Single Point Mutation Abolishes Water Capture in Germacradien-4-ol Synthase.

The high-fidelity sesquiterpene cyclase (-)-germacradien-4-ol synthase (GdolS) converts farnesyl diphosphate into the macrocyclic alcohol (-)-germacradien-4-ol. Site-directed mutagenesis was used to decipher the role of key residues in the water control mechanism. Replacement of Ala176, located in the G1/2 helix, with non-polar aliphatic residues of increasing size (valine, leucine, isoleucine and methionine) resulted in the accumulation of the non-hydroxylated products germacrene A and germacrene D. In contrast, hydroxylation was maintained when the polar residues threonine, glutamine or aspartate replaced Ala176. Additionally, although a contribution of His150 to the nucleophilic water addition could be ruled out, the imidazole ring of His150 appears to assist carbocation stabilisation. The results presented here shed light on how hydroxylating sesquiterpene synthases can be engineered to design modified sesquiterpene synthases to reduce the need for further steps in the biocatalytic production of oxygenated sesquiterpenoids.

Simulation-Guided Engineering Enables a Functional Switch in Selinadiene Synthase toward Hydroxylation.

Engineering sesquiterpene synthases to form predefined alternative products is a major challenge due to their diversity in cyclization mechanisms and our limited understanding of how amino acid changes affect the steering of these mechanisms. Here, we use a combination of atomistic simulation and site-directed mutagenesis to engineer a selina-4(15),7(11)-diene synthase (SdS) such that its final reactive carbocation is quenched by trapped active site water, resulting in the formation of a complex hydroxylated sesquiterpene (selin-7(11)-en-4-ol). Initially, the SdS G305E variant produced 20% selin-7(11)-en-4-ol. As suggested by modeling of the enzyme-carbocation complex, selin-7(11)-en-4-ol production could be further improved by varying the pH, resulting in selin-7(11)-en-4-ol becoming the major product (48%) at pH 6.0. We incorporated the SdS G305E variant along with genes from the mevalonate pathway into bacterial BL21(DE3) cells and demonstrated the production of selin-7(11)-en-4-ol at a scale of 10 mg/L in batch fermentation. These results highlight opportunities for the simulation-guided engineering of terpene synthases to produce predefined complex hydroxylated sesquiterpenes.

Chromosomal inversions harbour excess mutational load in the coral, Acropora kenti, on the Great Barrier Reef.

The future survival of coral reefs in the Anthropocene depends on the capacity of corals to adapt as oceans warm and extreme weather events become more frequent. Targeted interventions designed to assist evolutionary processes in corals require a comprehensive understanding of the distribution and structure of standing variation, however, efforts to map genomic variation in corals have so far focussed almost exclusively on SNPs, overlooking structural variants that have been shown to drive adaptive processes in other taxa. Here, we show that the reef-building coral, Acropora kenti, harbours at least five large, highly polymorphic structural variants, all of which exhibit signatures of strongly suppressed recombination in heterokaryotypes, a feature commonly associated with chromosomal inversions. Based on their high minor allele frequency, uniform distribution across habitats and elevated genetic load, we propose that these inversions in A. kenti are likely to be under balancing selection. An excess of SNPs with high impact on protein-coding genes within these loci elevates their importance both as potential targets for adaptive selection and as contributors to genetic decline if coral populations become fragmented or inbred in future.

Microbiome manipulation by corals and other Cnidaria via quorum quenching.

