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Abstract Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a “GG” dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases.

Activated protein C (APC) is a plasma serine protease with antithrombotic and cytoprotective functions. Here, the authors develop a monoclonal antibody that specifically inhibits APC’s anticoagulant function without compromising its cytoprotective function, and shows efficacy in animal models.

Neprilysin is a transmembrane zinc metallopeptidase that degrades a wide range of peptide substrates. It has received attention as a potential therapy for Alzheimer's disease due to its ability to degrade the peptide amyloid beta. However, its broad range of peptide substrates has the potential to limit its therapeutic use due to degradation of additional peptides substrates that tightly regulate many physiological processes. We sought to generate a soluble version of the ectodomain of neprilysin with improved activity and specificity towards amyloid beta as a potential therapeutic for Alzheimer's disease. Extensive amino acid substitutions were performed at positions surrounding the active site and inner surface of the enzyme and variants screened for activity on amyloid beta 1-40, 1-42 and a variety of other physiologically relevant peptides. We identified several mutations that modulated and improved both enzyme selectivity and intrinsic activity. Neprilysin variant G399V/G714K displayed an approximately 20-fold improved activity on amyloid beta 1-40 and up to a 3,200-fold reduction in activity on other peptides. Along with the altered peptide substrate specificity, the mutant enzyme produced a markedly altered series of amyloid beta cleavage products compared to the wild-type enzyme. Crystallisation of the mutant enzyme revealed that the amino acid substitutions result in alteration of the shape and size of the pocket containing the active site compared to the wild-type enzyme. The mutant enzyme offers the potential for the more efficient degradation of amyloid beta in vivo as a therapeutic for the treatment of Alzheimer's disease.

Protein turnover is highly energy consuming and overall relates to an organism’s growth performance varying largely between species, e.g., due to pre-adaptation to environmental characteristics such as temperature. Here, we determined protein synthesis rates and capacity of protein degradation in white muscle of the cold stenothermal Antarctic eelpout (Pachycara brachycephalum) and its closely related temperate counterpart, the eurythermal common eelpout (Zoarces viviparus). Both species were exposed to acute warming (P. brachycephalum, 0 °C + 2 °C day−1; Z. viviparus, 4 °C + 3 °C day−1). The in vivo protein synthesis rate (Ks) was monitored after injection of 13C-phenylalanine, and protein degradation capacity was quantified by measuring the activity of cathepsin D in vitro. Untargeted metabolic profiling by nuclear magnetic resonance (NMR) spectroscopy was used to identify the metabolic processes involved. Independent of temperature, the protein synthesis rate was higher in P. brachycephalum (Ks = 0.38–0.614 % day−1) than in Z. viviparus (Ks= 0.148–0.379% day−1). Whereas protein synthesis remained unaffected by temperature in the Antarctic species, protein synthesis in Z. viviparus increased to near the thermal optimum (16 °C) and tended to fall at higher temperatures. Most strikingly, capacities for protein degradation were about ten times higher in the Antarctic compared to the temperate species. These differences are mirrored in the metabolic profiles, with significantly higher levels of complex and essential amino acids in the free cytosolic pool of the Antarctic congener. Together, the results clearly indicate a highly cold-compensated protein turnover in the Antarctic eelpout compared to its temperate confamilial. Constant versus variable environments are mirrored in rigid versus plastic functional responses of the protein synthesis machinery.

Direct measurements of temperature-dependent weight gains are experimentally challenging and time-consuming in long-lived/slow-growing organisms such as Antarctic fish. Here, we reassess methodology to quantify the in vivo protein synthesis rate from amino acids, as a key component of growth. We tested whether it is possible to avoid hazardous radioactive materials and whether the analytical pathway chosen is robust against analytical errors. In the eelpout, Pachycara brachycephalum, 13C9H1115N1O2 phenylalanine was injected intraperitoneally and muscle tissue was sampled before injection and at 1.5 h time intervals up to 6 h thereafter. The incorporation of 13C15N-labeled-phenylalanine into muscle was monitored by quantification of bound and free phenylalanine through liquid chromatography–mass spectrometry. We found an increase in the pool of labeled, free phenylalanine in the cytosolic fraction that leveled off after 4.5 h. The labeled phenylalanine bound in the proteins increased linearly over time. The resulting protein synthesis rate (Ks) for P. brachycephalum was as low as 0.049 ± 0.021% day−1. This value and its variability were in good agreement with literature data obtained from studies using radioactive labels, indicating that this methodology is well suited for characterizing growth in polar fish under in situ conditions in remote areas or on research vessels.

