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Paper review summary

This review (Marine Drugs DOI 10.3390/md23090363) synthesizes experimental and bioinformatic evidence that many marine adhesive secretions (barnacle cement, cyprid footprints, sea urchin footprints, sea cucumber Cuvierian tubules, and some flatworm adhesives) contain amyloidlike assemblies (nanofibrils, cross-beta signatures, sawtooth AFM force curves, Thioflavin-T and Congo Red staining) that likely contribute cohesion, insolubility, sacrificial bonding, rapid curing, and wet stability, and that these functional amyloids are promising templates for biomimetic wet adhesives and hydrogels

Marine Drugs review on marine functional amyloids

Comprehensive review and critique

Scope and main claims

The authors review evidence that functional amyloidlike assemblies occur across multiple marine adhesives (permanent barnacle cement, cyprid footprints, echinoderm tubefoot adhesives and Cuvierian tubules, and some flatworm adhesives), and argue these assemblies contribute key mechanical properties (insolubility, cohesion, sacrificial bonds/selfhealing, rapid curing) and present attractive templates for biomimetic adhesives and hydrogels. The review synthesizes microscopy (AFM, SEM, TEM), spectroscopy (FTIR, CD, Raman), dye binding (ThioflavinT and Congo Red), singlemolecule force spectroscopy (sawtooth AFM curves), bioinformatics prediction of crossbeta motifs, and recombinant/peptide selfassembly studies to support this view.

Representative summary excerpt from the paper:

Functional amyloids overview from the review

Key evidence compiled by the authors

• AFM imaging: nanofibrillar and aggregated fibrillar morphologies in barnacle cyprid footprints, barnacle cement, and sea urchin footprints; authors specifically note aggregated fibrillar footprints and sawtooth forceextension curves indicative of modular unfolding and sacrificial bonds (cyprids and sea urchin) AFM and force spectroscopy evidence
• Dye binding: ThioflavinT and Congo Red positive staining reported for barnacle cement, sea urchin footprints, and some sea cucumber structures (authors caution dye specificity but cite multiple concordant methods) ThT and Congo Red staining reports
• Spectroscopy/secondary structure: CD and FTIR data indicating βsheet rich content in some cement extracts (percent β estimated variably across species) and Raman detection of βsheets in some adhesives.
• Recombinant and peptide work: recombinant expression of barnacle CP fragments and synthetic peptides demonstrate intrinsic amyloidogenic motifs in CPs and the ability of short peptides to selfassemble into amyloidlike fibrils in vitro (caveat: many sequences incomplete and PTMs missing).

Strengths of the review

• Comprehensive crossphyla synthesis bringing together orthogonal methods (imaging, spectroscopy, force spectroscopy, dyebinding, bioinformatics), increasing plausibility of functional amyloid occurrence across marine adhesives Crossphyla occurrence statement
• Clear translation emphasis: the review connects mechanistic signatures to biomimetic design principles (selfassembly, sacrificial bonds, water stability), which is useful to materials scientists.
• Critical discussion: authors note methodological limitations (difficulty extracting cured cement, incomplete sequences, variable detection specificity) and call for omics and highresolution structural work.

Weaknesses, limitations, and potential overinterpretations

1. Heterogeneous evidence quality across organisms: many claims rest on dye staining and AFM morphology which are suggestive but not definitive for crossβ architecture; the review correctly flags that FTIR/CD and ssNMR/cryoEM/atomicresolution data are sparse or absent for most native adhesives. This reduces the certainty that all observed fibrils possess the canonical crossβ core of amyloids rather than other beta rich aggregates Lack of highresolution structural data
2. Dye specificity caveat: ThioflavinT and Congo Red bind crossbeta but can also show fluorescence enhancement or birefringence with other ordered or hydrophobic aggregates; relying on dyes without corroborating ssNMR/cryoEM or Xray fiber diffraction risks false positives. The authors note this but sometimes present dye positivity alongside morphology as a decisive indicator.
3. Sequence incompleteness and PTMs: many barnacle CPs and other adhesive proteins are incompletely sequenced and lack native PTMs (glycosylation, phosphorylation, crosslinks), yet PTMs can profoundly change folding/assembly; recombinant fragments (often expressed in E coli without eukaryotic PTMs) may not replicate native behavior and can artificially expose amyloidogenic segments.
4. Publication and selection bias: barnacle adhesives are heavily studied for biofouling and biomaterials interest; other taxa may be underreported. The review compiles available literature but cannot correct field sampling bias.

Conclusions and confidence

Reasoned conclusion: There is moderate, convergent evidence that amyloidlike assemblies are a recurring structural solution in several marine adhesives, especially barnacle cement and some cyprid footprints, where multiple orthogonal measurements (AFM morphology, sawtooth force spectroscopy, spectral signatures, dye binding, peptide selfassembly) point to ordered beta rich nanofibrils contributing cohesion and curing. However, definitive atomiclevel confirmation (ssNMR or cryoEM of native fibrils or Xray fiber diffraction combined with complete native sequences and PTM maps) is missing for most systems; thus the hypothesis is plausible and promising but not yet proven universally across taxa Call for omics and atomic level data

What would disprove the main conclusion

• Highresolution structural studies (ssNMR, cryoEM, Xray fiber diffraction) of native adhesive fibrils showing alternative architectures lacking inregister crossβ stacking would falsify the universal amyloid interpretation.
• Comprehensive proteomics showing that the dominant solid phase of cured adhesive is composed of heavily crosslinked glycoproteins or polysaccharideprotein complexes lacking amyloidogenic sequences would change the interpretation.
• Demonstrations that ThT/Congo Red signal and AFM fibrils result from coaggregated nonprotein components or preparation artifacts would weaken the claim.

Suggested next experiments (concrete, testable)

1. Isolate native adhesive fibrils (barnacle cement, cyprid footprints, sea urchin footprints) under nativelike ionic and pH conditions, and perform ssNMR plus cryoEM and Xray fiber diffraction to test for inregister crossβ signatures and determine register and polymorphism. This would directly test the crossbeta architecture.
2. Integrative proteomics: couple LC-MS/MS proteomics of adhesive extracts with targeted glycoproteomics and PTM mapping to obtain full-length sequences and PTM maps of adhesive proteins; then perform in silico amyloid propensity scans and recapitulate assemblies using recombinant full length proteins expressed in eukaryotic systems to preserve PTMs.
3. Mechanics to structure mapping: perform correlated AFM force spectroscopy and cryoEM on the same sample regions to link sawtooth unfolding signatures to specific fibril morphologies and sequences.

Practical implications

If validated, marine functional amyloids provide a robust design motif for wet adhesives that combine surfacecoupling motifs (charged, hydroxylated residues), cohesive amyloid fibrillar cores for strength and insolubility, and sequence features enabling rapid assembly in response to ionic/pH triggers, enabling engineered peptides or recombinant proteins to be designed for biomedical adhesives, tissue engineering hydrogels, and environmentally friendly marine antifouling strategies Biomimetic application potential

Recommendation to readers

Read this review as a wellreferenced synthesis that convincingly compiles suggestive evidence for functional amyloid roles in marine adhesives, but interpret dye and morphology data cautiously and demand atomic resolution corroboration before assuming canonical amyloid crossbeta architecture and universal functionality across species.

Author reviews