A dynamic mucous layer containing numerous micro-organisms covers the surface of corals and has multiple functions including both removal of sediment and "food gathering."1 It is likely to also act as the primary barrier to infection; various proteins and compounds with antimicrobial activity have been identified in coral mucus, though these are thought to be largely or exclusively of microbial origin. As in Hydra,2 anti-microbial peptides (AMPs) are likely to play major roles in regulating the microbiomes of corals.3,4 Some eukaryotes employ a complementary but less obvious approach to manipulate their associated microbiome by interfering with quorum signaling, effectively preventing bacteria from coordinating gene expression across a population. Our investigation of immunity in the reef-building coral Acropora millepora,5 however, led to the discovery of a coral gene referred to here as AmNtNH1 that can inactivate a range of acyl homoserine lactones (AHLs), common bacterial quorum signaling molecules, and is induced on immune challenge of adult corals and expressed during the larval settlement process. Closely related proteins are widely distributed within the Scleractinia (hard corals) and some other cnidarians, with multiple paralogs in Acropora, but their closest relatives are bacterial, implying that these are products of one or more lateral gene transfer events post-dating the cnidarian-bilaterian divergence. The deployment by corals of genes used by bacteria to compete with other bacteria reflects a mechanism of microbiome manipulation previously unknown in Metazoa but that may apply more generally.

Production of non-natural terpenoids through chemoenzymatic synthesis using substrate analogs.

Chemoenzymatic synthesis of non-natural terpenes using the promiscuous activity of terpene synthases allows for the expansion of the chemical space of terpenoids with potentially new bioactivities. In this report, we describe protocols for the preparation of a novel aphid attractant, (S)-14,15-dimethylgermacrene D, by exploiting the promiscuity of (S)-germacrene D synthase from Solidago canadensis and using an engineered biocatalytic route to convert prenols to terpenoids. The method uses a combination of five enzymes to carry out the preparation of terpenoid semiochemicals in two steps: (1) diphosphorylation of five or six carbon precursors (prenol, isoprenol and methyl-isoprenol) catalyzed by Plasmodium falciparum choline kinase and Methanocaldococcus jannaschii isopentenyl phosphate kinase to form DMADP, IDP and methyl-IDP, and (2) chain elongation and cyclization catalyzed by Geobacillus stearothermophilus (2E,6E)-farnesyl diphosphate synthase and S. canadensis (S)-germacrene D synthase to produce (S)-germacrene D and (S)-14,15-dimethylgermacrene D. Using this method, new non-natural terpenoids are readily accessible and the approach can be adopted to produce different terpene analogs and terpenoid derivatives with potential novel applications.

Discovery and first-time disclosure of CVN766, an exquisitely selective orexin 1 receptor antagonist.

Modulators of orexin receptors are being developed for neurological illnesses such as sleep disorders, addictive behaviours and other psychiatric diseases. We herein describe the discovery of CVN766, a potent orexin 1 receptor antagonist that has greater than 1000-fold selectivity for the orexin 1 receptor over the orexin 2 receptor and demonstrates low off target hits in a diversity screen. In agreement with its in vitro ADME data, CVN766 demonstrated moderate in vivo clearance in rodents and displayed good brain permeability and target occupancy. This drug candidate is currently being investigated in clinical trials for schizophrenia and related psychiatric conditions.

Sperm in the Mammalian Female Reproductive Tract: Surfing Through the Tract to Try to Beat the Odds.

Mammalian sperm are deposited in the vagina or the cervix/uterus at coitus or at artificial insemination, and the fertilizing sperm move through the female reproductive tract to the ampulla of the oviduct, the site of fertilization. But the destination of most sperm is not the oviduct. Most sperm are carried by retrograde fluid flow to the vagina, are phagocytosed, and/or do not pass barriers on the pathway to the oviduct. The sperm that reach the site of fertilization are the exceptions and winners of one of the most stringent selection processes in nature. This review discusses the challenges sperm encounter and how the few sperm that reach the site of fertilization overcome them. The sperm that reach the goal must navigate viscoelastic fluid, swim vigorously and cooperatively along the walls of the female tract, avoid the innate immune system, and respond to potential cues to direct their movement.

Coupled Nanomechanical Graphene Resonators: A Promising Platform for Scalable NEMS Networks.

