Carbonic Anhydrase is Required for Statoconia Homeostasis in Organ Cultures of Statocysts from Aplysia californica

A novel organ culture system has been developed to study the regulation of statoconia production in the gravity sensing organ in Aplysia californica. Statocysts were cultured in Leibovitz (LI5) medium supplemented with salts and Aplysia haemolymph for four days at 17 C. The viability of the system was evaluated by examining four parameters: statocyst morphology, the activity of the mechanosensory cilia in the statocyst, production of new statoconia during culture and change in statoconia volume after culture. There were no morphological differences in statocysts before and after culture when ciliary beating was maintained. There was a 29% increase in the number of statoconia after four days in culture. Mean statocyst, statolith and statoconia volumes were not affected by culture conditions. The presence of carbonic anhydrase in the statocysts was shown using immunohistochemistry. When statocysts were cultured in the presence of 4.0 x 10(exp -4) M acetazolamide to inhibit the enzyme activity, there was a decrease in statoconia production and statoconia volume, indicating a role for this enzyme in statoconia homeostasis, potentially, via pH regulation. These studies are the first to report a novel system for the culture of statocysts and show that carbonic anhydrase is involved in the regulation of statoconia volume and production

Porta-enxertos e enxertia de castanheira-do-brasil pelo método da borbulhia em placa.

Grande parte da produção de castanha-do-brasil é oriunda do extrativismo em áreas nativas, sendo o sucesso da exploração em sistemas de cultivo altamente dependente de técnicas de propagação vegetativa, para manutenção das características genéticas e encurtamento do período juvenil de genótipos selecionados. Este estudo teve como objetivos determinar a idade ótima de porta-enxertos de castanheira para realização da enxertia por borbulhia em placa, bem como verificar a porcentagem de sobrevivência de enxertos e o crescimento inicial de brotações de diferentes genótipos em Roraima. Para isso foram instalados dois experimentos no delineamento de blocos casualizados, com quatro repetições. Os tratamentos foram arranjados em esquema de parcela subdividida no tempo, sendo genótipos as parcelas e tempos de avaliação as subparcelas. Foram avaliadas variáveis de crescimento dos porta-enxertos e porcentagem de enxertos vivos e variáveis de crescimento das brotações dos enxertos. Os porta-enxertos atingiram porte recomendado para enxertia entre os 26 e 34 meses após o plantio, indicando crescimento mais lento quando comparado com outros locais. Aos 1 20 dias após a enxertia foram obtidas porcentagens de sobrevivência de enxertos variando de 25% a 1 00%, dependendo do genótipo do enxerto. A porcentagem média geral de sobrevivência foi superior a 70%. O crescimento (altura, diâmetro e número de brotações laterais) das brotações observado para alguns genótipos é indicativo da adaptação desses às condições de cultivo consideradas. Como a castanheira é uma espécie perene, tanto o crescimento em idades mais avançadas, quanto a futura produtividade e longevidade das plantas deverão ser monitoradas

Caracterización clínicoepidemiológica de pacientes con enfermedades reumáticas de inicio juvenil en Paraguay (2011 – 2015)

Objective: Describe the frequency of juvenile onset rheumatologic inflammatory diseases (JIR ) in level III and IV complexity level hospitals in a quinquennium. Methods: Multicentric, descriptive and retrospective study, using CI E-10 coding of hospitals’ files in Asunción and Central Department. Results: Four hospitalary records were included. 382 patients followed inclusion criteria. Sex ratio (M:F) of the population was 1:1,6, with an average age at captation of 11.6 years (SD±4.5). Kawsaki’s disease group has an average age of 5.4 years (SD±3.7) and patients with systemic lupus erythematosus (SLE) were presented with 13.1 years on average (SD±3.9). The 68,6% of patients were found through follow up specialized consults. Idiopathic juvenile arthritis was the most frequently diagnosed disease (n=167 cases, 43,7% of the total), followed by SLE (n=130, 34,0%). Most of the cases (n=290, 75,9%) were patients from (place of birth) Asuncion or Central Department. Conclusion: The spectrum of JIR diseases is broad with variable distribution in each included hospital. This study orients to know the burden of disease from JIR diseases in Paraguay.Objetivo: Describir la frecuencia de las enfermedades reumatológicas inflamatorias de inicio juvenil (RI J) en hospitales de nivel de complejidad III y IV en un quinquenio. Método: Estudio multicéntrico, descriptivo, retrospectivo utilizando códigos CI E-10 de los archivos de hospitales de Asunción y del departamento central. Resultados: Cuatro archivos hospitalarios fueron incluidos. En total 382 pacientes cumplían los criterios de inclusión. El sex ratio (M: F) de la población fue de 1:1.6, edad promedio a la captación de 11.6 años (DE±4.5). El grupo de pacientes con Enfermedad de Kawasaki tenían edad promedio de 5.4 años (± 3.7 DE) y los pacientes con Lupus Eritematoso Sistémico (LES) se presentaron con 13.1 años (± 3.9 DE). El 68,6% de los pacientes fueron captados por consulta especializada de seguimiento. La artritis idiopática juvenil fue la enfermedad RIJ más frecuentemente diagnosticada (n=167 casos, 43,7% del total), seguida por el LES (n=130, 34,0%). La mayoría de los casos (75.9%, n=290) eran pacientes procedentes (al nacimiento) de Asunción o del departamento central. Conclusión: El espectro de las enfermedades RI J es amplio con distribución variable según los hospitales incluidos. Este estudio orienta a conocer la “carga de enfermedad” por enfermedades RI J en Paraguay

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field