Summary Expansion of a hexanucleotide repeat GGGGCC (G4C2) in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Transcripts carrying (G4C2) expansions undergo unconventional, non-ATG-dependent translation, generating toxic dipeptide repeat (DPR) proteins thought to contribute to disease. Here we identify the interactome of all DPRs and find that arginine-containing DPRs, polyGly-Arg (GR) and polyPro-Arg (PR), interact with RNA-binding proteins and proteins with low complexity sequence domains (LCDs) that often mediate the assembly of membrane-less organelles. Indeed, most GR/PR interactors are components of membrane-less organelles such as nucleoli, the nuclear pore complex and stress granules. Genetic analysis in Drosophila demonstrated the functional relevance of these interactions to DPR toxicity. Furthermore, we show that GR and PR altered phase separation of LCD-containing proteins, insinuating into their liquid assemblies and changing their material properties, resulting in perturbed dynamics and/or functions of multiple membrane-less organelles.

GGGGCC (G4C2) repeat expansion in a noncoding region of C9ORF72 is the most common cause of sporadic and familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)1,2. The basis for pathogenesis is unknown. To capture the consequences of G4C2 repeat expansion in a tractable genetic system, we generated transgenic fly lines expressing 8, 28 or 58 G4C2 repeat-containing transcripts that do not have a translation start site (AUG) but contain an open-reading frame for green fluorescent protein (GFP) to detect repeat-associated non-AUG (RAN) translation. These transgenic animals show dosage-dependent, repeat length-dependent degeneration in neuronal tissues and RAN translation of dipeptide repeat (DPR) proteins as observed in patients. This model was used in a large-scale, unbiased genetic screen ultimately leading to the identification of 18 genetic modifiers that encode components of the nuclear pore complex (NPC) as well as the machinery that coordinates the export of nuclear RNA and the import of nuclear proteins. Consistent with these results we found morphological abnormalities in the architecture of the nuclear envelope in cells expressing expanded G4C2 repeats in vitro and in vivo. Moreover, we identified a substantial defect in RNA export resulting in retention of RNA in the nuclei of Drosophila cells expressing expanded G4C2 repeats and also in mammalian cells, including aged iPSC-derived neurons from C9ORF72 patients. These studies show that a primary consequence of G4C2 repeat expansion is the compromise of nucleocytoplasmic transport through the nuclear pore, revealing a novel mechanism of neurodegeneration.

Survival of free-living and symbiotic dinoflagellates (Symbiodinium spp.) in coral reefs is critical to the maintenance of a healthy coral community. Most coral reefs exist in oligotrophic waters, and their survival strategy in such nutrient-depleted waters remains largely unknown. In this study, we found that two strains of Symbiodinium spp. cultured from the environment and acquired from the tissues of the coral Alveopora japonica had the ability to feed heterotrophically. Symbiodinium spp. fed on heterotrophic bacteria, cyanobacteria (Synechococcus spp.), and small microalgae in both nutrient-replete and nutrient-depleted conditions. Cultured free-living Symbiodinium spp. displayed no autotrophic growth under nitrogen-depleted conditions, but grew when provided with prey. Our results indicate that Symbiodinium spp.'s mixotrophic activity greatly increases their chance of survival and their population growth under nitrogen-depleted conditions, which tend to prevail in coral habitats. In particular, free-living Symbiodinium cells acquired considerable nitrogen from algal prey, comparable to or greater than the direct uptake of ammonium, nitrate, nitrite, or urea. In addition, freeliving Symbiodinium spp. can be a sink for planktonic cyanobacteria (Synechococcus spp.) and remove substantial portions of Synechococcus populations from coral reef waters. Our discovery of Symbiodinium's feeding alters our conventional views of the survival strategies of photosynthetic Symbiodinium and corals. mixotrophy | zooxanthella | coral bleaching | food web | Heterosigma