The potential of marine dissolved organic matter (DOM) for free radical scavenging has been extensively evaluated, however, the quantitative assessment of the antioxidant potential has been recently measured for the first time. The linkage of the DOM antioxidant potential to its molecular composition has not yet been examined. Following this line, this article takes a step forward by assessing, throughout a polarity-mediated fractionation, (1) the antioxidant capacity and phenolic content and (2) the molecular characterization of DOM in a more exhaustive manner. (3) The DOM antioxidant potential and phenolic content was linked to the molecular composition of DOM, which was molecularly characterized using ultrahigh resolution Fourier transform Ion Cyclotron Resonance mass spectrometry (FT-ICR MS). Antioxidant activity and phenolic content were quantified by the free radical 2,2’-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS⋅) and the Folin-Ciocalteu methods, respectively. We considered three types of different natural DOM samples: the deep North Pacific Ocean, the oligotrophic surface of the North Pacific Ocean and porewater from the sulfidic tidal flats of the Wadden Sea. Bulk porewater and its individual polarity fractions presented the highest antioxidant activity and phenolic content. DOM from the water column samples had lower antioxidant activity and phenolic content than porewater, but exceeded what it is commonly found in macroalgae, microalgae, fruits and vegetables with cosmeceutical purposes. Our values were similar to published values for terrestrial DOM. The variations in bioactivity were dependent on polarity and molecular composition. The high resolution and high mass accuracy used to determine the molecular composition of marine DOM and the chemometric and multistatistical analyses employed have allowed to distinguish molecular categories that are related to the bioactive potential. As a future perspective, we performed cytotoxicity tests with human cells and propose marine DOM as a natural ingredient for the development of cosmeceutical products.

The dinoflagellate genus Prorocentrum is globally represented by a wide variety of species found upon benthic and/or epiphytic substrates. Many epibenthic Prorocentrum species produce lipophilic polyether toxins, some of which act as potent protein phosphatase inhibitors and tumor-promoters associated with Diarrheic Shellfish Poisoning (DSP). Most members of the Prorocentrum lima species complex (PLSC) commonly found in the tropics and sub-tropics are toxigenic. Epiphytic and planktonic bacteria co-occur with toxigenic Prorocentrum but reciprocal allelochemical interactions are under-investigated. The aim of the present study was to identify the culturable bacteria collected together with isolates of the PLSC from seagrass (Thalassia testudinum) and macroalgae along tropical Atlantic coasts of Mexico, and to explore potential species interactions with selected isolates. Twenty-one bacterial genera belonging to Proteobacteria, Actinobacteria, and Bacteroidetes were identified by amplification of the 16S rRNA gene marker from nine clonal Prorocentrum cultures, with γ-proteobacteria comprising the dominant class. A positive correlation was found between the bacterial genera associated with two Prorocentrum clones and the esterified toxin analog DTX1a-D8, but there was no apparent correlation between the other PLSC clones and their associated bacteria with the other five DSP toxins detected. No bacteriostatic or allelochemical response was found for cell- and culture medium extracts of five Prorocentrum isolates assayed for bioactivity against Staphylococcus sp. DMBS2 and Vibrio sp. HEL66. Bulk cell-washing of Prorocentrum PA1, followed by growth with antibiotics, was only effective in reducing bacterial load in the initial growth stages, but did not yield axenic cultures or lower bacterial cell densities throughout the culture cycle. Antibiotic treatment did not impair growth or survival of the dinoflagellate, or apparently affect DSP toxin production. There was no significant correlation between Prorocentrum cell volume, growth rate, bacterial cell counts, or cellular toxin concentration over the entire time-series culture cycle. Benthic Prorocentrum and associated bacterial communities comprise highly diverse and characteristic microbiomes upon substrates, and among compartments in culture, but this study provides little evidence that allelochemical interactions among Prorocentrum cells and associated bacteria originating from epibenthic substrates play a definable role in growth and toxigenicity.

