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Oxidases are found to play a growing role in providing functional chemistry to marine adhesives for the permanent attachment of macrofouling organisms. Here, we demonstrate active peroxidase and lysyl oxidase enzymes in the adhesive layer of adult Amphibalanus amphitrite barnacles through live staining, proteomic analysis, and competitive enzyme assays on isolated cement. A novel full-length peroxinectin (AaPxt-1) secreted by barnacles is largely responsible for oxidizing phenolic chemistries; AaPxt-1 is driven by native hydrogen peroxide in the adhesive and oxidizes phenolic substrates typically preferred by phenoloxidases (POX) such as laccase and tyrosinase. A major cement protein component AaCP43 is found to contain ketone/aldehyde modifications via 2,4-dinitrophenylhydrazine (DNPH) derivatization, also called Brady's reagent, of cement proteins and immunoblotting with an anti-DNPH antibody. Our work outlines the landscape of molt-related oxidative pathways exposed to barnacle cement proteins, where ketone- and aldehyde-forming oxidases use peroxide intermediates to modify major cement components such as AaCP43.

We report the development of a quantum dot (QD)–peptide–fullerene (C60) electron transfer (ET)-based nanobioconjugate for the visualization of membrane potential in living cells. The bioconjugate is composed of (1) a central QD electron donor, (2) a membrane-inserting peptidyl linker, and (3) a C60 electron acceptor. The photoexcited QD donor engages in ET with the C60 acceptor, resulting in quenching of QD photoluminescence (PL) that tracks positively with the number of C60 moieties arrayed around the QD. The nature of the QD-capping ligand also modulates the quenching efficiency; a neutral ligand coating facilitates greater QD quenching than a negatively charged carboxylated ligand. Steady-state photophysical characterization confirms an ET-driven process between the donor–acceptor pair. When introduced to cells, the amphiphilic QD–peptide–C60 bioconjugate labels the plasma membrane by insertion of the peptide–C60 portion into the hydrophobic bilayer, while the hydrophilic QD sits on the exofacial side of the membrane. Depolarization of cellular membrane potential augments the ET process, which is manifested as further quenching of QD PL. We demonstrate in HeLa cells, PC12 cells, and primary cortical neurons significant QD PL quenching (ΔF/F0 of 2–20% depending on the QD–C60 separation distance) in response to membrane depolarization with KCl. Further, we show the ability to use the QD–peptide–C60 probe in combination with conventional voltage-sensitive dyes (VSDs) for simultaneous two-channel imaging of membrane potential. In in vivo imaging of cortical electrical stimulation, the optical response of the optimal QD–peptide–C60 configuration exhibits temporal responsivity to electrical stimulation similar to that of VSDs. Notably, however, the QD–peptide–C60 construct displays 20- to 40-fold greater ΔF/F0 than VSDs. The tractable nature of the QD–peptide–C60 system offers the advantages of ease of assembly, large ΔF/F0, enhanced photostability, and high throughput without the need for complicated organic synthesis or genetic engineering, respectively, that is required of traditional VSDs and fluorescent protein constructs.

Cell encapsulation is critical for many biotechnology applications including environmental remediation, bioreactors, and regenerative medicine. Here, the development of biohybrid microfibers comprised of encapsulated bacteria in hydrogel matrices produced on‐chip using microfluidics is reported. The fiber production process utilizes hydrodynamic shaping of a cell‐laden core fluid by a miscible sheath fluid. Production of the fibers containing viable bacteria was continuous in contrast to the more typical methods in which cells infiltrated or were attached to prepared fibers. The biohybrid fibers were composed of poly (ethylene glycol dimethacrylate) matrices and individually both E. coli and B. cereus were explored as model cellular payloads. Post processing growth curves (24 h) of bacteria within fibers were in excellent agreement with that of controls suggesting minimal impact. Finally, the biohybrid fibers showed even distribution of encapsulated cells and >90% cell viability.

BackgroundA complete understanding of barnacle adhesion remains elusive as the process occurs within and beneath the confines of a rigid calcified shell. Barnacle cement is mainly proteinaceous and several individual proteins have been identified in the hardened cement at the barnacle-substrate interface. Little is known about the molt- and tissue-specific expression of cement protein genes but could offer valuable insight into the complex multi-step processes of barnacle growth and adhesion.MethodsThe main body and sub-mantle tissue of the barnacle Amphibalanus amphitrite (basionym Balanus amphitrite) were collected in pre- and post-molt stages. RNA-seq technology was used to analyze the transcriptome for differential gene expression at these two stages and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) was used to analyze the protein content of barnacle secretions.ResultsWe report on the transcriptomic analysis of barnacle cement gland tissue in pre- and post-molt growth stages and proteomic investigation of barnacle secretions. While no significant difference was found in the expression of cement proteins genes at pre- and post-molting stages, expression levels were highly elevated in the sub-mantle tissue (where the cement glands are located) compared to the main barnacle body. We report the discovery of a novel 114kD cement protein, which is identified in material secreted onto various surfaces by adult barnacles and with the encoding gene highly expressed in the sub-mantle tissue. Further differential gene expression analysis of the sub-mantle tissue samples reveals a limited number of genes highly expressed in pre-molt samples with a range of functions including cuticular development, biominerialization, and proteolytic activity.ConclusionsThe expression of cement protein genes appears to remain constant through the molt cycle and is largely confined to the sub-mantle tissue. Our results reveal a novel and potentially prominent protein to the mix of cement-related components in A. amphitrite. Despite the lack of a complete genome, sample collection allowed for extended transcriptomic analysis of pre- and post-molt barnacle samples and identified a number of highly-expressed genes. Our results highlight the complexities of this sessile marine organism as it grows via molt cycles and increases the area over which it exhibits robust adhesion to its substrate.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2076-1) contains supplementary material, which is available to authorized users.