Fluorescent proteins generate a genetic color polymorphism and counteract oxidative stress in intertidal sea anemones.

Fluorescent proteins (FPs) are ubiquitous tools in research, yet their endogenous functions in nature are poorly understood. In this work, we describe a combination of functions for FPs in a clade of intertidal sea anemones whose FPs control a genetic color polymorphism together with the ability to combat oxidative stress. Focusing on the underlying genetics of a fluorescent green "Neon" color morph, we show that allelic differences in a single FP gene generate its strong and vibrant color, by increasing both molecular brightness and FP gene expression level. Natural variation in FP sequences also produces differences in antioxidant capacity. We demonstrate that these FPs are strong antioxidants that can protect live cells against oxidative stress. Finally, based on structural modeling of the responsible amino acids, we propose a model for FP antioxidant function that is driven by molecular surface charge. Together, our findings shed light on the multifaceted functions that can co-occur within a single FP and provide a framework for studying the evolution of fluorescence as it balances spectral and physiological functions in nature.

Chemoenzymatic Isolation and Characterization of High Purity Mammalian Melanin.

Although melanin is one of the most ubiquitous polymers in living systems, our understanding of its molecular structure, biosynthesis and biophysical properties has been limited to only a small number of organisms other than humans. This is in part due to the difficulty associated with isolating pure melanin. While purification methods exist, they typically involve harsh treatments with strong acid/base conditions combined with elevated temperatures that can lead to the polymer backbone degradation. To be successful, a viable isolation method must deliver a selective, yet complete degradation of non-melanin biopolymers as well as remove small molecule metabolites that are not integrative to the melanin backbone. Here, we demonstrate the use of chemoenzymatic processing guided by fluorescent probes for the purification and isolation of native mammalian melanin without significant induction of chemical degradation. This multi-step purification-tracking methodology enables quantitative isolation of pure melanin from mammalian tissue for spectroscopic characterization.

Fate plasticity and reprogramming in genetically distinct populations of Danio leucophores.

Understanding genetic and cellular bases of adult form remains a fundamental goal at the intersection of developmental and evolutionary biology. The skin pigment cells of vertebrates, derived from embryonic neural crest, are a useful system for elucidating mechanisms of fate specification, pattern formation, and how particular phenotypes impact organismal behavior and ecology. In a survey of Danio fishes, including the zebrafish Danio rerio, we identified two populations of white pigment cells-leucophores-one of which arises by transdifferentiation of adult melanophores and another of which develops from a yellow-orange xanthophore or xanthophore-like progenitor. Single-cell transcriptomic, mutational, chemical, and ultrastructural analyses of zebrafish leucophores revealed cell-type-specific chemical compositions, organelle configurations, and genetic requirements. At the organismal level, we identified distinct physiological responses of leucophores during environmental background matching, and we showed that leucophore complement influences behavior. Together, our studies reveal independently arisen pigment cell types and mechanisms of fate acquisition in zebrafish and illustrate how concerted analyses across hierarchical levels can provide insights into phenotypes and their evolution.

Unraveling the Structure and Function of Melanin through Synthesis.

Melanin is ubiquitous in living organisms across different biological kingdoms of life, making it an important, natural biomaterial. Its presence in nature from microorganisms to higher animals and plants is attributed to the many functions of melanin, including pigmentation, radical scavenging, radiation protection, and thermal regulation. Generally, melanin is classified into five types-eumelanin, pheomelanin, neuromelanin, allomelanin, and pyomelanin-based on the various chemical precursors used in their biosynthesis. Despite its long history of study, the exact chemical makeup of melanin remains unclear, and it moreover has an inherent diversity and complexity of chemical structure, likely including many functions and properties that remain to be identified. Synthetic mimics have begun to play a broader role in unraveling structure and function relationships of natural melanins. In the past decade, polydopamine, which has served as the conventional form of synthetic eumelanin, has dominated the literature on melanin-based materials, while the synthetic analogues of other melanins have received far less attention. In this perspective, we will discuss the synthesis of melanin materials with a special focus beyond polydopamine. We will emphasize efforts to elucidate biosynthetic pathways and structural characterization approaches that can be harnessed to interrogate specific structure-function relationships, including electron paramagnetic resonance (EPR) and solid-state nuclear magnetic resonance (ssNMR) spectroscopy. We believe that this timely Perspective will introduce this class of biopolymer to the broader chemistry community, where we hope to stimulate new opportunities in novel, melanin-based poly-functional synthetic materials.

Acquisition of obligate mutualist symbionts during the larval stage is not beneficial for a coral host.