Arrays of coupled nanoelectromechanical resonators are a promising foundation for implementing large-scale network applications, such as mechanical-based information processing and computing, but their practical realization remains an outstanding challenge. In this work, we demonstrate a scalable platform of suspended graphene resonators, such that neighboring resonators are persistently coupled mechanically. We provide evidence of strong coupling between neighboring resonators using two different tuning methods. Additionally, we provide evidence of inter-resonator coupling of higher-order modes, demonstrating the rich dynamics that can be accessed with this platform. Our results establish this platform as a viable option for realizing large-scale programmable networks, enabling applications such as phononic circuits, tunable waveguides, and reconfigurable metamaterials.

Active Site Loop Engineering Abolishes Water Capture in Hydroxylating Sesquiterpene Synthases.

Terpene synthases (TS) catalyze complex reactions to produce a diverse array of terpene skeletons from linear isoprenyl diphosphates. Patchoulol synthase (PTS) from Pogostemon cablin converts farnesyl diphosphate into patchoulol. Using simulation-guided engineering, we obtained PTS variants that eliminate water capture. Further, we demonstrate that modifying the structurally conserved Hα-1 loop also reduces hydroxylation in PTS, as well as in germacradiene-11-ol synthase (Gd11olS), leading to cyclic neutral intermediates as products, including α-bulnesene (PTS) and isolepidozene (Gd11olS). Hα-1 loop modification could be a general strategy for engineering sesquiterpene synthases to produce complex cyclic hydrocarbons without the need for structure determination or modeling.

Bathymetric evolution of black corals through deep time.

Deep-sea lineages are generally thought to arise from shallow-water ancestors, but this hypothesis is based on a relatively small number of taxonomic groups. Anthozoans, which include corals and sea anemones, are significant contributors to the faunal diversity of the deep sea, but the timing and mechanisms of their invasion into this biome remain elusive. Here, we reconstruct a fully resolved, time-calibrated phylogeny of 83 species in the order Antipatharia (black coral) to investigate their bathymetric evolutionary history. Our reconstruction indicates that extant black coral lineages first diversified in continental slope depths (∼250-3000 m) during the early Silurian (∼437 millions of years ago (Ma)) and subsequently radiated into, and diversified within, both continental shelf (less than 250 m) and abyssal (greater than 3000 m) habitats. Ancestral state reconstruction analysis suggests that the appearance of morphological features that enhanced the ability of black corals to acquire nutrients coincided with their invasion of novel depths. Our findings have important conservation implications for anthozoan lineages, as the loss of 'source' slope lineages could threaten millions of years of evolutionary history and confound future invasion events, thereby warranting protection.

Validation of key sponge symbiont pathways using genome-centric metatranscriptomics.

The sponge microbiome underpins host function through provision and recycling of essential nutrients in a nutrient poor environment. Genomic data suggest that carbohydrate degradation, carbon fixation, nitrogen metabolism, sulphur metabolism and supplementation of B-vitamins are central microbial functions. However, validation beyond the genomic potential of sponge symbiont pathways is rarely explored. To evaluate metagenomic predictions, we sequenced the metagenomes and metatranscriptomes of three common coral reef sponges: Ircinia ramosa, Ircinia microconulosa and Phyllospongia foliascens. Multiple carbohydrate active enzymes were expressed by Poribacteria, Bacteroidota and Cyanobacteria symbionts, suggesting these lineages have a central role in assimilating dissolved organic matter. Expression of entire pathways for carbon fixation and multiple sulphur compound transformations were observed in all sponges. Gene expression for anaerobic nitrogen metabolism (denitrification and nitrate reduction) were more common than aerobic metabolism (nitrification), where only the I. ramosa microbiome expressed the nitrification pathway. Finally, while expression of the biosynthetic pathways for B-vitamins was common, the expression of additional transporter genes was far more limited. Overall, we highlight consistencies and disparities between metagenomic and metatranscriptomic results when inferring microbial activity, while uncovering new microbial taxa that contribute to the health of their sponge host via nutrient exchange.

