Diversity and toxicity of Pseudo-nitzschia species in Monterey Bay: Perspectives from targeted and adaptive sampling.

Monterey Bay, California experiences near-annual blooms of Pseudo-nitzschia that can affect marine animal health and the economy, including impacts to tourism and commercial/recreational fisheries. One species in particular, P. australis, has been implicated in the most toxic of events, however other species within the genus can contribute to widespread variability in community structure and associated toxicity across years. Current monitoring methods are limited in their spatial coverage as well as their ability to capture the full suite of species present, thereby hindering understanding of HAB events and limiting predictive accuracy. An integrated deployment of multiple in situ platforms, some with autonomous adaptive sampling capabilities, occurred during two divergent bloom years in the bay, and uncovered detailed aspects of population and toxicity dynamics. A bloom in 2013 was characterized by spatial differences in Pseudo-nitzschia populations, with the low-toxin producer P. fraudulenta dominating the inshore community and toxic P. australis dominating the offshore community. An exceptionally toxic bloom in 2015 developed as a diverse Pseudo-nitzschia community abruptly transitioned into a bloom of highly toxic P. australis within the time frame of a week. Increases in cell density and proliferation coincided with strong upwelling of nutrients. High toxicity was driven by silicate limitation of the dense bloom. This temporal shift in species composition mirrored the shift observed further north in the California Current System off Oregon and Washington. The broad scope of sampling and unique platform capabilities employed during these studies revealed important patterns in bloom formation and persistence for Pseudo-nitzschia. Results underscore the benefit of expanded biological observing capabilities and targeted sampling methods to capture more comprehensive spatial and temporal scales for studying and predicting future events.

Strength in numbers: Collaborative science for new experimental model systems.

Our current understanding of biology is heavily based on a small number of genetically tractable model organisms. Most eukaryotic phyla lack such experimental models, and this limits our ability to explore the molecular mechanisms that ultimately define their biology, ecology, and diversity. In particular, marine protists suffer from a paucity of model organisms despite playing critical roles in global nutrient cycles, food webs, and climate. To address this deficit, an initiative was launched in 2015 to foster the development of ecologically and taxonomically diverse marine protist genetic models. The development of new models faces many barriers, some technical and others institutional, and this often discourages the risky, long-term effort that may be required. To lower these barriers and tackle the complexity of this effort, a highly collaborative community-based approach was taken. Herein, we describe this approach, the advances achieved, and the lessons learned by participants in this novel community-based model for research.

A MALDI-TOF MS analysis study of the binding of 4-(N,N-dimethylamino)pyridine to amine-bis(phenolate) chromium(III) chloride complexes: mechanistic insight into differences in catalytic activity for CO2/epoxide copolymerization.

Amine-bis(phenolato)chromium(III) chloride complexes, [LCrCl], are capable of catalyzing the copolymerization of cyclohexene oxide with carbon dioxide to give poly(cyclohexane) carbonate. When combined with 4-(N,N-dimethylamino)pyridine (DMAP) these catalyst systems yield low molecular weight polymers with moderately narrow polydispersities. The coordination chemistry of DMAP with five amine-bis(phenolato)chromium(III) chloride complexes was studied by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The amine-bis(phenolato) ligands were varied in the nature of their neutral pendant donor-group and include oxygen-containing tetrahydrofurfuryl and methoxyethyl moieties, or nitrogen-containing N,N-dimethylaminoethyl or 2-pyridyl moieties. The relative abundance of mono and bis(DMAP) adducts, as well as DMAP-free ions is compared under various DMAP : Cr complex ratios. The [LCr](+) cations show the ability to bind two DMAP molecules to form six-coordinate complex ions in all cases, except when the pendant group is N,N-dimethylaminoethyl (compound ). Even in the presence of a 4 : 1 ratio of DMAP to Cr, no ions corresponding to [L3Cr(DMAP)2](+) were observed for the complex containing the tertiary sp(3)-hybridized amino donor in the pendant arm. The difference in DMAP-binding ability of these compounds results in differences in catalytic activity for alternating copolymerization of CO2 and cyclohexene oxide. Kinetic investigations by infrared spectroscopy of compounds 2 and 3 show that polycarbonate formation by 3 is twice as fast as that of compound 2 and that no initiation time is observed.

Impact of IL-1 signalling on experimental uveitis and arthritis.

