Winter Flounder Research Articles

Molecular insights into the synergistic inhibition mechanisms of antifreeze protein and methanol on carbon dioxide hydrate growth

The formation of CO2 hydrates during CO2 transportation and deep-sea injection poses a significant risk of pipeline blockage. Combining kinetic (KHIs) and thermodynamic hydrate inhibitors (THIs) serves as a promising efficient and economical strategy to mitigate this issue, whose performance and microscopic mechanisms yet are still poorly understood. This study employs molecular dynamics simulations to explore the effects of the combination of winter flounder antifreeze protein (wf-AFP) as a KHI and methanol as a THI on CO2 hydrate growth. We find that methanol and wf-AFP exhibit an intriguing synergistic inhibition performance on hydrate growth. In the AFP-only system, AFP serves as a spatial hindrance near the hydrate growth interface. However, in the AFP + methanol system, AFP changes its position due to the attractive force of methanol. Part of the AFP fragment remains on the hydrate interface, while the rest surrounds the CO2 droplet. Methanol, in addition to disrupting the water structure, combines with AFP to act as a double barrier to CO2 dissolution into the aqueous solution, thereby significantly reducing the gas source for hydrate growth. These findings provide molecular-level insights into hydrate inhibition mechanisms and guide the design of efficient inhibitors for safe CO2 transportation and injection.

Diverse Origins of Near-Identical Antifreeze Proteins in Unrelated Fish Lineages Provide Insights Into Evolutionary Mechanisms of New Gene Birth and Protein Sequence Convergence.

Determining the origins of novel genes and the mechanisms driving the emergence of new functions is challenging yet crucial for understanding evolutionary innovations. Recently evolved fish antifreeze proteins (AFPs) offer a unique opportunity to explore these processes, particularly the near-identical type I AFP (AFPI) found in four phylogenetically divergent fish taxa. This study tested the hypothesis of protein sequence convergence beyond functional convergence in three unrelated AFPI-bearing fish lineages. Through comprehensive comparative analyses of newly sequenced genomes of winter flounder and grubby sculpin, along with available high-quality genomes of cunner and 14 other related species, the study revealed that near-identical AFPI proteins originated from distinct genetic precursors in each lineage. Each lineage independently evolved a de novo coding region for the novel ice-binding protein while repurposing fragments from their respective ancestors into potential regulatory regions, representing partial de novo origination-a process that bridges de novo gene formation and the neofunctionalization of duplicated genes. The study supports existing models of new gene origination and introduces new ones: the innovation-amplification-divergence model, where novel changes precede gene duplication; the newly proposed duplication-degeneration-divergence model, which describes new functions arising from degenerated pseudogenes; and the duplication-degeneration-divergence gene fission model, where each new sibling gene differentially degenerates and renovates distinct functional domains from their parental gene. These findings highlight the diverse evolutionary pathways through which a novel functional gene with convergent sequences at the protein level can evolve across divergent species, advancing our understanding of the mechanistic intricacies in new gene formation.

Patterns of persistence: Genetic and behavioral population complexity of winter flounder amid population declines.

Winter flounder Pseudopleuronectes americanus (Walbaum 1792) are a coastal flatfish species of economic and cultural importance that have dwindled to <15, % of their historic abundance in the southern New England/Mid-Atlantic region of the United States, with evidence indicating near-extirpation of certain local populations. This species exhibits intricate behaviors in spawning and migration that contribute to population complexity and resilience. These behaviors encompass full or partial philopatry to natal estuaries, the generation of multiple pulses of larval delivery, and partial migration. The patterns of genetic diversity within and among estuaries and cohorts presented here carry important implications in understanding the susceptibility to demographic shocks, even if the full extent of genetic diversity within and among winter flounder stocks on the US East Coast remains unresolved. Our findings reveal connectivity between estuaries in Long Island, New York, suggesting the potential for genetic rescue of depleted subpopulations. Family reconstruction and relatedness analysis indicate that split cohorts and migration contingents are not the result of genetically distinct lineages. We found no evidence for genetic structure separating these groups, and in some instances, we were able to detect closely related individuals that belonged to different migratory contingents or cohorts. Characterizing the spatial and behavioral organization of this species at the population level is crucial for comprehending its potential for recovery, not only in terms of biomass but also in reinstating the complex population structure that supports resilience. The search for generality in winter flounder spawning and migration behavior remains elusive, but perhaps the lack of generalities within this species is what has allowed it to persist in the face of decades of environmental and anthropogenic stressors.