An oviduct glycan increases sperm lifespan by diminishing the production of ubiquinone and reactive oxygen species†.

Sperm storage by females after mating for species-dependent periods is used widely among animals with internal fertilization to allow asynchrony between mating and ovulation. Many mammals store sperm in the lower oviduct where specific glycans on oviduct epithelial cells retain sperm to form a reservoir. Binding to oviduct cells suppresses sperm intracellular Ca2+ and increases sperm longevity. We investigated the mechanisms by which a specific oviduct glycan, 3-O-sulfated Lewis X trisaccharide (suLeX), prolongs the lifespan of porcine sperm. Using targeted metabolomics, we found that binding to suLeX diminishes the abundance of 4-hydroxybenzoic acid, the precursor to ubiquinone (also known as Coenzyme Q), 30 min after addition. Ubiquinone functions as an electron acceptor in the electron transport chain (ETC). 3-O-sulfated Lewis X trisaccharide also suppressed the formation of fumarate. A component of the citric acid cycle, fumarate is synthesized by succinate-coenzyme Q reductase, which employs ubiquinone and is also known as Complex II in the ETC. Consistent with the reduced activity of the ETC, the production of harmful reactive oxygen species (ROS) was diminished. The enhanced sperm lifespan in the oviduct may be because of suppressed ROS production because high ROS concentrations have toxic effects on sperm.

Direct laser writing of 3D electrodes on flexible substrates.

This report describes a 3D microelectrode array integrated on a thin-film flexible cable for neural recording in small animals. The fabrication process combines traditional silicon thin-film processing techniques and direct laser writing of 3D structures at micron resolution via two-photon lithography. Direct laser-writing of 3D-printed electrodes has been described before, but this report is the first to provide a method for producing high-aspect-ratio structures. One prototype, a 16-channel array with 300 µm pitch, demonstrates successful electrophysiological signal capture from bird and mouse brains. Additional devices include 90 µm pitch arrays, biomimetic mosquito needles that penetrate through the dura of birds, and porous electrodes with enhanced surface area. The rapid 3D printing and wafer-scale methods described here will enable efficient device fabrication and new studies examining the relationship between electrode geometry and electrode performance. Applications include small animal models, nerve interfaces, retinal implants, and other devices requiring compact, high-density 3D electrodes.

Research priorities for the sustainability of coral-rich western Pacific seascapes.

Nearly a billion people depend on tropical seascapes. The need to ensure sustainable use of these vital areas is recognised, as one of 17 policy commitments made by world leaders, in Sustainable Development Goal (SDG) 14 ('Life below Water') of the United Nations. SDG 14 seeks to secure marine sustainability by 2030. In a time of increasing social-ecological unpredictability and risk, scientists and policymakers working towards SDG 14 in the Asia-Pacific region need to know: (1) How are seascapes changing? (2) What can global society do about these changes? and (3) How can science and society together achieve sustainable seascape futures? Through a horizon scan, we identified nine emerging research priorities that clarify potential research contributions to marine sustainability in locations with high coral reef abundance. They include research on seascape geological and biological evolution and adaptation; elucidating drivers and mechanisms of change; understanding how seascape functions and services are produced, and how people depend on them; costs, benefits, and trade-offs to people in changing seascapes; improving seascape technologies and practices; learning to govern and manage seascapes for all; sustainable use, justice, and human well-being; bridging communities and epistemologies for innovative, equitable, and scale-crossing solutions; and informing resilient seascape futures through modelling and synthesis. Researchers can contribute to the sustainability of tropical seascapes by co-developing transdisciplinary understandings of people and ecosystems, emphasising the importance of equity and justice, and improving knowledge of key cross-scale and cross-level processes, feedbacks, and thresholds.

Sperm selection by the oviduct: perspectives for male fertility and assisted reproductive technologies†.