Speculation surrounds the importance of ecologically cryptic Symbiodinium spp. (dinoflagellates) that occur at low abundances in reef-building corals and in the surrounding environment. Evidence acquired from extensive sampling, long-term monitoring, and experimental manipulation can allow us to deduce the ecology and functional significance of these populations and whether they might contribute to the response of coral-dinoflagellate mutualisms to climate change. Quantitative PCR was used here to diagnose the prevalence, seasonal variation, and abundances of Symbiodinium spp. within and between colonies of the coral, Alveopora japonica. Consistent with broader geographic sampling, only one species comprised 99.9 %, or greater, the population of symbionts in every sample. However, other Symbiodinium including the non-mutualistic species, Symbiodinium voratum, were often detected, but at estimated cell densities thousands-fold less than the dominant symbiont. The temporal variation in prevalence and abundances of these "background" Symbiodinium could not be definitively related to any particular environmental factor including seasonality and water chemistry. The prevalence (proportion detected among host samples), but not abundance, of S. voratum may weakly correspond to increases in environmental inorganic silica (SiO2) and possibly nitrogen (NO3). When multiple background Symbiodinium occurred within an individual polyp, the average cell densities were positively correlated, suggesting non-specific processes of cell sorting and retention by the animal. While these findings substantiate the existence of a broader, yet uncharacterized, diversity of Symbiodinium, we conclude that only those species which can occur in high abundance and are temporally stable are ultimately important to coral-dinoflagellate mutualisms. Many transient Symbiodinium spp., which occur only at trace abundances in the coral's microbiome, belong to different functional guilds and likely have little, if any, importance to a coral's physiology. The successful integration between host and symbiont into a stable functional unit should therefore be considered when defining host-symbiont specificity.

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Probiotics offer various health benefits. Lactobacillus plantarum has been used for decades to enhance human intestinal mucosal immunity and improve skin barrier integrity. Extracellular vesicles (EVs) derived from eukaryotic or prokaryotic cells have been recognized as efficient carriers for delivery of biomolecules to recipient cells, and to efficiently regulate human pathophysiology. However, the mechanism underlying the beneficial effects of probiotic bacteriaderived EVs on human skin is unclear. Herein, we investigated how L. plantarum-derived EVs (LEVs) exert beneficial effects on human skin by examining the effect of LEVs on cutaneous immunity, particularly on macrophage polarization. LEVs promoted differentiation of human monocytic THP1 cells towards an anti-inflammatory M2 phenotype, especially M2b, by inducing biased expression of cell-surface markers and cytokines associated with M2 macrophages. Pre-or post-treatment with LEVs under inflammatory M1 macrophage-favouring conditions, induced by LPS and interferon-γ, inhibited M1-associated surface marker, HLA-DRα expression. Moreover, LEV treatment significantly induced expression of macrophage-characteristic cytokines, IL-1β, GM-CSF and the representative anti-inflammatory cytokine, IL-10, in human skin organ cultures. Hence, LEVs can trigger M2 macrophage polarization in vitro, and induce an anti-inflammatory phenomenon in the human skin, and may be a potent anti-inflammatory strategy to alleviate hyperinflammatory skin conditions.

Woloszynskia species are dinoflagellates in the order Suessiales inhabiting marine or freshwater environments; their ecophysiology has not been well investigated, in particular, their trophic modes have yet to be elucidated. Previous studies have reported that all Woloszynskia species are photosynthetic, although their mixotrophic abilities have not been explored. We isolated a dinoflagellate from coastal waters in western Korea and established clonal cultures of this dinoflagellate. On the basis of morphology and analyses of the small/large subunit rRNA gene (GenBank accession number=FR690459), we identified this dinoflagellate as Woloszynskia cincta. We further established that this dinoflagellate is a mixotrophic species. We found that W. cincta fed on algal prey using a peduncle. Among the diverse prey provided, W. cincta ingested those algal species that had equivalent spherical diameters (ESDs) ≤12.6 μm, exceptions being the diatom Skeletonema costatum and the dinoflagellate Prorocentrum minimum. However, W. cincta did not feed on larger algal species that had ESDs≥15 μm. The specific growth rates for W. cincta increased continuously with increasing mean prey concentration before saturating at a concentration of ca. 134 ng C/ml (1,340 cells/ml) when Heterosigma akashiwo was used as food. The maximum specific growth rate (i.e. mixotrophic growth) of W. cincta feeding on H. akashiwo was 0.499 d(-1) at 20 °C under illumination of 20 μE/m(2) /s on a 14:10 h light-dark cycle, whereas its growth rate (i.e. phototrophic growth) under the same light conditions without added prey was 0.040 d(-1). The maximum ingestion and clearance rates of W. cincta feeding on H. akashiwo were 0.49 ng C/grazer/d (4.9 cells/grazer/d) and 1.9 μl/grazer/h, respectively. The calculated grazing coefficients for W. cincta on co-occurring H. akashiwo were up to 1.1 d(-1). The results of the present study suggest that grazing by W. cincta can have a potentially considerable impact on prey algal populations.