Diverse origins of near-identical antifreeze proteins in unrelated fish lineages provide insights into evolutionary mechanisms of new gene birth and protein sequence convergence.

Determining the origins of novel genes and the genetic mechanisms underlying the emergence of new functions is challenging yet crucial for understanding evolutionary innovations. The convergently evolved fish antifreeze proteins provide excellent opportunities to investigate evolutionary origins and pathways of new genes. Particularly notable is the near-identical type I antifreeze proteins (AFPI) in four phylogenetically divergent fish taxa. This study tested the hypothesis of protein sequence convergence beyond functional convergence in three unrelated AFPI-bearing fish lineages, revealing different paths by which a similar protein arose from diverse genomic resources. Comprehensive comparative analyses of de novo sequenced genome of the winter flounder and grubby sculpin, available high-quality genome of the cunner and 14 other relevant species found that the near-identical AFPI originated from a distinct genetic precursor in each lineage. Each independently evolved a coding region for the novel ice-binding protein while retaining sequence identity in the regulatory regions with their respective ancestor. The deduced evolutionary processes and molecular mechanisms are consistent with the Innovation-Amplification-Divergence (IAD) model applicable to AFPI formation in all three lineages, a new Duplication-Degeneration-Divergence (DDD) model we propose for the sculpin lineage, and a DDD model with gene fission for the cunner lineage. This investigation illustrates the multiple ways by which a novel functional gene with sequence convergence at the protein level could evolve across divergent species, advancing our understanding of the mechanistic intricacies in new gene formation.

Investigation of the synergistic antimicrobial effect of winter flounder peptides using molecular dynamics simulations

Investigation of the synergistic antimicrobial effect of winter flounder peptides using molecular dynamics simulations

From antimicrobial to anticancer: unraveling the potential of pleurocidin and pleurocidin-derived peptides in the treatment of cancers.

Antimicrobial peptides (AMPs), commonly referred to as host defense peptides, are found in a wide range of organisms, including bacteria, plants, and both vertebrate and invertebrate animals. They function as an initial defense mechanism against pathogenic microorganisms, modulate immune responses, and in specific instances, confer protection against the onset of cancer. Pleurocidin (Ple) is a linear antimicrobial peptide with amphipathic α-helical conformation, isolated originally from the winter flounder (Pleuronectes americanus), notable for its wide-ranging effectiveness against both bacteria and fungi. While the majority of research on pleurocidin's biological characteristics has primarily focused on deciphering its mechanisms of interaction with the biological membranes of pathogenic bacteria and host cells, as well as investigating its modes of killing activities, there is a growing body of evidence suggesting that pleurocidin and pleurocidin-derived analogs might be effectively employed as anti-cancer agents against breast carcinoma and leukemia due to their potent cytotoxic properties and selectivity towards cancer cells. Notably, some characteristics of pleurocidin observed in microbiological investigations of this compound could be effectively applied in examining the anti-cancer capabilities of Ple-like derivatives. This review provides a comprehensive overview of the literature on the biological activities of pleurocidin, pleurocidin-derived peptides, pleurocidin-containing hybrid peptides, and nanosystems. The primary emphasis is on elucidating the range of activities exhibited by these compounds, evaluating their potential therapeutic applications, assessing their safety profile, and identifying any limits observed thus far. This paper will also discuss potential areas for further investigation into the anti-cancer effects of Ple and its derivatives, drawing insights from microbiological research.

Emergent conformational and aggregation properties of synergistic antimicrobial peptide combinations.

Synergy between antimicrobial peptides (AMPs) may be the key to their evolutionary success and could be exploited to develop more potent antibacterial agents. One of the factors thought to be essential for AMP potency is their conformational flexibility, but characterising the diverse conformational states of AMPs experimentally remains challenging. Here we introduce a method for characterising the conformational flexibility of AMPs and provide new insights into how the interplay between conformation and aggregation in synergistic AMP combinations yields emergent properties. We use unsupervised learning and molecular dynamics simulations to show that mixing two AMPs from the Winter Flounder family (pleurocidin (WF2) & WF1a) constrains their conformational space, reducing the number of distinct conformations adopted by the peptides, most notably for WF2. The aggregation behaviour of the peptides is also altered, favouring the formation of higher-order aggregates upon mixing. Critically, the interaction between WF1a and WF2 influences the distribution of WF2 conformations within aggregates, revealing how WF1a can modulate WF2 behaviour. Our work paves the way for deeper understanding of the synergy between AMPs, a fundamental process in nature.