Gymnodimines and spirolides are cyclic imine phycotoxins and known antagonists of nicotinic acetylcholine receptors (nAChRs). We investigated the effect of gymnodimine A (GYM A) and 13-desmethyl spirolide C (SPX 1) from Alexandrium ostenfeldii on rat pheochromocytoma (PC12) cells by monitoring intracellular calcium levels ([Ca]i). Using whole cells, the presence of 0.5 µM of GYM A or SPX 1 induced an increase in [Ca]i mediated by acetylcholine receptors (AChRs) and inhibited further activation of AChRs by acetylcholine (ACh). To differentiate the effects of GYM A or SPX 1, the toxins were applied to cells with pharmacologically isolated nAChRs and muscarinic AChRs (mAChRs) as mediated by the addition of atropine and tubocurarine, respectively. GYM A and SPX 1 activated nAChRs and inhibited the further activation of nAChRs by ACh, indicating that both toxins mimicked the activity of ACh. Regarding mAChRs, a differential response was observed between the two toxins. Only GYM A activated mAChRs, resulting in elevated [Ca]i, but both toxins prevented a subsequent activation by ACh. The absence of the triketal ring system in GYM A may provide the basis for a selective activation of mAChRs. GYM A and SPX 1 induced no changes in [Ca]i when nAChRs and mAChRs were inhibited simultaneously, indicating that both toxins target AChRs.

Spirolides belong to a group of marine phycotoxins produced by the marine planktonic dinophyte Alexandrium ostenfeldii. Composed of an imine moiety and a spiroketal ring system within a macrocylcle, spirolides are highly diverse with toxin types that vary among different strains. This study aims to characterize the spirolides from clonal A. ostenfeldii strains collected from The Netherlands, Greenland and Norway by mass spectral techniques. The structural characterization of unknown spirolides as inferred from high-resolution mass spectrometry (HR-MS) and collision induced dissociation (CID) spectra revealed the presence of nine novel spirolides that have the pseudo-molecular ions m/z 670 (1), m/z 666 (2), m/z 696 (3), m/z 678 (4), m/z 694 (5), m/z 708 (6), m/z 720 (7), m/z 722 (8) and m/z 738 (9). Of the nine new spirolides proposed in this study, compound 1 was suggested to have a truncated side chain in lieu of the commonly observed butenolide ring in spirolides. Moreover, there is indication that compound 5 might belong to new spirolide subclasses with a trispiroketal ring configuration having a 6:5:6 trispiroketal ring system. On the other hand, the other compounds were proposed as C- and G-type SPX, respectively. Compound 7 is proposed as the first G-type SPX with a 10-hydroxylation as usually observed in C-type SPX. This mass spectrometry-based study thus demonstrates that structural variability of spirolides is larger than previously known and does not only include the presence or absence of certain functional groups but also involves the triketal ring system.

Only few naturally occurring cyclic imines have been fully structurally elucidated or synthesized to date. The configuration at the C-4 carbon plays a pivotal role in the neurotoxicity of many of these metabolites, for example, gymnodomines (GYMs) and spirolides (SPXs). However, the stereochemistry at this position is not accessible by nuclear Overhauser effect—nuclear magnetic resonance spectroscopy (NOE-NMR) due to unconstrained rotation of the single carbon bond between C-4 and C-5. Consequently, the relative configuration of GYMs and SPXs at C-4 and its role in protein binding remains elusive. Here, we determined the stereochemical configuration at carbon C-4 in the butenolide ring of spirolide- and gymnodimine-phycotoxins by comparison of measured 13C NMR shifts with values obtained in silico using force field, semiempirical and density functional theory methods. This comparison demonstrated that modeled data support S configuration at C-4 for all studied SPXs and GYMs, suggesting a biosynthetically conserved relative configuration at carbon C-4 among these toxins.

Amphidinols are polyketides produced by dinoflagellates suspected of causing fish kills. Here, we demonstrate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the identification and quantification of amphidinols (AM). Novel AM were detected by neutral loss (NL) scan and then quantified together with known AM by selection reaction monitoring (SRM). With the new method, AM were detected in four of eight analyzed strains with a maximum of 3680 fg toxin content per cell. In total, sixteen novel AM were detected by NL scan and characterized via their fragmentation patterns. Of these, two substances are glycosylated forms. This is the first detection of glycosylated AM.