Theory suggests that the direct transmission of beneficial endosymbionts (mutualists) from parents to offspring (vertical transmission) in animal hosts is advantageous and evolutionarily stable, yet many host species instead acquire their symbionts from the environment (horizontal acquisition). An outstanding question in marine biology is why some scleractinian corals do not provision their eggs and larvae with the endosymbiotic dinoflagellates that are necessary for a juvenile's ultimate survival. We tested whether the acquisition of photosynthetic endosymbionts (family Symbiodiniaceae) during the planktonic larval stage was advantageous, as is widely assumed, in the ecologically important and threatened Caribbean reef-building coral Orbicella faveolata. Following larval acquisition, similar changes occurred in host energetic lipid use and gene expression regardless of whether their symbionts were photosynthesizing, suggesting the symbionts did not provide the energetic benefit characteristic of the mutualism in adults. Larvae that acquired photosymbionts isolated from conspecific adults on their natal reef exhibited a reduction in swimming, which may interfere with their ability to find suitable settlement substrate, and also a decrease in survival. Larvae exposed to two cultured algal species did not exhibit differences in survival, but decreased their swimming activity in response to one species. We conclude that acquiring photosymbionts during the larval stage confers no advantages and can in fact be disadvantageous to this coral host. The timing of symbiont acquisition appears to be a critical component of a host's life history strategy and overall reproductive fitness, and this timing itself appears to be under selective pressure.

Before platelets: the production of platelet-activating factor during growth and stress in a basal marine organism.

Corals and humans represent two extremely disparate metazoan lineages and are therefore useful for comparative evolutionary studies. Two lipid-based molecules that are central to human immunity, platelet-activating factor (PAF) and Lyso-PAF were recently identified in scleractinian corals. To identify processes in corals that involve these molecules, PAF and Lyso-PAF biosynthesis was quantified in conditions known to stimulate PAF production in mammals (tissue growth and exposure to elevated levels of ultraviolet light) and in conditions unique to corals (competing with neighbouring colonies over benthic space). Similar to observations in mammals, PAF production was higher in regions of active tissue growth and increased when corals were exposed to elevated levels of ultraviolet light. PAF production also increased when corals were attacked by the stinging cells of a neighbouring colony, though only the attacked coral exhibited an increase in PAF. This reaction was observed in adjacent areas of the colony, indicating that this response is coordinated across multiple polyps including those not directly subject to the stress. PAF and Lyso-PAF are involved in coral stress responses that are both shared with mammals and unique to the ecology of cnidarians.

Combined effects of water flow and copper concentration on the feeding behavior, growth rate, and accumulation of copper in tissue of the infaunal polychaete Polydora cornuta.

We performed an experiment in a laboratory flume to test the effects of water flow speed and the concentration of aqueaous copper on the feeding behavior, growth rate, and accumulation of copper in the tissues of juvenile polychaetes Polydora cornuta. The experiment included two flow speeds (6 or 15 cm/s) and two concentrations of added copper (0 or 85 µg/L). Worms grew significantly faster in the faster flow and in the lower copper concentration. In the slower flow, the total time worms spent feeding decreased significantly as copper concentration increased, but copper did not significantly affect the time worms spent feeding in the faster flow. Across all treatments, there was a significant, positive relationship between the time individuals spent feeding and their relative growth rate. Worms were observed suspension feeding significantly more often in the faster flow and deposit feeding significantly more often in the slower flow, but copper concentration did not affect the proportion of time spent in either feeding mode. The addition of 85 µg/L copper significantly increased copper accumulation in P. cornuta tissue, but the accumulation did not differ significantly due to flow speed. There was a significant interaction between copper and flow; the magnitude of the difference in copper accumulation between the 0 and 85 µg/L treatments was greater in the faster flow than in the slower flow. In slow flows that favor deposit feeding, worms grow slowly and accumulate less copper in their tissue than in faster flows that favor suspension feeding and faster growth.

Capillary foams: stabilization and functionalization of porous liquids and solids.

Liquid foams are two-phase systems in which a large volume of gas is dispersed as bubbles in a continuous liquid phase. These foams are ubiquitous in nature. In addition, they are found in industrial applications, such as pharmaceutical formulation, food processing, wastewater treatment, construction, and cosmetics. Recently, we reported a new type of foam material, capillary foam, which is stabilized by the synergistic action of particles and a small amount of an immiscible secondary liquid. In this study, we explore in more detail the foam preparation routes. To illustrate some of the potential applications, we create vividly colored wet and dried foams, which are difficult to prepare using traditional methods, and load-bearing porous solids. The combined action of particles and immiscible secondary fluid confers exceptional stability to capillary foams and many options for functionalization, suggesting a wide range of possible applications.