The effect of winter flounder antifreeze protein and its mutants on methane hydrate growth: A molecular dynamic simulation study

The effect of winter flounder antifreeze protein and its mutants on methane hydrate growth: A molecular dynamic simulation study

Paradoxical effects on ice nucleation are intrinsic to a small winter flounder antifreeze protein

Antifreeze proteins (AFPs) bind to ice in solutions, resulting in non-colligative freezing point depression; however, their effects on ice nucleation are not well understood. The predominant plasma AFP of winter flounder (Pseudopleuronectes americanus) is AFP6, which is an amphiphilic alpha helix. In this study, AFP6 and modified constructs were produced as fusion proteins in Escherichia coli, subjected to proteolysis when required and purified prior to use. AFP6 and its recombinant fusion precursor generated similar thermal hysteresis and bipyramidal ice crystals, whereas an inactive mutant AFP6 produced hexagonal crystals and no hysteresis. Circular dichroism spectra of the wild-type and mutant AFP6 were consistent with an alpha helix. The effects of these proteins on ice nucleation were investigated alongside non-AFP proteins using a standard droplet freezing assay. In the presence of nucleating AgI, modest reductions in the nucleation temperature occurred with the addition of mutant AFP6, and several non-AFPs, suggesting non-specific inhibition of AgI-induced ice nucleation. In these experiments, both AFP6 and its recombinant precursor resulted in lower nucleation temperatures, consistent with an additional inhibitory effect. Conversely, in the absence of AgI, AFP6 induced ice nucleation, with no other proteins showing this effect. Nucleation by AFP6 was dose-dependent, reaching a maximum at 1.5 mM protein. Nucleation by AFP6 also required an ice-binding site, as the inactive mutant had no effect. Furthermore, the absence of nucleation by the recombinant precursor protein suggested that the fusion moiety was interfering with the formation of a surface capable of nucleating ice.

Isotopic Turnover and Fractionation of δ15N and δ13C in Captive Pseudopleuronectes americanus (Walbaum)

Stable isotope ratios of nitrogen (δ15N) and carbon (δ13C) are ubiquitous ecological tracers used to elucidate an organism’s diet and habitat. However, the application of stable isotope ratios to reconstruct a consumer’s ecology relies upon accurate rates for isotopic turnover at both a tissue and species-specific level. This study estimated isotope turnover rates and trophic discrimination factors in four different tissues (liver, digestive tissue, muscle, and skin) with variable metabolic activity in winter flounder Pseudopleuronectes americanus using a controlled diet-switch experiment. Differences in half-lives were noted among the tissues and between the experimental diets for both δ15N and δ13C. The experimental diets of krill and mysis had variability in nutritional composition, resulting in similar turnovers in δ15N but slower turnovers in δ13C for fish fed krill. Turnovers in both δ15N and δ13C were strongly influenced by metabolism, with the contribution reaching up to 98%, as fish exhibited minimal overall growth. The results of this study demonstrate the importance of considering differences in the catabolic activity of tissue maintenance for fish exhibiting minimal growth, as well as differences in metabolic assimilation of dietary sources that vary in their protein and lipid contents.

Synergy between Winter Flounder antimicrobial peptides

Some antimicrobial peptides (AMPs) have potent bactericidal activity and are being considered as potential alternatives to classical antibiotics. In response to an infection, such AMPs are often produced in animals alongside other peptides with low or no perceivable antimicrobial activity, whose role is unclear. Here we show that six AMPs from the Winter Flounder (WF) act in synergy against a range of bacterial pathogens and provide mechanistic insights into how this increases the cooperativity of the dose-dependent bactericidal activity and potency that enable therapy. Only two WF AMPs have potent antimicrobial activity when used alone but we find a series of two-way combinations, involving peptides which otherwise have low or no activity, yield potent antimicrobial activity. Weakly active WF AMPs modulate the membrane interactions of the more potent WF AMPs and enable therapy in a model of Acinetobacter baumannii burn wound infection. The observed synergy and emergent behaviour may explain the evolutionary benefits of producing a family of related peptides and are attractive properties to consider when developing AMPs towards clinical applications.