The ethanol extract of Pseudoalteromonas strain J010, isolated from the surface of the crustose coralline alga Neogoniolithon fosliei, yielded thirteen natural products. These included a new bromopyrrole, 4′-((3,4,5-tribromo-1H-pyrrol-2-yl) methyl)phenol (1) and five new korormicins G–K (2–6). Also isolated was the known inducer of coral larval metamorphosis, tetrabromopyrrole (TBP), five known korormicins (A–E, previously named 1, 1a–c and 3) and bromoalterochromide A (BAC-A). Structures of the new compounds were elucidated through interpretation of spectra obtained after extensive NMR and MS investigations and comparison with literature values. The antibacterial, antifungal and antiprotozoal potential of 1–6, TBP and BAC-A was assessed. Compounds 1–6 showed antibacterial activity while BAC-A exhibited antiprotozoal properties against Tetrahymena pyriformis. TBP was found to have broad-spectrum activity against all bacteria, the protozoan and the fungus Candida albicans.

Bacterial communities associated with healthy corals produce antimicrobial compounds that inhibit the colonization and growth of invasive microbes and potential pathogens. To date, however, bacteria-derived antimicrobial molecules have not been identified in reef-building corals. Here, we report the isolation of an antimicrobial compound produced by Pseudovibrio sp. P12, a common and abundant coral-associated bacterium. This strain was capable of metabolizing dimethylsulfoniopropionate (DMSP), a sulfur molecule produced in high concentrations by reef-building corals and playing a role in structuring their bacterial communities. Bioassay-guided fractionation coupled with nuclear magnetic resonance (NMR) and mass spectrometry (MS), identified the antimicrobial as tropodithietic acid (TDA), a sulfur-containing compound likely derived from DMSP catabolism. TDA was produced in large quantities by Pseudovibrio sp., and prevented the growth of two previously identified coral pathogens, Vibrio coralliilyticus and V. owensii, at very low concentrations (0.5 μg/mL) in agar diffusion assays. Genome sequencing of Pseudovibrio sp. P12 identified gene homologs likely involved in the metabolism of DMSP and production of TDA. These results provide additional evidence for the integral role of DMSP in structuring coral-associated bacterial communities and underline the potential of these DMSP-metabolizing microbes to contribute to coral disease prevention.

Surface-associated marine bacteria often produce secondary metabolites with antagonistic activities. In this study, tropodithietic acid (TDA) was identified to be responsible for the antibacterial activity of the marine epiphytic bacterium Pseudovibrio sp. D323 and related strains. Phenol was also produced by these bacteria but was not directly related to the antibacterial activity. TDA was shown to effectively inhibit a range of marine bacteria from various phylogenetic groups. However TDA-producers themselves were resistant and are likely to possess resistance mechanism preventing autoinhibition. We propose that TDA in isolate D323 and related eukaryote-associated bacteria plays a role in defending the host organism against unwanted microbial colonisation and, possibly, bacterial pathogens.

Cyclic imine toxins are neurotoxic, macrocyclic compounds produced by marine dinoflagellates. Mass spectrometric screenings of extracts from natural plankton assemblages revealed a high chemical diversity among this toxin class, yet only few toxins are structurally known. Here we report the structural characterization of four novel cyclic-imine toxins (two gymnodimines (GYMs) and two spirolides (SPXs)) from cultures of Alexandrium ostenfeldii. A GYM with m/z 510 (1) was identified as 16-desmethylGYM D. A GYM with m/z 526 was identified as the hydroxylated degradation product of (1) with an exocyclic methylene at C-17 and an allylic hydroxyl group at C-18. This compound was named GYM E (2). We further identified a SPX with m/z 694 as 20-hydroxy-13,19-didesmethylSPX C (10) and a SPX with m/z 696 as 20-hydroxy-13,19-didesmethylSPX D (11). This is the first report of GYMs without a methyl group at ring D and SPXs with hydroxyl groups at position C-20. These compounds can be conceived as derivatives of the same nascent polyketide chain, supporting the hypothesis that GYMs and SPXs are produced through common biosynthetic genes. Both novel GYMs 1 and 2 were detected in significant amounts in extracts from natural plankton assemblages (1: 447 pg; 2: 1250 pg; 11: 40 pg per mL filtered seawater respectively).

The early post-settlement stage is the most sensitive during the life history of reef building corals. However, few studies have examined the factors that influence coral mortality during this period. Here, the impact of fouling on the survival of newly settled coral spat of Acropora millepora was investigated by manipulating the extent of fouling cover on settlement tiles using non-toxic, wax antifouling coatings. Survival of spat on coated tiles was double that on control tiles. Moreover, there was a significant negative correlation between percentage cover of fouling and spat survival across all tiles types, suggesting that fouling in direct proximity to settled corals has detrimental effects on early post-settlement survival. While previous studies have shown that increased fouling negatively affects coral larval settlement and health of juvenile and adult corals, to the best of our knowledge, this is the first study to show a direct relationship between fouling and early post-settlement survival for a broadcast spawning scleractinian coral. The negative effects of fouling on this sensitive life history stage may become more pronounced in the future as coastal eutrophication increases. Our results further suggest that targeted seeding of coral spat on artificial surfaces in combination with fouling control could prove useful to improve the efficiency of sexual reproduction-based coral propagation for reef rehabilitation.

