Structure-function relationships in ShKT domain peptides: ShKT-Ts1 from the sea anemone Telmatactis stephensoni.

Diverse structural scaffolds have been described in peptides from sea anemones, with the ShKT domain being a common scaffold first identified in ShK toxin from Stichodactyla helianthus. ShK is a potent blocker of voltage-gated potassium channels (KV 1.x), and an analog, ShK-186 (dalazatide), has completed Phase 1 clinical trials in plaque psoriasis. The ShKT domain has been found in numerous other species, but only a tiny fraction of ShKT domains has been characterized functionally. Despite adopting the canonical ShK fold, some ShKT peptides from sea anemones inhibit KV 1.x, while others do not. Mutagenesis studies have shown that a Lys-Tyr (KY) dyad plays a key role in KV 1.x blockade, although a cationic residue followed by a hydrophobic residue may also suffice. Nevertheless, ShKT peptides displaying an ShK-like fold and containing a KY dyad do not necessarily block potassium channels, so additional criteria are needed to determine whether new ShKT peptides might show activity against potassium channels. In this study, we used a combination of NMR and molecular dynamics (MD) simulations to assess the potential activity of a new ShKT peptide. We determined the structure of ShKT-Ts1, from the sea anemone Telmatactis stephensoni, examined its tissue localization, and investigated its activity against a range of ion channels. As ShKT-Ts1 showed no activity against KV 1.x channels, we used MD simulations to investigate whether solvent exposure of the dyad residues may be informative in rationalizing and potentially predicting the ability of ShKT peptides to block KV 1.x channels. We show that either a buried dyad that does not become exposed during MD simulations, or a partially exposed dyad that becomes buried during MD simulations, correlates with weak or absent activity against KV 1.x channels. Therefore, structure determination coupled with MD simulations, may be used to predict whether new sequences belonging to the ShKT family may act as potassium channel blockers.

Equinins as Novel Broad-Spectrum Antimicrobial Peptides Isolated from the Cnidarian Actinia equina (Linnaeus, 1758).

Sea anemones are valuable for therapeutic research as a diversified source of bioactive molecules, due to their diverse bioactive molecules linked to predation and defence mechanisms involving toxins and antimicrobial peptides. Acid extracts from Actinia equina tentacles and body were examined for antibacterial activity against Gram-positive, Gram-negative bacteria, and fungi. The peptide fractions showed interesting minimum inhibitory concentration (MIC) values (up to 0.125 µg/mL) against the tested pathogens. Further investigation and characterization of tentacle acid extracts with significant antimicrobial activity led to the purification of peptides through reverse phase chromatography on solid phase and HPLC. Broad-spectrum antimicrobial peptide activity was found in 40% acetonitrile fractions. The resulting peptides had a molecular mass of 2612.91 and 3934.827 Da and MIC ranging from 0.06 to 0.20 mg/mL. Sequencing revealed similarities to AMPs found in amphibians, fish, and Cnidaria, with anti-Gram+, Gram-, antifungal, candidacidal, anti-methicillin-resistant Staphylococcus aureus, carbapenemase-producing, vancomycin-resistant bacteria, and multi-drug resistant activity. Peptides 6.2 and 7.3, named Equinin A and B, respectively, were synthesized and evaluated in vitro towards the above-mentioned bacterial pathogens. Equinin B exerted interesting antibacterial activity (MIC and bactericidal concentrations of 1 mg/mL and 0.25 mg/mL, respectively) and gene organization supporting its potential in applied research.

Pore-forming moss protein bryoporin is structurally and mechanistically related to actinoporins from evolutionarily distant cnidarians.