Thirty years of the NaCl cotransporter: from cloning to physiology and structure.

The primary structure of the thiazide-sensitive NaCl cotransporter (NCC) was resolved 30 years ago by the molecular identification of the cDNA encoding this cotransporter, from the winter's flounder urinary bladder, following a functional expression strategy. This review outlines some aspects of how the knowledge about thiazide diuretics and NCC evolved, the history of the cloning process, and the expansion of the SLC12 family of electroneutral cotransporters. The diseases associated with activation or inactivation of NCC are discussed, as well as the molecular model by which the activity of NCC is regulated. The controversies in the field are discussed as well as recent publication of the three-dimensional model of NCC obtained by cryo-electron microscopy, revealing not only the amino acid residues critical for Na+ and Cl- translocation but also the residues critical for polythiazide binding to the transporter, opening the possibility for a new era in thiazide diuretic therapy.

Anti-Protozoan Activities of Polar Fish-Derived Polyalanine Synthetic Peptides.

Chagas disease, sleeping sickness and malaria are infectious diseases caused by protozoan parasites that kill millions of people worldwide. Here, we performed in vitro assays of Pa-MAP, Pa-MAP1.9, and Pa-MAP2 synthetic polyalanine peptides derived from the polar fish Pleuronectes americanus toward Trypanosoma cruzi, T. brucei gambiense and Plasmodium falciparum activities. We demonstrated that the peptides Pa-MAP1.9 and Pa-MAP2 were effective to inhibit T. brucei growth. In addition, structural analyses using molecular dynamics (MD) studies showed that Pa-MAP2 penetrates deeper into the membrane and interacts more with phospholipids than Pa-MAP1.9, corroborating the previous in vitro results showing that Pa-MAP1.9 acts within the cell, while Pa-MAP2 acts via membrane lysis. In conclusion, polyalanine Pa-MAP1.9 and Pa-MAP2 presented activity against bloodstream forms of T. b. gambiense, thus encouraging further studies on the application of these peptides as a treatment for sleeping sickness.

De novo designed ice-binding proteins from twist-constrained helices

Attaining molecular-level control over solidification processes is a crucial aspect of materials science. To control ice formation, organisms have evolved bewildering arrays of ice-binding proteins (IBPs), but these have poorly understood structure-activity relationships. We propose that reverse engineering using de novo computational protein design can shed light on structure-activity relationships of IBPs. We hypothesized that the model alpha-helical winter flounder antifreeze protein uses an unusual undertwisting of its alpha-helix to align its putative ice-binding threonine residues in exactly the same direction. We test this hypothesis by designing a series of straight three-helix bundles with an ice-binding helix projecting threonines and two supporting helices constraining the twist of the ice-binding helix. Our findings show that ice-recrystallization inhibition by the designed proteins increases with the degree of designed undertwisting, thus validating our hypothesis, and opening up avenues for the computational design of IBPs.

The Structures of the Demersal Fish Communities of New Bedford and Gloucester Harbors, Two Massachusetts Urban Estuaries

We sampled the inshore fish communities of New Bedford and Gloucester harbors synoptically using identical gear for 12 months during 1998–1999. Although the 2 harbors are only 110 km apart, they are separated by Cape Cod, which is the transition between the southern New England (New Bedford) and the Gulf of Maine (Gloucester) biogeographic regions. The 2 fish communities were 69% dissimilar from each other using the Bray–Curtis dissimilarity index (B–C). Stenotomus chrysops (Scup) composed 80% of the catch in New Bedford Harbor, did not occur in Gloucester Harbor, and contributed 7.40% to the total dissimilarity between the harbors. B–C identified 2 seasonal groups in the New Bedford Harbor fish community: May–October and November–April. Leucoraja spp. (skates) comprised the most numerous taxon captured in Gloucester Harbor, accounting for 24.90% of the total catch followed by Pseudopleuronectes americanus (Winter Flounder; 24.16%), and Gadus morhua (Atlantic Cod; 22.52%). These taxa contributed 7.23%, 3.88%, and 6.16% respectively to the total dissimilarity between harbors. B–C identified 4 seasonal groups in Gloucester Harbor: January, February and March, April and May, and June–December. These data provide an important description of the fish communities in the 2 harbors in different biogeographic regions at the end of the 20th century. We expect differences between the communities to decrease with increasing water temperature due to climate change.

