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MicroRNAs have been associated with many different biological functions, but little is known about their roles in conditioned behavior. We demonstrate that Drosophila miR-980 is a memory suppressor gene functioning in multiple regions of the adult brain. Memory acquisition and stability were both increased by miR-980 inhibition. Whole cell recordings and functional imaging experiments indicated that miR-980 regulates neuronal excitability. We identified the autism susceptibility gene, A2bp1, as an mRNA target for miR-980. A2bp1 levels varied inversely with miR-980 expression; memory performance was directly related to A2bp1 levels. In addition, A2bp1 knockdown reversed the memory gains produced by miR-980 inhibition, consistent with A2bp1 being a downstream target of miR-980 responsible for the memory phenotypes. Our results indicate that miR-980 represses A2bp1 expression to tune the excitable state of neurons, and the overall state of excitability translates to memory impairment or improvement.

Naegleria fowleri is an amoeboflagellate responsible for the fatal central nervous system (CNS) disease primary amoebic meningoencephalitis (PAM). This amoeba gains access to the CNS by invading the olfactory mucosa and crossing the cribriform plate. Studies using a mouse model of infection have shown that the host secretes mucus during the very early stages of infection, and this event is followed by an infiltration of neutrophils into the nasal cavity. In this study, we investigated the role of IV. fowleri trophozoites in inducing the expression and secretion of airway mucin and pro-inflammatory mediators. Using the human mucoepidermal cell line NCI-H292, we demonstrated that N. fowleri induced the expression of the MUC5AC gene and protein and the pro-inflammatory mediators interleukin-8 (IL-8) and interleukin-1[beta] (IL-1[beta]), but not tumour necrosis factor-[alpha] or chemokine c-c motif ligand 11 (eotaxin). Since the production of reactive oxygen species (ROS) is a common phenomenon involved in the signalling pathways of these molecules, we analysed if trophozoites were capable of causing ROS production in NCI-H292 cells by detecting oxidation of the fluorescent probe 2,7-dichlorofluorescein diacetate. NCl-H292 cells generated ROS after 15-30 min of trophozoite stimulation. Furthermore, the expression of MUC5AC, IL-8 and IL-1[beta] was inhibited in the presence of the ROS scavenger DMSO. In addition, the use of an epidermal growth factor receptor inhibitor decreased the expression of MUCSAC and IL-8, but not IL-1[beta]. We conclude that N. fowleri induces the expression of some host innate defence mechanisms, such as mucin secretion (MUC5AC) and local inflammation (IL-8 and IL-1,8) in respiratory epithelial cells via ROS production and suggest that these innate immune mechanisms probably prevent most PAM infections. DOI 10.1099/mic.0.030635-0

Naegleria fowleri is the aetiological agent of primary amoebic meningoencephalitis (PAM). This parasite invades its host by penetrating the olfactory mucosa. During the initial stages of infection, the host response is initiated by the secretion of mucus that traps the trophozoites. Despite this response, some trophozoites are able to reach, adhere to and penetrate the epithelium. In the present work, we evaluated the effect of mucins on amoebic adherence and cytotoxicity to Madin--Darby canine kidney (MDCK) cells and the MUC5AC-inducing cell line NCI-H292. We showed that mucins inhibited the adhesion of amoebae to both cell lines; however, this inhibition was overcome in a time-dependent manner. N. fowleri re-established the capacity to adhere faster than N. gruberi. Moreover, mucins reduced the cytotoxicity to target cells and the progression of the illness in mice. In addition, we demonstrated mucinolytic activity in both Naegleria strains and identified a 37 kDa protein with mucinolytic activity. The activity of this protein was inhibited by cysteine protease inhibitors. Based on these results, we suggest that mucus, including its major mucin component, may act as an effective protective barrier that prevents most cases of PAM; however, when the number of amoebae is sufficient to overwhelm the innate immune response, the parasites may evade the mucus by degrading mucins via a proteolytic mechanism.

System consolidation, as opposed to cellular consolidation, is defined as the relatively slow process of reorganizing the brain circuits that maintain long-term memory. This concept is founded in part on observations made in mammals that recently formed memories become progressively independent of brain regions initially involved in their acquisition and retrieval and dependent on other brain regions for their long-term storage. Here we present evidence that olfactory appetitive and aversive memories in Drosophila evolve using a system-like consolidation process. We show that all three classes of mushroom body neurons (MBNs) are involved in the retrieval of short- and intermediate-term memory. With the passage of time, memory retrieval becomes independent of α'/β' and γ MBNs, and long-term memory becomes completely dependent on α/β MBNs. This shift in neuronal dependency for behavioral performance is paralleled by shifts in the activity of the relevant neurons during the retrieval of short-term versus long-term memories. Moreover, transient neuron inactivation experiments using flies trained to have both early and remote memories showed that the α'/β' MBNs have a time-limited role in memory processing. These results argue that system consolidation is not a unique feature of the mammalian brain and memory systems, but rather a general and conserved feature of how different temporal memories are encoded from relatively simple to complex brains. [ABSTRACT FROM AUTHOR]

Protein retention expansion microscopy (ExM) retains genetically encoded fluorescent proteins or antibody-conjugated fluorescent probes in fixed tissue and isotropically expands the tissue through a swellable polymer network to allow nanoscale (<70 nm) resolution on diffraction-limited confocal microscopes. Despite numerous advantages ExM brings to biological studies, the full protocol is time-consuming and can take multiple days to complete. Here, we adapted the ExM protocol to the vibratome-sectioned brain tissue of Xenopus laevis tadpoles and implemented a microwave-assisted protocol to reduce the workflow from days to hours. In addition to the significantly accelerated processing time, our microwave-assisted ExM ( M/W ExM) protocol maintains the superior resolution and signal-to-noise ratio of the original ExM protocol. Furthermore, the M/W ExM protocol yields higher magnitude of expansion, suggesting that in addition to accelerating the process through increased diffusion rate of reagents, microwave radiation may also facilitate the expansion process. To demonstrate the applicability of this method to other specimens and protocols, we adapted the microwave-accelerated protocol to whole mount adult brain tissue of Drosophila melanogaster fruit flies, and successfully reduced the total processing time of a widely-used Drosophila IHC-ExM protocol from 6 days to 2 days. Our results demonstrate that with appropriate adjustment of the microwave parameters (wattage, pulse duration, interval, and number of cycles), this protocol can be readily adapted to different model organisms and tissue types to greatly increase the efficiency of ExM experiments., Competing Interests: Declarations of Interest The authors declare no competing interests.

The protozoon Naegleria fowleri (N. fowleri) is a free-living amoeba that produces primary amoebic meningoencephalitis (PAM), which is an acute and frequently fatal infection of the central nervous system. We characterized the strains of N. fowleri isolated from the cerebrospinal fluid (CSF) of two cases presented in northwestern Mexico. The strains were isolated and cultured in 2% bactocasitone medium. Enflagellation assays, ultrastructural analysis, protein and protease electrophoresis patterns, and PCR were performed as confirmatory tests. Virulence tests were done using in Balb/c mice. Light microscopy analysis of brain tissue showed amoebae with abundant inflammatory reaction and extensive necrotic and hemorrhagic areas. The enflagellation assay was positive and the electron microscopy showed trophozoites with morphologic features typical of the genus. Protein and protease profiles of the isolated strains were identical to the reference strain. Finally, a 1500-bp PCR product was found in all three strains. Based on all the analyses performed, we concluded that the etiologic agent of both PAM cases was N. fowleri. The need for better epidemiological information and educational programs about basic clinical and pathological aspects of free-living amoebae provided by the health authorities are emphasized.