Pore-forming proteins perforate lipid membranes and consequently affect their integrity and cell fitness. Therefore, it is not surprising that many of these proteins from bacteria, fungi, or certain animals act as toxins. While pore-forming proteins have also been found in plants, there is little information about their molecular structure and mode of action. Bryoporin is a protein from the moss Physcomitrium patens, and its corresponding gene was found to be upregulated by various abiotic stresses, especially dehydration, as well as upon fungal infection. Based on the amino acid sequence, it was suggested that bryoporin was related to the actinoporin family of pore-forming proteins, originally discovered in sea anemones. Here, we provide the first detailed structural and functional analysis of this plant cytolysin. The crystal structure of monomeric bryoporin is highly similar to those of actinoporins. Our cryo-EM analysis of its pores showed an actinoporin-like octameric structure, thereby revealing a close kinship of proteins from evolutionarily distant organisms. This was further confirmed by our observation of bryoporin's preferential binding to and formation of pores in membranes containing animal sphingolipids, such as sphingomyelin and ceramide phosphoethanolamine; however, its binding affinity was weaker than that of actinoporin equinatoxin II. We determined bryoporin did not bind to major sphingolipids found in fungi or plants, and its membrane-binding and pore-forming activity was enhanced by various sterols. Our results suggest that bryoporin could represent a part of the moss defense arsenal, acting as a pore-forming toxin against membranes of potential animal pathogens, parasites, or predators.

Cloning and structural basis of fluorescent protein color variants from identical species of sea anemone, Diadumene lineata.

Diadumene lineata is a colorful sea anemone with orange stripe tissue of the body column and plain tentacles with red lines. We subjected Diadumene lineata to expression cloning and obtained genes encoding orange (OFP: DiLiFP561) and red fluorescent proteins (RFPs: DiLiFP570 and DiLiFP571). These proteins formed obligatory tetramers. All three proteins showed bright fluorescence with the brightness of 58.3 mM-1·cm-1 (DiLiFP561), 43.9 mM-1·cm-1 (DiLiFP570), and 31.2 mM-1·cm-1 (DiLiFP571), which were equivalent to that of commonly used red fluorescent proteins. Amplitude-weighted average fluorescence lifetimes of DiLiFP561, DiLiFP570 and DiLiFP571 were determined as 3.7, 3.6 and 3.0 ns. We determined a crystal structure of DiLiFP570 at 1.63 Å resolution. The crystal structure of DiLiFP570 revealed that the chromophore has an extended π-conjugated structure similar to that of DsRed. Most of the amino acid residues surrounding the chromophore were common between DiLiFP570 and DiLiFP561, except M159 of DiLiFP570 (Lysine in DiLiFP561), which is located close to the chromophore hydroxyl group. Interestingly, a similar K-to-M substitution has been reported in a red-shifted variant of DsRed (mRFP1). It is a striking observation that the naturally evolved color-change variants are consistent with the mutation induced via protein engineering processes. The newly cloned proteins are promising as orange and red fluorescent markers for imaging with long fluorescence lifetime.

Interaction of the Inhibitory Peptides ShK and HmK with the Voltage-Gated Potassium Channel KV1.3: Role of Conformational Dynamics.

Peptide toxins that adopt the ShK fold can inhibit the voltage-gated potassium channel KV1.3 with IC50 values in the pM range and are therefore potential leads for drugs targeting autoimmune and neuroinflammatory diseases. Nuclear magnetic resonance (NMR) relaxation measurements and pressure-dependent NMR have shown that, despite being cross-linked by disulfide bonds, ShK itself is flexible in solution. This flexibility affects the local structure around the pharmacophore for the KV1.3 channel blockade and, in particular, the relative orientation of the key Lys and Tyr side chains (Lys22 and Tyr23 in ShK) and has implications for the design of KV1.3 inhibitors. In this study, we have performed molecular dynamics (MD) simulations on ShK and a close homologue, HmK, to probe the conformational space occupied by the Lys and Tyr residues, and docked the different conformations with a recently determined cryo-EM structure of the KV1.3 channel. Although ShK and HmK have 60% sequence identity, their dynamic behaviors are quite different, with ShK sampling a broad range of conformations over the course of a 5 µs MD simulation, while HmK is relatively rigid. We also investigated the importance of conformational dynamics, in particular the distance between the side chains of the key dyad Lys22 and Tyr23, for binding to KV1.3. Although these peptides have quite different dynamics, the dyad in both adopts a similar configuration upon binding, revealing a conformational selection upon binding to KV1.3 in the case of ShK. Both peptides bind to KV1.3 with Lys22 occupying the pore of the channel. Intriguingly, the more flexible peptide, ShK, binds with significantly higher affinity than HmK.

