Specific calpain activity evaluation in Plasmodium parasites

In the intraerythrocytic trophozoite stages of Plasmodium falciparum, the calcium-dependent cysteine protease calpain (Pf-calpain) has an important role in the parasite calcium modulation and cell development. We established specific conditions to follow by confocal microscopy and spectrofluorimetry measurements the intracellular activity of Pf-calpain in live cells. The catalytic activity was measured using the fluorogenic Z-Phe-Arg-MCA (where Z is carbobenzoxy and MCA is 4-methylcoumaryl-7-amide). The calmodulin inhibitor calmidazolium and the sarcoplasmic reticulum calcium ATPase inhibitor thapsigargin were used for modifications in the cytosolic calcium concentrations that persisted in the absence of extracellular calcium. The observed calcium-dependent peptidase activity was greatly inhibited by specific cysteine protease inhibitor E-64 and by the selective calpain inhibitor ALLN (N-acetyl-l-leucyl-l-leucyl-l-norleucinal). Taken together, we observed that intracellular Pf-calpain can be selectively detected and is the main calcium-dependent protease in the intraerythrocytic stages of the parasite. The method described here can be helpful in cell metabolism studies and antimalarial drug screening.     

RUN and FYVE domain–containing protein 4 enhances autophagy and lysosome tethering in response to Interleukin-4

Autophagy is a key degradative pathway coordinated by external cues, including starvation, oxidative stress, or pathogen detection. Rare are the molecules known to contribute mechanistically to the regulation of autophagy and expressed specifically in particular environmental contexts or in distinct cell types. Here, we unravel the role of RUN and FYVE domain–containing protein 4 (RUFY4) as a positive molecular regulator of macroautophagy in primary dendritic cells (DCs). We show that exposure to interleukin-4 (IL-4) during DC differentiation enhances autophagy flux through mTORC1 regulation and RUFY4 induction, which in turn actively promote LC3 degradation, Syntaxin 17–positive autophagosome formation, and lysosome tethering. Enhanced autophagy boosts endogenous antigen presentation by MHC II and allows host control of Brucella abortus replication in IL-4–treated DCs and in RUFY4-expressing cells. RUFY4 is therefore the first molecule characterized to date that promotes autophagy and influences endosome dynamics in a subset of immune cells.     

Dusky damselfish Stegastes fuscus relational learning: evidences from associative and spatial tasks

This study investigated the ability of the dusky damselfish Stegastes fuscus to associate conditioned and unconditioned stimuli (single CS–US) and to find a specific place in a clueless ambiece (spatial learning). After tested for colour preference and showing no specific colour attractively, the fish were trained to associate a colour cue with a stimulus fish (conspecific). Fish were then challenged to locate the exact place where the stimulus fish was presented. Stegastes fuscus spent most time close to the zone where stimulus was presented, even without obvious marks for orientation. The results confirm that S. fuscus show single CS–US learning and suggest the fish ability for spatial orientation. Stegastes fuscus appears to use multiple senses (sight and lateral line) for cues association and recall, and appear to perform relational learning similar to mammals. These data suggest the importance of cognitive skill for reef fishes that may have contributed to their establishment and evolutionary success in such complex environment.