Structure-function relationships in the sodium chloride cotransporter.

The thiazide sensitive Na+:Cl- cotransporter (NCC) is the principal via for salt reabsorption in the apical membrane of the distal convoluted tubule (DCT) in mammals and plays a fundamental role in managing blood pressure. The cotransporter is targeted by thiazide diuretics, a highly prescribed medication that is effective in treating arterial hypertension and edema. NCC was the first member of the electroneutral cation-coupled chloride cotransporter family to be identified at a molecular level. It was cloned from the urinary bladder of the Pseudopleuronectes americanus (winter flounder) 30years ago. The structural topology, kinetic and pharmacology properties of NCC have been extensively studied, determining that the transmembrane domain (TM) coordinates ion and thiazide binding. Functional and mutational studies have discovered residues involved in the phosphorylation and glycosylation of NCC, particularly on the N-terminal domain, as well as the extracellular loop connected to TM7-8 (EL7-8). In the last decade, single-particle cryogenic electron microscopy (cryo-EM) has permitted the visualization of structures at high atomic resolution for six members of the SLC12 family (NCC, NKCC1, KCC1-KCC4). Cryo-EM insights of NCC confirm an inverted conformation of the TM1-5 and TM6-10 regions, a characteristic also found in the amino acid-polyamine-organocation (APC) superfamily, in which TM1 and TM6 clearly coordinate ion binding. The high-resolution structure also displays two glycosylation sites (N-406 and N-426) in EL7-8 that are essential for NCC expression and function. In this review, we briefly describe the studies related to the structure-function relationship of NCC, beginning with the first biochemical/functional studies up to the recent cryo-EM structure obtained, to acquire an overall view enriched with the structural and functional aspects of the cotransporter.

The Role of Fishery‐Independent Bottom Trawl Surveys in Providing Regional and Temporal Context to Offshore Wind Farm Monitoring Studies

AbstractBottom trawl surveys are commonly used to examine potential effects on fishes and invertebrates from offshore wind (OSW) farms in Europe and in the northeastern United States. Because OSW surveys typically occur over a limited spatial footprint, comparison of OSW monitoring results to long‐term fishery‐independent surveys may provide a regional and temporal context for OSW data sets. We compared results of the Block Island Wind Farm (BIWF) bottom trawl survey (2013–2019) to three fishery‐independent bottom trawl surveys (Northeast Area Monitoring and Assessment Program, Northeast Fisheries Science Center, and Rhode Island Department of Environmental Management [RIDEM]) using catch rates of 12 federally managed species. We evaluated temporal trends in annual residual catches for each species calculated within each survey as the difference between the mean annual biomass per trawl and the long‐term mean. Regional consistency in relative catches was apparent for species exhibiting synchronous interannual variability among surveys (Black Sea Bass Centropristis striata, Scup Stenotomus chrysops, Summer Flounder Paralichthys dentatus, and Winter Flounder Pseudopleuronectes americanus) or a decreasing trend in residual catch rates across the 7‐year study period (Little Skate Leucoraja erinacea, longfin inshore squid Doryteuthis pealeii, and Winter Skate L. ocellata). For other species, catches among surveys were asynchronous (Atlantic Herring Clupea harengus, Butterfish Peprilus triacanthus, and Windowpane Scophthalmus aquosus) or anomalous catches in a single year affected the results (Red Hake Urophycis chuss and Silver Hake Merluccius bilinearis). Monitoring of BIWF occurred during a period with lower‐than‐average historical catches in a 32‐year RIDEM data set for Atlantic Herring, Butterfish, Little Skate, longfin inshore squid, Red Hake, Silver Hake, and Winter Flounder and higher‐than‐average catches for Black Sea Bass, Scup, and Summer Flounder. There was no evidence that variation in catches near BIWF differed from regional trends in a way consistent with a detrimental impact of OSW farm operation. The regional context provided from multiple bottom trawl surveys varies by species and thus may be limited for interpreting OSW monitoring results.