Regulation of Green Fluorescent Proteins by Sea Anemones (Anthopleura spp.) in Response to Light.

Green Fluorescent Proteins (GFPs) are a family of proteins with a disjunct systematic distribution; their biological functions remain speculative for the most part. Here we report studies of 3 closely related species of green sea anemones (Anthopleura) that express GFPs throughout their ectoderm. Individuals of these species maintain facultative symbiosis with zooxanthellae in their endoderm and inhabit the rocky intertidal or shallow subtidal. Thus, they depend on exposure to light to maintain photosynthesis of their symbionts, and simultaneously need to manage stresses associated with this exposure. We present experimental evidence that these sea anemones regulate the amount of GFP in their bodies in response to the surrounding light environment: they increase or reduce GFP when exposed to brighter or dimmer light, respectively, yet they maintain some GFP while in darkness, for surprisingly long periods.

Not so biodegradable: Polylactic acid and cellulose/plastic blend textiles lack fast biodegradation in marine waters.

The resistance of plastic textiles to environmental degradation is of major concern as large portions of these materials reach the ocean. There, they persist for undefined amounts of time, possibly causing harm and toxicity to marine ecosystems. As a solution to this problem, many compostable and so-called biodegradable materials have been developed. However, to undergo rapid biodegradation, most compostable plastics require specific conditions that are achieved only in industrial settings. Thus, industrially compostable plastics might persist as pollutants under natural conditions. In this work, we tested the biodegradability in marine waters of textiles made of polylactic acid, a diffused industrially compostable plastic. The test was extended also to cellulose-based and conventional non-biodegradable oil-based plastic textiles. The analyses were complemented by bio-reactor tests for an innovative combined approach. Results show that polylactic acid, a so-called biodegradable plastic, does not degrade in the marine environment for over 428 days. This was also observed for the oil-based polypropylene and polyethylene terephthalate, including their portions in cellulose/oil-based plastic blend textiles. In contrast, natural and regenerated cellulose fibers undergo complete biodegradation within approximately 35 days. Our results indicate that polylactic acid resists marine degradation for at least a year, and suggest that oil-based plastic/cellulose blends are a poor solution to mitigate plastic pollution. The results on polylactic acid further stress that compostability does not imply environmental degradation and that appropriate disposal management is crucial also for compostable plastics. Referring to compostable plastics as biodegradable plastics is misleading as it may convey the perception of a material that degrades in the environment. Conclusively, advances in disposable textiles should consider the environmental impact during their full life cycle, and the existence of environmentally degradable disposal should not represent an alibi for perpetuating destructive throw-away behaviors.

Spectrally resolved chemiluminescent probes for sensitive multiplex molecular quantification.

Luminophores are frequently utilized probe labels for detecting biological analytes. Multiple fluorescent luminophores, or fluorophores, can be readily distinguished from one another based on different energy excitation and emission wavelengths and lifetimes. However, suitable methods and reagents for distinguishing multiples of the much more sensitive chemically initiated luminophores have been limited. Herein we describe a new class of hybrid luminophore probes that emit light of distinct wavelength ranges and intensities upon energy transfer (ET) from an in-common, acridinium ester chemiluminophore to a covalently conjugated fluorophore. This format supports rapid, rational design of spectrally resolvable, chemically initiated probes. Time-resolved spectrographic and luminescence characterizations indicate that ET is not dependent on overlap in the emission spectrum of the luminophore and the absorption spectra of acceptors, suggesting a non-Förster resonance ET mechanism. Analysis of a combination of the chemiluminophore and new chemiluminophore-acceptor conjugate probes demonstrates the benefits of their use in sensitive, multiplex quantification of nucleic acid sequences indicative of environmentally relevant microbes without prior enzymatic amplification.

Simultaneous quantification of multiple nucleic acid targets using chemiluminescent probes.

A novel method is described for simultaneous detection and quantification of attomoles or a few femtomoles of two (or potentially more) nucleic acid targets, without need for amplification. The technique depends on spectral-temporal resolution of chemiluminescence emitted from independent hybridization-induced chemiluminescent signal probes. The probes are internally quenched except in the presence of their specific targets, thereby allowing detection limits up to 10,000 times lower than with fluorescent probes. This is sufficient to obviate the need for amplification in many cases. The utility of the technique has been demonstrated by use of resolvable N-linked acridinium and 2,7-dimethoxyacridinium ester labeled probes in a homogeneous assay for sensitive and simultaneous independent quantification of pan-bacterial and pan-fungal target sequences in seawater.