Host-dependent nitrogen recycling as a mechanism of symbiont control in Aiptasia.

The metabolic symbiosis with photosynthetic algae allows corals to thrive in the oligotrophic environments of tropical seas. Different aspects of this relationship have been investigated using the emerging model organism Aiptasia. However, many fundamental questions, such as the nature of the symbiotic relationship and the interactions of nutrients between the partners remain highly debated. Using a meta-analysis approach, we identified a core set of 731 high-confidence symbiosis-associated genes that revealed host-dependent recycling of waste ammonium and amino acid synthesis as central processes in this relationship. Subsequent validation via metabolomic analyses confirmed that symbiont-derived carbon enables host recycling of ammonium into nonessential amino acids. We propose that this provides a regulatory mechanism to control symbiont growth through a carbon-dependent negative feedback of nitrogen availability to the symbiont. The dependence of this mechanism on symbiont-derived carbon highlights the susceptibility of this symbiosis to changes in carbon translocation, as imposed by environmental stress.

Genomic Structure of Two Kv1.3 Channel Blockers from Scorpion Mesobuthus eupeus and Sea Anemone Stichodactyla haddoni and Construction of their Chimeric Peptide as a Novel Blocker.

Different toxins acting on Kv1.3 channel have been isolated from animal venom. MeuKTX toxin from Mesobuthus eupeus phillipsi scorpion and shtx-k toxin from Stichodactyla haddoni sea anemone have been identified as two effective Kv1.3 channel blockers. In this work, we characterized the genomic organization of both toxins. MeuKTX gene contains one intron and two exons, similar to the most scorpion toxins. There are a few reports of genomic structure of sea anemone toxins acting on Kv channels. The sequence encoding mature peptide of shtx-k was located in an exon separated by an intron from the coding exon of the propeptide and signal region. In order to make a peptide with more affinity for Kv1.3 channel and greater stability, the shtx-k/ MeuKTX chimeric peptide was designed and constructed using splicing by overlap extension-PCR (SOE-PCR) method. MeuKTX, shtx-k, and shtx-k/MeuKTX were cloned and the expression of the soluble proteins in E. coli was determined. Molecular docking studies indicated more inhibitory effect of shtx-k/MeuKTX on Kv1.3 channel compared to shtx-k and MeuKTX toxins. Key amino acids binding channel from both toxins, also involved in interaction of chimeric peptide with channel. Our results showed that the fusion peptide, shtx-k/MeuKTX could be an effective agent to target Kv1.3 channel.

Kunitz-Type Peptides from Sea Anemones Protect Neuronal Cells against Parkinson's Disease Inductors via Inhibition of ROS Production and ATP-Induced P2X7 Receptor Activation.

Parkinson's disease (PD) is a socially significant disease, during the development of which oxidative stress and inflammation play a significant role. Here, we studied the neuroprotective effects of four Kunitz-type peptides from Heteractis crispa and Heteractis magnifica sea anemones against PD inductors. The peptide HCIQ1c9, which was obtained for the first time, inhibited trypsin less than other peptides due to unfavorable interactions of Arg17 with Lys43 in the enzyme. Its activity was reduced by up to 70% over the temperature range of 60-100 °C, while HCIQ2c1, HCIQ4c7, and HMIQ3c1 retained their conformation and stayed active up to 90-100 °C. All studied peptides inhibited paraquat- and rotenone-induced intracellular ROS formation, in particular NO, and scavenged free radicals outside the cells. The peptides did not modulate the TRPV1 channels but they affected the P2X7R, both of which are considered therapeutic targets in Parkinson's disease. HMIQ3c1 and HCIQ4c7 almost completely inhibited the ATP-induced uptake of YO-PRO-1 dye in Neuro-2a cells through P2X7 ion channels and significantly reduced the stable calcium response in these cells. The complex formation of the peptides with the P2X7R extracellular domain was determined via SPR analysis. Thus, these peptides may be considered promising compounds to protect neuronal cells against PD inductors, which act as ROS production inhibitors and partially act as ATP-induced P2X7R activation inhibitors.

