyloid-like deposits. Depletion of SERF pro-teins ameliorates this toxic process in worm and human cell models for diseases. Whether SERF modifies amyloid pathology in mammalian brain, however, has remained unknown. Here, we generated conditional Serf2 knockout mice and found that full -body deletion of Serf2 delayed embryonic development, causing premature birth and perinatal lethality. Brain-specific Serf2 knockout mice, on the other hand, were viable, and showed no major behavioral or cognitive abnormalities. In a mouse model for amyloid-beta aggregation, brain depletion of Serf2 altered the binding of structure-specific amyloid dyes, previously used to distinguish amyloid polymorphisms in the human brain. These results suggest that Serf2 depletion changed the structure of amyloid deposits, which was further supported by scanning transmission electron microscopy, but further study will be required to confirm this observation. Altogether, our data reveal the pleiotropic functions of SERF2 in embryonic development and in the brain and support the existence of modifying factors of amyloid deposition in mammalian brain, which offer possi-bilities for polymorphism-based interventions