Bioluminescent signals spatially amplified by wavelength-specific diffusion through the shell of a marine snail.

Some living organisms produce visible light (bioluminescence) for intra- or interspecific visual communication. Here, we describe a remarkable bioluminescent adaptation in the marine snail Hinea brasiliana. This species produces a luminous display in response to mechanical stimulation caused by encounters with other motile organisms. The light is produced from discrete areas on the snail's body beneath the snail's shell, and must thus overcome this structural barrier to be viewed by an external receiver. The diffusion and transmission efficiency of the shell is greater than a commercial diffuser reference material. Most strikingly, the shell, although opaque and pigmented, selectively diffuses the blue-green wavelength of the species bioluminescence. This diffusion generates a luminous display that is enlarged relative to the original light source. This unusual shell thus allows spatially amplified outward transmission of light communication signals from the snail, while allowing the animal to remain safely inside its hard protective shell.

Increased bioavailability of mercury in the lagoons of Lomb, Togo: the possible role of dredging.

Surface sediments of the lagoons of Lomé, Togo, were analyzed for mercury, methylmercury, and trace elements. Concentrations were greater than typical for natural lagoon sediments, and with greater variability within the Eastern lagoon compared to the Western one. The Eastern lagoon is larger and has been dredged in the past, while the Western lagoon, which also receives major waste inputs, has not been dredged and shows less tidal flushing. Accordingly, one naturally believes that the Eastern lagoon is cleaner and probably safe to use due to its natural resources, including fishes to eat. Unexpectedly, we describe here that mercury methylation was greater in the Eastern lagoon, indicating increased bioavailability of mercury, as probably facilitated by past dredging that decreased solid-phase retention of inorganic mercury. Urbanization has historically been more developed in the southern part of the lagoons, which is still reflected in contamination levels of sediment despite dredging, probably because sources of contamination are still more important there today. Such urban contamination emphasizes the need to regulate waste discharges and possible airborne contamination in growing cities of developing countries, and implements environmental and public health monitoring, especially in relation to misbelieves systematically associated with the cleansing effect of dredging activity.

Polycyclic aromatic hydrocarbons and trace metal contamination of coastal sediment and biota from Togo.

The state of contamination of tropical environments, particularly in Africa, remains a relatively under explored subject. Here, we determined polycyclic aromatic hydrocarbon (PAH) and trace metal concentrations in coastal sediment and biota samples (fish and mussels) from Togo (West Africa). In the sediments, the ∑21 PAH concentrations ranged from <4 ng g(-1) to 257 ng g(-1), averaging 92 ng g(-1). Concentration ratios of low molecular weight PAHs (2-3 rings) versus high molecular weight PAHs (≥4 rings) were always lower than 1 (ranging from 0.08 to 0.46) indicating that high molecular weight PAHs were dominant in all sediment samples, and that PAHs originated mainly from anthropogenic combustion activities. The sediments were also analyzed for major elements and a total of 15 trace metals, which were found in elevated concentrations. The calculated enrichment factor (EF) values relative to the Earth's crust show that the contamination is extremely severe for Cd (EF = 191), severe for Cr (EF = 18) and U (EF = 17.8), moderately severe for Zr (EF = 8.8), for Ni (EF = 6.8), Sr (EF = 5.9) and Ba (EF = 5.4), and moderate for V (EF = 3.6) and Zn (EF = 3.4). Sediments sampled in areas affected by the dumping of phosphorite mine tailings showed particularly high concentrations of trace metals. Overall, concentrations of both PAHs and trace metals in sediment tend to increase from the coastline to the open sea (2 km offshore). This is attributable to the increasingly finer texture of coastal sediment found offshore, which has a terrigenous origin and appears loaded with various contaminants through adsorption processes. Such high loads of trace metals were also found in the biota (fish and mussels). The ratio of measured trace metal concentrations in biota to threshold limits set by the World Health Organization herein defined as relative health factor (RHF) was high. Average RHF values in fish were highest for Se (470), As (250), Ag (97), Ni (78), Mn (63), Fe (53), Pb (36), Cd (10), and Cr (7) while lowest for Cu (0.08) and Zn (0.03). Cd and Al did not bioaccumulate in the analyzed fish species. In mussels, the RHF values were highest for Fe (9,108), As (295), Pb (276), Se (273), Mn (186), Ni (71), Ag (70), Cd (14), and Cu (4).

Degradation of synthetic and wood-based cellulose fabrics in the marine environment: Comparative assessment of field, aquarium, and bioreactor experiments.