The morphogenetic transition of motile coral larvae into sessile primary polyps is triggered and genetically programmed upon exposure to environmental biomaterials, such as crustose coralline algae (CCA) and bacterial biofilms. Although the specific chemical cues that trigger coral larval morphogenesis are poorly understood there is much more information available on the genes that play a role in this early life phase. Putative chemical cues from natural biomaterials yielded defined chemical samples that triggered different morphogenetic outcomes: an extract derived from a CCA-associated Pseudoalteromonas bacterium that induced metamorphosis, characterized by non-attached metamorphosed juveniles; and two fractions of the CCA Hydrolithon onkodes (Heydrich) that induced settlement, characterized by attached metamorphosed juveniles. In an effort to distinguish the genes involved in these two morphogenetic transitions, competent larvae of the coral Acropora millepora were exposed to these predictable cues and the expression profiles of 47 coral genes of interest (GOI) were investigated after only 1 hour of exposure using multiplex RT-qPCR. Thirty-two GOI were differentially expressed, indicating a putative role during the early regulation of morphogenesis. The most striking differences were observed for immunity-related genes, hypothesized to be involved in cell recognition and adhesion, and for fluorescent protein genes. Principal component analysis of gene expression profiles resulted in separation between the different morphogenetic cues and exposure times, and not only identified those genes involved in the early response but also those which influenced downstream biological changes leading to larval metamorphosis or settlement.

Biofilms of the bacterium Pseudoalteromonas induce metamorphosis of acroporid coral larvae. The bacterial metabolite tetrabromopyrrole (TBP), isolated from an extract of Pseudoalteromonas sp. associated with the crustose coralline alga (CCA) Neogoniolithon fosliei, induced coral larval metamorphosis (100%) with little or no attachment (0-2%). To better understand the molecular events and mechanisms underpinning the induction of Acropora millepora larval metamorphosis, including cell proliferation, apoptosis, differentiation, migration, adhesion and biomineralisation, two novel coral gene expression assays were implemented. These involved the use of reverse-transcriptase quantitative PCR (RT-qPCR) and employed 47 genes of interest (GOI), selected based on putative roles in the processes of settlement and metamorphosis. Substantial differences in transcriptomic responses of GOI were detected following incubation of A. millepora larvae with a threshold concentration and 10-fold elevated concentration of TBP-containing extracts of Pseudoalteromonas sp. The notable and relatively abrupt changes of the larval body structure during metamorphosis correlated, at the molecular level, with significant differences (p

The induction of larval attachment and metamorphosis of benthic marine invertebrates is widely considered to rely on habitat specific cues. While microbial biofilms on marine hard substrates have received considerable attention as specific signals for a wide and phylogenetically diverse array of marine invertebrates, the presumed chemical settlement signals produced by the bacteria have to date not been characterized. Here we isolated and fully characterized the first chemical signal from bacteria that induced larval metamorphosis of acroporid coral larvae (Acropora millepora). The metamorphic cue was identified as tetrabromopyrrole (TBP) in four bacterial Pseudoalteromonas strains among a culture library of 225 isolates obtained from the crustose coralline algae Neogoniolithon fosliei and Hydrolithon onkodes. Coral planulae transformed into fully developed polyps within 6 h, but only a small proportion of these polyps attached to the substratum. The biofilm cell density of the four bacterial strains had no influence on the ratio of attached vs. non-attached polyps. Larval bioassays with ethanolic extracts of the bacterial isolates, as well as synthetic TBP resulted in consistent responses of coral planulae to various doses of TBP. The lowest bacterial density of one of the Pseudoalteromonas strains which induced metamorphosis was 7,000 cells mm(-2) in laboratory assays, which is on the order of 0.1-1% of the total numbers of bacteria typically found on such surfaces. These results, in which an actual cue from bacteria has been characterized for the first time, contribute significantly towards understanding the complex process of acroporid coral larval settlement mediated through epibiotic microbial biofilms on crustose coralline algae.