The contribution of sperm to embryogenesis is gaining attention with up to 50% of infertility cases being attributed to a paternal factor. The traditional methods used in assisted reproductive technologies for selecting and assessing sperm quality are mainly based on motility and viability parameters. However, other sperm characteristics, including deoxyribonucleic acid integrity, have major consequences for successful live birth. In natural reproduction, sperm navigate the male and female reproductive tract to reach and fertilize the egg. During transport, sperm encounter many obstacles that dramatically reduce the number arriving at the fertilization site. In humans, the number of sperm is reduced from tens of millions in the ejaculate to hundreds in the Fallopian tube (oviduct). Whether this sperm population has higher fertilization potential is not fully understood, but several studies in animals indicate that many defective sperm do not advance to the site of fertilization. Moreover, the oviduct plays a key role in fertility by modulating sperm transport, viability, and maturation, providing sperm that are ready to fertilize at the appropriate time. Here we present evidence of sperm selection by the oviduct with emphasis on the mechanisms of selection and the sperm characteristics selected. Considering the sperm parameters that are essential for healthy embryonic development, we discuss the use of novel in vitro sperm selection methods that mimic physiological conditions. We propose that insight gained from understanding how the oviduct selects sperm can be translated to assisted reproductive technologies to yield high fertilization, embryonic development, and pregnancy rates.

An oviduct glycan increases sperm lifespan by diminishing ubiquinone and production of reactive oxygen species.

Sperm storage by females after mating for species-dependent periods is used widely among animals with internal fertilization to allow asynchrony between mating and ovulation. Many mammals store sperm in the lower oviduct where specific glycans on epithelial cells retain sperm to form a reservoir. Binding to oviduct cells suppresses sperm intracellular Ca 2+ and increases sperm longevity. We investigated the mechanisms by which a specific oviduct glycan, 3-O-sulfated Lewis X trisaccharide (suLe X ), prolongs the lifespan of porcine sperm. Using targeted metabolomics, we report that binding to suLe X diminishes the abundance of the precursor to ubiquinone and suppresses formation of fumarate, a specific citric acid cycle component, diminishing the activity of the electron transport chain and reducing the production of harmful reactive oxygen species (ROS). The enhanced sperm lifespan in the oviduct may be due to suppressed ROS production as many reports have demonstrated toxic effects of high ROS concentrations on sperm.

Multiple opsins in a reef-building coral, Acropora millepora.

Opsins, light-sensitive G protein-coupled receptors, have been identified in corals but their properties are largely unknown. Here, we identified six opsin genes (acropsins 1-6) from a coral species Acropora millepora, including three novel opsins (acropsins 4-6), and successfully characterized the properties of four out of the six acropsins. Acropsins 1 and 6 exhibited light-dependent cAMP increases in cultured cells, suggesting that the acropsins could light-dependently activate Gs-type G protein like the box jellyfish opsin from the same opsin group. Spectral sensitivity curves having the maximum sensitivities at ~ 472 nm and ~ 476 nm were estimated for acropsins 1 and 6, respectively, based on the light wavelength-dependent cAMP increases in these opsins-expressing cells (heterologous action spectroscopy). Acropsin 2 belonging to the same group as acropsins 1 and 6 did not induce light-dependent cAMP or Ca2+ changes. We then successfully estimated the acropsin 2 spectral sensitivity curve having its maximum value at ~ 471 nm with its chimera mutant which possessed the third cytoplasmic loop of the Gs-coupled jellyfish opsin. Acropsin 4 categorized as another group light-dependently induced intracellular Ca2+ increases but not cAMP changes. Our results uncovered that the Acropora coral possesses multiple opsins coupling two distinct cascades, cyclic nucleotide and Ca2+signaling light-dependently.

Evolutionary responses of a reef-building coral to climate change at the end of the last glacial maximum.