BACKGROUND: Uveitis, or inflammatory eye disease, is a common extra-articular manifestation of many systemic autoinflammatory diseases involving the joints. Anakinra (recombinant interleukin (IL)-1 receptor antagonist (Ra)) is an effective therapy in several arthritic diseases; yet, few studies have investigated the extent to which IL-1 signalling or IL-1Ra influences the onset and/or severity of uveitis. OBJECTIVE: To seek possible links between arthritis and uveitis pathogenesis related to IL-1 signalling. METHODS: The eyes of IL-1Ra-deficient BALB/c mice were monitored histologically and by intravital videomicroscopy to determine if uveitis developed along with the expected spontaneous arthritis in ankles and knees. Expression levels of IL-1R and its negative regulators (IL-1Ra, IL-1RII, IL-1RAcP and single Ig IL-1R-related molecule) in eye and joint tissues were compared. Differences in uveitis induced by intraocular injection of lipopolysaccharide (LPS) in mice lacking IL-1R or IL-1Ra were assessed. RESULTS: Deficiency in IL-1Ra predisposes to spontaneous arthritis, which is exacerbated by previous systemic LPS exposure. The eye, however, does not develop inflammatory disease despite the progressive arthritis or LPS exposure. Organ-specific expression patterns for IL-1Ra and negative regulators of IL-1 activity were observed that appear to predict predisposition to inflammation in each location in IL-1Ra knockout mice. The eye is extremely sensitive to locally administered LPS, and IL-1Ra deficiency markedly exacerbates the resulting uveitis. CONCLUSION: This study demonstrates that IL-1Ra plays an important role in suppressing local responses in eyes injected with LPS and that there is discordance between murine eyes and joints in the extent to which IL-1Ra protects against spontaneous inflammation.

The NLRP3 inflammasome is active but not essential in endotoxin-induced uveitis.

OBJECTIVE: The inflammasome complex involving caspase-1 and nucleotide-binding domain, leucine-rich repeat containing protein (NLRP)3, also known as NALP3 or cryopyrin is important for host responses to microbial pathogens and several autoinflammatory diseases. We investigated the extent to which NLRP3 and caspase-1 control ocular interleukin (IL)-1ß production and severity of uveitis (intraocular inflammatory disease) in an established, acute inflammatory uveitis model, endotoxin-induced uveitis (EIU). METHODS: Expression of NLRP3, its adaptor molecule ASC, also known as PYCARD (PYD and CARD domain containing), and caspase-1 were examined by immunoblotting. IL-1ß production was measured by enzyme-linked immunosorbent assay (ELISA). Using knockout mice, roles for caspase-1 and NLRP3 were examined in uveitis induced by intraocular injection of Escherichia coli lipopolysaccharide (LPS). RESULTS: NLRP3, ASC, and caspase-1 proteins are constitutively expressed in eye tissue. During EIU, IL-1ß protein production increases; this requires the presence of both caspase-1 and NLRP3. However, severity of EIU is not altered by deficiency in either caspase-1 or NLRP3, as assessed by both intravital microscopy and histology. CONCLUSIONS: These data identify the importance of the NLRP3 inflammasome for IL-1ß production in the eye, yet indicate that its participation in EIU is nonessential.

Contrasting ocular effects of local versus systemic endotoxin.

PURPOSE. A marked cellular infiltrate has been observed when endotoxin (lipopolysaccharide [LPS]) is injected into the mouse eye, but systemically injected LPS does not produce a comparable effect. Several hypotheses were tested to reconcile this discordance. METHODS. BALB/c mice were injected intravitreally (ivt) or intraperitoneally (ip) with Escherichia coli LPS. Uveitis was assessed by traditional and intravital microscopy. Cytokine levels in the eye, plasma, or spleen were measured by single or multiplex ELISA assays. RESULTS. The eye's higher sensitivity was confirmed to local LPS exposure, as 250 ng ivt LPS produced a brisk leukocytic infiltrate whereas ip injection of 100 µg LPS did not. The hypothesis was tested that the lack of a cellular infiltrate after ip LPS is explained by less induction of cytokines in the eye, but surprisingly, ip LPS resulted in comparable cytokine levels to ivt LPS. The hypothesis was disproved that the eye's sensitivity to local LPS is due to lack of expression of intracellular inhibitors of LPS such as A20, IRAK-M, or SARM. Finally, the hypothesis that systemic LPS inhibits diapedesis was tested by injection of LPS ip and ivt simultaneously, a strategy that did not significantly reduce leukocyte rolling or sticking in iris vessels but blocked the cellular infiltrate normally seen with ivt LPS. CONCLUSIONS. Systemic and local LPS exposures produce discordant effects within the murine eye. The hypothesis that systemic LPS desensitizes leukocytes to the stimuli responsible for transmigration offers a plausible explanation for this discordance.

The structure of corepressor Dax-1 bound to its target nuclear receptor LRH-1.

The Dax-1 protein is an enigmatic nuclear receptor that lacks an expected DNA binding domain, yet functions as a potent corepressor of nuclear receptors. Here we report the structure of Dax-1 bound to one of its targets, liver receptor homolog 1 (LRH-1). Unexpectedly, Dax-1 binds to LRH-1 using a new module, a repressor helix built from a family conserved sequence motif, PCFXXLP. Mutations in this repressor helix that are linked with human endocrine disorders dissociate the complex and attenuate Dax-1 function. The structure of the Dax-1:LRH-1 complex provides the molecular mechanism for the function of Dax-1 as a potent transcriptional repressor.