Oligomerization of Sticholysins from Förster Resonance Energy Transfer.

Sticholysins are pore-forming toxins produced by sea anemones that are members of the actinoporin family. They exert their activity by forming pores on membranes, provided they have sphingomyelin. To assemble into pores, specific recognition, binding, and oligomerization are required. While recognition and binding have been extensively studied, delving into the oligomerization process and the stoichiometry of the pores has been more difficult. Here, we present evidence that these toxins are capable of oligomerizing in solution and suggesting that the interaction of sticholysin II (StnII) with its isoform sticholysin I (StnI) is stronger than that of StnI with itself. We also show that the stoichiometry of the final, thermodynamically stable StnI pores is, at least, heptameric. Furthermore, our results indicate that this association maintains its oligomerization number when StnII is included, indicating that the stoichiometry of StnII is also of that order, and not tetrameric, as previously thought. These results are compatible with the stoichiometry observed for the crystallized pore of FraC, another very similar actinoporin produced by a different sea anemone species. Our results also indicate that the stoichiometry of actinoporin pores in equilibrium is conserved regardless of the particular composition of a given pore ensemble, which we have shown for mixed sticholysin pores.

A disulfide-stabilised helical hairpin fold in acrorhagin I: An emerging structural motif in peptide toxins.

Acrorhagin I (U-AITX-Aeq5a) is a disulfide-rich peptide identified in the aggressive organs (acrorhagi) of the sea anemone Actinia equina. Previous studies (Toxicon 2005, 46:768-74) found that the peptide is toxic in crabs, although the structural and functional properties of acrorhagin I have not been reported. In this work, an Escherichia coli (BL21 strain) expression system was established for the preparation of 13C,15N-labelled acrorhagin I, and the solution structure was determined using NMR spectroscopy. Structurally, acrorhagin I is similar to B-IV toxin from the marine worm Cerebratulus lacteus (PDB id 1VIB), with a well-defined helical hairpin structure stabilised by four intramolecular disulfide bonds. The recombinant peptide was tested in patch-clamp electrophysiology assays against voltage-gated potassium and sodium channels, and in bacterial and fungal growth inhibitory assays and haemolytic assays. Acrorhagin I was not active against any of the ion channels tested and showed no activity in functional assays, indicating that this peptide may possess a different biological function. Metal ion interaction studies using NMR spectroscopy showed that acrorhagin I bound zinc and nickel, suggesting that its function might be modulated by metal ions or that it may be involved in regulating metal ion levels and their transport. The similarity between the structure of acrorhagin I and that of B-IV toxin from a marine worm suggests that this fold may prove to be a recurring motif in disulfide-rich peptides from marine organisms.

Common mechanism of thermostability in small α- and ß-proteins studied by molecular dynamics.