Climate change threatens the survival of coral reefs on a global scale, primarily through mass bleaching and mortality as a result of marine heatwaves. While these short-term effects are clear, predicting the fate of coral reefs over the coming century is a major challenge. One way to understand the longer-term effects of rapid climate change is to examine the response of coral populations to past climate shifts. Coastal and shallow-water marine ecosystems such as coral reefs have been reshaped many times by sea-level changes during the Pleistocene, yet, few studies have directly linked this with its consequences on population demographics, dispersal, and adaptation. Here we use powerful analytical techniques, afforded by haplotype phased whole-genomes, to establish such links for the reef-building coral, Acropora digitifera. We show that three genetically distinct populations are present in northwestern Australia, and that their rapid divergence since the last glacial maximum (LGM) can be explained by a combination of founder-effects and restricted gene flow. Signatures of selective sweeps, too strong to be explained by demographic history, are present in all three populations and overlap with genes that show different patterns of functional enrichment between inshore and offshore habitats. In contrast to rapid divergence in the host, we find that photosymbiont communities are largely undifferentiated between corals from all three locations, spanning almost 1000 km, indicating that selection on host genes and not acquisition of novel symbionts, has been the primary driver of adaptation for this species in northwestern Australia.

Newly Discovered Peptides from the Coral Heliofungia actiniformis Show Structural and Functional Diversity.

Scleractinian corals are crucially important to the health of some of the world's most biodiverse, productive, and economically important marine habitats. Despite this importance, analysis of coral peptidomes is still in its infancy. Here we show that the tentacle extract from the stony coral Heliofungia actiniformis is rich in peptides with diverse and novel structures. We have characterized the sequences and three-dimensional structures of four new peptides, three of which have no known homologues. We show that a 2 kDa peptide, Hact-2, promotes significant cell proliferation on human cells and speculate this peptide may be involved in the remarkable regenerative capacity of corals. We found a 3 kDa peptide, Hact-3, encoded within a fascin-like domain, and homologues of Hact-3 are present in the genomes of other coral species. Two additional peptides, Hact-4 and Hact-SCRiP1, with limited sequence similarity, both contain a beta-defensin-like fold and highlight a structural link with the small cysteine-rich proteins (SCRiP) family of proteins found predominantly in corals. Our results provide a first glimpse into the remarkable and unexplored structural diversity of coral peptides, providing insight into their diversity and putative functions and, given the ancient lineage of corals, potential insight into the evolution of structural motifs.

Hyperactivation is sufficient to release porcine sperm from immobilized oviduct glycans.

Fertilizing sperm are retained by adhesion to specific glycans on the epithelium of the oviduct forming a reservoir before sperm are released from the reservoir so fertilization can ensue. Capacitated sperm lose affinity for the oviduct epithelium but the components of capacitation that are important for sperm release are uncertain. One important correlate of capacitation is the development of hyperactivated motility. Hyperactivation is characterized by asymmetrical flagellar beating with high beat amplitude. We tested whether the development of full-type asymmetrical motility was sufficient to release sperm from immobilized oviduct glycans. Sperm hyperactivation was induced by four different compounds, a cell-permeable cAMP analog (cBiMPS), CatSper activators (4-aminopyridine and procaine), and an endogenous steroid (progesterone). Using standard analysis (CASA) and direct visualization with high-speed video microscopy, we first confirmed that all four compounds induced hyperactivation. Subsequently, sperm were allowed to bind to immobilized oviduct glycans, and compounds or vehicle controls were added. All compounds caused sperm release from immobilized glycans, demonstrating that hyperactivation was sufficient to release sperm from oviduct cells and immobilized glycans. Pharmacological inhibition of the non-genomic progesterone receptor and CatSper diminished sperm release from oviduct glycans. Inhibition of the proteolytic activities of the ubiquitin-proteasome system (UPS), implicated in the regulation of sperm capacitation, diminished sperm release in response to all hyperactivation inducers. In summary, induction of sperm hyperactivation was sufficient to induce sperm release from immobilized oviduct glycans and release was dependent on CatSper and the UPS.