Partial characterization of digestive proteases in Totoaba (Totoaba macdonaldi)

ABSTRACT

The objective of this study was to characterize the digestive proteases of totoaba (Totoaba macdonaldi). Fish were sacrificed to obtain the multienzymatic extracts from the stomach and intestine, and determine the stability and optimum pH and temperature values. Residual activity and number of isoforms were determined with some inhibitors. Optimal pH of stomach proteases was 2, with stability above 100% at that same pH. Optimum pH of intestinal proteases was between 9 and 11, with stability above 100% between 8-12. Optimum temperature for stomach proteases was 35°C and remained highly stable, while optimum temperature for intestinal proteases was 45°C, with high stability between 35-55°C. Pepstatin A totally inhibited acid protease activity and revealed a single band. SDS-PAGE zymogram revealed 8 bands in the intestine, where phenanthroline inhibited 80% of the total activity. The digestive capacity of T. macdonaldi is characteristic of a strict carnivore, similar to other marine fish species.     

Vulnerability of microcrustaceans to predation by the invasive filter-feeding mussel Limnoperna fortunei (Dunker)

Limnoperna fortunei is an Asian mussel introduced to South America around 1990. One of the most important impacts of this invader is probably its grazing on the plankton. In this study we evaluate the vulnerability of several planktonic microcrustaceans from the Paraná River floodplain to predation by adult L. fortunei. We conducted 2-h laboratory feeding experiments where the bivalves were offered microcrustaceans differing in overall body shape, size, and locomotive abilities. Ingestion and clearance rates for each taxon were estimated. Results suggest that, in addition to detritus and phytoplankton, microcrustaceans may be a very important food item for this invasive mollusc. Limnoperna fortunei can prey on larger organisms (up to 1100?µm) than Dreissena polymorpha, the European and North American invasive mussel.     

Mangifera indica L. extract (QF808) reduces ischaemia-induced neuronal loss and oxidative damage in the gerbil brain

The effect of oral administration of Mangifera indica L. extract (QF808) on ischemia-reperfusion-induced neuronal death in the gerbil hippocampal CA1 sector was examined. Oral administration of QF808 for 7 days dose-dependently protected against neuronal cell death following transient ischaemia and reperfusion as assessed by histopathology. In addition, locomotor activity assessment prior to ischaemia and 7 days after correlated well with the histological results. To evaluate redox alterations by reactive oxygen species, total sulfhydryl, non-protein sulfhydryl groups (NPSH), malondialdehyde+4-hydroxyalkenals and total nitrogen oxide levels were assayed in hippocampus and cortex homogenates. QF808 treatment attenuated NPSH loss, nitrogen oxide levels and lipid peroxidation in the hippocampus. These results suggest that orally administered QF808 is absorbed across the blood-brain barrier and attenuates neuronal death of the hippocampal CA1 area after ischaemia-reperfusion. These protective effects are most likely due to the antioxidant activity of QF808.     

Alkaline phosphatase dual‐binding sites for collagen dictate cell migration and microvessel assembly in vitro

Interactions between cell types, growth factors, and extracellular matrix components involved in angiogenesis are crucial for new vessel formation leading to tissue regeneration. This study investigated whether cocultures of fibroblasts and endothelial cells (ECs; from macro‐ or microvasculature) play a role in the formation of microvessel‐like structures by ECs, as well as modulate fibroblast differentiation and growth factors production (vascular endothelial cell growth factor, basic fibroblast growth factor, active transforming growth factor‐β1, and interleukin‐8), which are important for vessel sprouting and maturation. Data obtained revealed that in vitro coculture systems of fibroblasts and human ECs stimulate collagen synthesis and growth factors production by fibroblasts that ultimately affect the formation and distribution of microvessel‐like structures in cell cultures. In this study, areas with activated fibroblasts and high alkaline phosphatase (ALP) activity were also observed in cocultures. Molecular docking assays revealed that ALP has two binding positions for collagen, suggesting its impact in collagen proteins’ aggregation, cell migration, and microvessel assembly. These findings indicate that bioinformatics and coculture systems are complementary tools for investigating the participation of proteins, like collagen and ALP in angiogenesis.     

High-content imaging-based pooled CRISPR screens in mammalian cells

CRISPR (clustered regularly interspaced short palindromic repeats)-based gene inactivation provides a powerful means for linking genes to particular cellular phenotypes. CRISPR-based screening typically uses large genomic pools of single guide RNAs (sgRNAs). However, this approach is limited to phenotypes that can be enriched by chemical selection or FACS sorting. Here, we developed a microscopy-based approach, which we name optical enrichment, to select cells displaying a particular CRISPR-induced phenotype by automated imaging-based computation, mark them by photoactivation of an expressed photoactivatable fluorescent protein, and then isolate the fluorescent cells using fluorescence-activated cell sorting (FACS). A plugin was developed for the open source software μManager to automate the phenotypic identification and photoactivation of cells, allowing ∼1.5 million individual cells to be screened in 8 h. We used this approach to screen 6,092 sgRNAs targeting 544 genes for their effects on nuclear size regulation and identified 14 bona fide hits. These results present a scalable approach to facilitate imaging-based pooled CRISPR screens.