Protein thermostability is important to evolution, diseases, and industrial applications. Proteins use diverse molecular strategies to achieve stability at high temperature, yet reducing the entropy of unfolding seems required. We investigated five small α-proteins and five ß-proteins with known, distinct structures and thermostability (Tm ) using multi-seed molecular dynamics simulations at 300, 350, and 400 K. The proteins displayed diverse changes in hydrogen bonding, solvent exposure, and secondary structure with no simple relationship to Tm . Our dynamics were in good agreement with experimental B-factors at 300 K and insensitive to force-field choice. Despite the very distinct structures, the native-state (300 + 350 K) free-energy landscapes (FELs) were significantly broader for the two most thermostable proteins and smallest for the three least stable proteins in both the α- and ß-group and with both force fields studied independently (tailed t-test, 95% confidence level). Our results suggest that entropic ensembles stabilize proteins at high temperature due to reduced entropy of unfolding, viz., ΔG = ΔH - TΔS. Supporting this mechanism, the most thermostable proteins were also the least kinetically stable, consistent with broader FELs, typified by villin headpiece and confirmed by specific comparison to a mesophilic ortholog of Thermus thermophilus apo-pyrophosphate phosphohydrolase. We propose that molecular strategies of protein thermostabilization, although diverse, tend to converge toward highest possible entropy in the native state consistent with the functional requirements. We speculate that this tendency may explain why many proteins are not optimally structured and why molten-globule states resemble native proteins so much.

Structural and functional characterization of sticholysin III: A newly discovered actinoporin within the venom of the sea anemone Stichodactyla helianthus.

Actinoporins are a family of pore-forming toxins produced by sea anemones as part of their venomous cocktail. These proteins remain soluble and stably folded in aqueous solution, but when interacting with sphingomyelin-containing lipid membranes, they become integral oligomeric membrane structures that form a pore permeable to cations, which leads to cell death by osmotic shock. Actinoporins appear as multigenic families within the genome of sea anemones: several genes encoding very similar actinoporins are detected within the same species. The Caribbean Sea anemone Stichodactyla helianthus produces three actinoporins (sticholysins I, II and III; StnI, StnII and StnIII) that differ in their toxic potency. For example, StnII is about four-fold more effective than StnI against sheep erythrocytes in causing hemolysis, and both show synergy. However, StnIII, recently discovered in the S. helianthus transcriptome, has not been characterized so far. Here we describe StnIII's spectroscopic and functional properties and show its potential to interact with the other Stns. StnIII seems to maintain the well-preserved fold of all actinoporins, characterized by a high content of ß-sheet, but it is significantly less thermostable. Its functional characterization shows that the critical concentration needed to form active pores is higher than for either StnI or StnII, suggesting differences in behavior when oligomerizing on membrane surfaces. Our results show that StnIII is an interesting and unexpected piece in the puzzle of how this Caribbean Sea anemone species modulates its venomous activity.

Marine Toxins Targeting Kv1 Channels: Pharmacological Tools and Therapeutic Scaffolds.

Toxins from marine animals provide molecular tools for the study of many ion channels, including mammalian voltage-gated potassium channels of the Kv1 family. Selectivity profiling and molecular investigation of these toxins have contributed to the development of novel drug leads with therapeutic potential for the treatment of ion channel-related diseases or channelopathies. Here, we review specific peptide and small-molecule marine toxins modulating Kv1 channels and thus cover recent findings of bioactives found in the venoms of marine Gastropod (cone snails), Cnidarian (sea anemones), and small compounds from cyanobacteria. Furthermore, we discuss pivotal advancements at exploiting the interaction of κM-conotoxin RIIIJ and heteromeric Kv1.1/1.2 channels as prevalent neuronal Kv complex. RIIIJ's exquisite Kv1 subtype selectivity underpins a novel and facile functional classification of large-diameter dorsal root ganglion neurons. The vast potential of marine toxins warrants further collaborative efforts and high-throughput approaches aimed at the discovery and profiling of Kv1-targeted bioactives, which will greatly accelerate the development of a thorough molecular toolbox and much-needed therapeutics.

Functional and Structural Variation among Sticholysins, Pore-Forming Proteins from the Sea Anemone Stichodactyla helianthus.