Cumulative effects of long-term anthropogenic impacts on resident fish communities in subarctic fishing harbours

Ecosystems perceived to have sustained historical human pressure from multiple stressors are often considered to be degraded. Diversity and abundance of marine macroflora and macrofauna were observed at 17 harbour sites in Newfoundland (Canada) over four consecutive years (2008–2011), to determine cumulative effects of long-term anthropogenic impacts on coastal marine biota in small fishing harbours. A Principal Component Analysis was applied to evaluate the relative effects of physical harbour characteristics (harbour shape, size, and orientation), and those directly associated with anthropogenic influence (current and historical human population size, human landscape “footprint”, and operating capacity of the local fishing industry), on marine vegetation, fish, and invertebrate macrofauna. The first three principal components explained 77% of the cumulative variance of the physical harbour and anthropogenic variables. PC1 was dominated by variables associated with human use, and site depth; PC2 was associated with physical site orientation and the cumulative wharf length; and PC3 was associated with long-term use and harbour area. The densities of cod (Gadus spp.) and crab species increased significantly along the gradient of human impact metrics, while winter flounder (Pseudopleuronectes americanus), cunner (Tautogolaborus adspersus) and shorthorn sculpin (Myoxocephalus scorpius) decreased. Sessile fauna diversity and density, and flora percent cover correlated with long-term use and harbour area. Species densities changed in response to cumulative effects of long term human settlement near harbours, but prominent species were ubiquitous in these coastal ecosystems.

Truncated Pleurocidin Derivative with High Pepsin Hydrolysis Resistance to Combat Multidrug-Resistant Pathogens.

The global prevalence of antimicrobial resistance calls for the development of novel antimicrobial agents, particularly for these orally available drugs. Structural modifications of the natural antimicrobial peptides (AMPs) provide a straightforward approach to develop potent antimicrobial agents with high specificity and low toxicity. In this study, we truncated 11-amino-acids at the C-terminus of Pleurocidin, an AMP produced by Pleuronectes americanus, and obtained four peptide analogues termed GK-1, GK-2, GK-3 and GK-4. Minimum inhibitory concentration (MIC) tests showed that GK-1 obtained by direct truncation of Pleurocidin has no antibacterial activity, while GK-2, GK-3 and GK-4 show considerable antibacterial activity with Pleurocidin. Notably, GK-4 displays rapid bacteriostatic activity, great stability and low hemolysis, as well as enhanced hydrolytic resistance to pepsin treatment. Mechanistic studies showed that GK-4 induces membrane damage by interacting with bacterial membrane-specific components, dissipates bacterial membrane potential and promotes the generation of ROS. SEM and CD analysis further confirmed the ability of GK-4 to resist pepsin hydrolysis, which may be attributed to its stable helicity structure. Collectively, our findings reveal that GK-4 is a potential orally available candidate to treat infections caused by multidrug-resistant pathogens.

Interaction of curcumin with a winter flounder alpha-helical antifreeze protein

The winter flounder, Pseudopleuronectes americanus, synthesizes a variety of alpha-helical antifreeze proteins (AFPs) that adhere to ice and inhibit its growth. The best studied of these is AFP6, which is a 37-residue protein abundant in the flounder blood plasma during winter. Curcumin from the turmeric plant (Curcuma longa) was found to interact with AFP6 in aqueous solutions, resulting in measurable changes in the curcumin, but not in the protein. Specifically, the secondary structure and unfolding of synthetic AFP6, shown by circular dichroism, appeared to be unaffected by curcumin. In contrast, the peak absorbance of curcumin shifted and increased in the presence of AFP6, and the maximum fluorescence emission was greater and blue shifted. These results also suggested the possibility of AFP6 detection by curcumin fluorescence. Synthetic AFP6 did not interact with Coomassie blue, silver or a commercial fluorescent stain following electrophoresis; however, the change in curcumin fluorescence upon binding to electrophoresed AFP6 resulted in a fluorescent signal, which was also detected upon interaction with purified natural AFP and flounder blood plasma containing the protein. Thus, aqueous curcumin can be used for the direct detection of AFP6 and curcumin binding could provide new avenues for the study of this protein.