As global production of textiles rapidly grows, there is urgency to understand the persistence of fabrics in the marine environment, particularly from the microfibers they shed during wearing and washing. Here, we show that fabrics containing polyester (one of the most common plastics) remained relatively intact (viz., with a limited biofilm) after >200 days in seawater off the Scripps Oceanography pier (La Jolla, CA), in contrast to wood-based cellulose fabrics that fell apart within 30 days. We also show similar results under experimental aquaria (in open circuit with the pier waters) as well as bioreactor settings (in close circuit, using microbial inoculum from the North Sea, off Belgium), using nonwoven fabrics and individual fibers, respectively. The fact that fibers released from synthetic textiles remain persistent and non-biodegradable despite their small (invisible) size, highlights concern for the growing industry that uses polyester from recycled plastics to make clothing.

Green fluorescent protein regulation in the coral Acropora yongei during photoacclimation.

Reef-building corals inhabit high light environments and are dependent on photosynthetic endosymbiotic dinoflagellates for nutrition. While photoacclimation responses of the dinoflagellates to changes in illumination are well understood, host photoacclimation strategies are poorly known. This study investigated fluorescent protein expression in the shallow-water coral Acropora yongei during a 30 day laboratory photoacclimation experiment in the context of its dinoflagellate symbionts. Green fluorescent protein (GFP) concentration measured by Western blotting changed reversibly with light intensity. The first 15 days of the photoacclimation experiment led to a ∼1.6 times increase in GFP concentration for high light corals (900 µmol quanta m⁻² s⁻¹) and a ∼4 times decrease in GFP concentration for low light corals (30 µmol quanta m⁻² s⁻¹) compared with medium light corals (300 µmol quanta m⁻² s⁻¹). Green fluorescence increased ∼1.9 times in high light corals and decreased ∼1.9 times in low light corals compared with medium light corals. GFP concentration and green fluorescence intensity were significantly correlated. Typical photoacclimation responses in the dinoflagellates were observed including changes in density, photosynthetic pigment concentration and photosynthetic efficiency. Although fluorescent proteins are ubiquitous and abundant in scleractinian corals, their functions remain ambiguous. These results suggest that scleractinian corals regulate GFP to modulate the internal light environment and support the hypothesis that GFP has a photoprotective function. The success of photoprotection and photoacclimation strategies, in addition to stress responses, will be critical to the fate of scleractinian corals exposed to climate change and other stressors.

Limitations on electromagnetic communication by vibrational resonances in biological systems.

Previous research in biology and physics speculates that high-frequency electromagnetic fields may be an unexplored method of cellular and subcellular communication. The predominant theory for generating electric fields in the cell is mechanical vibration of charged or polar biomolecules such as cell membranes or microtubules. The challenge to this theory is explaining how high-frequency vibrations would not be overdamped by surrounding biological media. As many of these suspected resonators are too large for atomistic molecular dynamics simulations, accurately modeling biological resonators remains an ongoing challenge. While many resonators have been studied and simulated, the general limitations on communication imposed by energy transfer arguments have not been considered. Starting with energy transfer expressions from coupled-mode theory, we derive expressions for the minimum quality factor (Q factor) required to sustain communication for both near- and far-field interactions. We compare previous simulation studies and our theory. We determine the flexing mode of microtubules as an identified resonance in the literature which meets our criteria. Our results suggest the major obstacle to meeting our criteria for effective electromagnetic communication is the trade-off between the Q factor and the plasma frequency: Resonators must be large enough to have a large Q factor, but small enough to resonate at frequencies greater than the plasma frequency.

Iron redox pathway revealed in ferritin via electron transfer analysis.

Ferritin protein is involved in biological tissues in the storage and management of iron - an essential micro-nutrient in the majority of living systems. While there are extensive studies on iron-loaded ferritin, its functionality in iron delivery is not completely clear. Here, for the first time, differential pulse voltammetry (DPV) has been successfully adapted to address the challenge of resolving a cascade of fast and co-occurring redox steps in enzymatic systems such as ferritin. Using DPV, comparative analysis of ferritins from two evolutionary-distant organisms has allowed us to propose a stepwise resolution for the complex mix of concurrent redox steps that is inherent to ferritins and to fine-tune the structure-function relationship of each redox step. Indeed, the cyclic conversion between Fe3+ and Fe2+ as well as the different oxidative steps of the various ferroxidase centers already known in ferritins were successfully discriminated, bringing new evidence that both the 3-fold and 4-fold channels can be functional in ferritin.