Venoms constitute complex mixtures of many different molecules arising from evolution in processes driven by continuous prey-predator interactions. One of the most common compounds in these venomous cocktails are pore-forming proteins, a family of toxins whose activity relies on the disruption of the plasmatic membranes by forming pores. The venom of sea anemones, belonging to the oldest lineage of venomous animals, contains a large amount of a characteristic group of pore-forming proteins known as actinoporins. They bind specifically to sphingomyelin-containing membranes and suffer a conformational metamorphosis that drives them to make pores. This event usually leads cells to death by osmotic shock. Sticholysins are the actinoporins produced by Stichodactyla helianthus. Three different isotoxins are known: Sticholysins I, II, and III. They share very similar amino acid sequence and three-dimensional structure but display different behavior in terms of lytic activity and ability to interact with cholesterol, an important lipid component of vertebrate membranes. In addition, sticholysins can act in synergy when exerting their toxin action. The subtle, but important, molecular nuances that explain their different behavior are described and discussed throughout the text. Improving our knowledge about sticholysins behavior is important for eventually developing them into biotechnological tools.

New Sea Anemone Toxin RTX-VI Selectively Modulates Voltage-Gated Sodium Channels.

A new neurotoxin RTX-VI that modulates the voltage-gated sodium channels (NaV) was isolated from the ethanolic extract of the sea anemone Heteractis crispa. Its amino acid sequence was determined using the combination of Edman degradation and tandem mass spectrometry. RTX-VI turned out to be an unusual natural analogue of the previously described sea anemone toxin RTX-III. The RTX-VI molecule consists of two disulfide-linked peptide chains and is devoid of Arg13, which is important for the selectivity and affinity of such peptides for the NaV channels. Electrophysiological screening of RTV-VI on NaV channel subtypes showed its selective interaction with the central nervous system (NaV1.2, NaV1.6) and insect (BgNaV1, VdNaV1) sodium channels.

Evolution of cnidarian trans-defensins: Sequence, structure and exploration of chemical space.

Many of the small, cysteine-rich ion-channel modulatory peptides found in Cnidaria are distantly related to vertebrate defensins (of the trans-defensin superfamily). Transcriptomic and proteomic studies of the endemic Australian speckled sea anemone (Oulactis sp.) yielded homologous peptides to known defensin sequences. We extended these data using existing and custom-built hidden Markov models to extract defensin-like families from the transcriptomes of seven endemic Australian cnidarian species. Newly sequenced transcriptomes include three species of Actiniaria (true sea anemones); the speckled anemone (Oulactis sp.), Oulactis muscosa, Dofleinia cf. armata and a species of Corallimorpharia, Rhodactis sp. We analyzed these novel defensin-like sequences along with published homologues to study the evolution of their physico-chemical properties in vertebrate and invertebrate fauna. The cnidarian trans-defensins form a distinct cluster within the chemical space of the superfamily, with a unique set of motifs and biophysical properties. This cluster contains identifiable subgroups, whose distribution in chemical space also correlates with the divergent evolution of their structures. These sequences, currently restricted to cnidarians, form an evolutionarily distinct clade within the trans-defensin superfamily.

PHAB toxins: a unique family of predatory sea anemone toxins evolving via intra-gene concerted evolution defines a new peptide fold.

Sea anemone venoms have long been recognized as a rich source of peptides with interesting pharmacological and structural properties, but they still contain many uncharacterized bioactive compounds. Here we report the discovery, three-dimensional structure, activity, tissue localization, and putative function of a novel sea anemone peptide toxin that constitutes a new, sixth type of voltage-gated potassium channel (KV) toxin from sea anemones. Comprised of just 17 residues, κ-actitoxin-Ate1a (Ate1a) is the shortest sea anemone toxin reported to date, and it adopts a novel three-dimensional structure that we have named the Proline-Hinged Asymmetric ß-hairpin (PHAB) fold. Mass spectrometry imaging and bioassays suggest that Ate1a serves a primarily predatory function by immobilising prey, and we show this is achieved through inhibition of Shaker-type KV channels. Ate1a is encoded as a multi-domain precursor protein that yields multiple identical mature peptides, which likely evolved by multiple domain duplication events in an actinioidean ancestor. Despite this ancient evolutionary history, the PHAB-encoding gene family exhibits remarkable sequence conservation in the mature peptide domains. We demonstrate that this conservation is likely due to intra-gene concerted evolution, which has to our knowledge not previously been reported for toxin genes. We propose that the concerted evolution of toxin domains provides a hitherto unrecognised way to circumvent the effects of the costly evolutionary arms race considered to drive toxin gene evolution by ensuring efficient secretion of ecologically important predatory toxins.

Sea Anemone Toxins: A Structural Overview.

Sea anemones produce venoms of exceptional molecular diversity, with at least 17 different molecular scaffolds reported to date. These venom components have traditionally been classified according to pharmacological activity and amino acid sequence. However, this classification system suffers from vulnerabilities due to functional convergence and functional promiscuity. Furthermore, for most known sea anemone toxins, the exact receptors they target are either unknown, or at best incomplete. In this review, we first provide an overview of the sea anemone venom system and then focus on the venom components. We have organised the venom components by distinguishing firstly between proteins and non-proteinaceous compounds, secondly between enzymes and other proteins without enzymatic activity, then according to the structural scaffold, and finally according to molecular target.

Magnificamide, a ß-Defensin-Like Peptide from the Mucus of the Sea Anemone Heteractis magnifica, Is a Strong Inhibitor of Mammalian α-Amylases.

Sea anemones' venom is rich in peptides acting on different biological targets, mainly on cytoplasmic membranes and ion channels. These animals are also a source of pancreatic α-amylase inhibitors, which have the ability to control the glucose level in the blood and can be used for the treatment of prediabetes and type 2 diabetes mellitus. Recently we have isolated and characterized magnificamide (44 aa, 4770 Da), the major α-amylase inhibitor of the sea anemone Heteractis magnifica mucus, which shares 84% sequence identity with helianthamide from Stichodactyla helianthus. Herein, we report some features in the action of a recombinant analog of magnificamide. The recombinant peptide inhibits porcine pancreatic and human saliva α-amylases with Ki's equal to 0.17 ± 0.06 nM and 7.7 ± 1.5 nM, respectively, and does not show antimicrobial or channel modulating activities. We have concluded that the main function of magnificamide is the inhibition of α-amylases; therefore, its functionally active recombinant analog is a promising agent for further studies as a potential drug candidate for the treatment of the type 2 diabetes mellitus.

An adaptable chromosome preparation methodology for use in invertebrate research organisms.

BACKGROUND: The ability to efficiently visualize and manipulate chromosomes is fundamental to understanding the genome architecture of organisms. Conventional chromosome preparation protocols developed for mammalian cells and those relying on species-specific conditions are not suitable for many invertebrates. Hence, a simple and inexpensive chromosome preparation protocol, adaptable to multiple invertebrate species, is needed. RESULTS: We optimized a chromosome preparation protocol and applied it to several planarian species (phylum Platyhelminthes), the freshwater apple snail Pomacea canaliculata (phylum Mollusca), and the starlet sea anemone Nematostella vectensis (phylum Cnidaria). We demonstrated that both mitotically active adult tissues and embryos can be used as sources of metaphase chromosomes, expanding the potential use of this technique to invertebrates lacking cell lines and/or with limited access to the complete life cycle. Simple hypotonic treatment with deionized water was sufficient for karyotyping; growing cells in culture was not necessary. The obtained karyotypes allowed the identification of differences in ploidy and chromosome architecture among otherwise morphologically indistinguishable organisms, as in the case of a mixed population of planarians collected in the wild. Furthermore, we showed that in all tested organisms representing three different phyla this protocol could be effectively coupled with downstream applications, such as chromosome fluorescent in situ hybridization. CONCLUSIONS: Our simple and inexpensive chromosome preparation protocol can be readily adapted to new invertebrate research organisms to accelerate the discovery of novel genomic patterns across the branches of the tree of life.