Barnacle cement (BC) was beneficially applied on stainless steel (SS) to serve as the initiator anchor for surface-initiated polymerization. The amine and hydroxyl moieties of barnacle cement reacted with 2-bromoisobutyryl bromide to provide the alkyl halide initiator for the surface-initiated atom transfer radical polymerization (ATRP) of 2-hydroxyethyl methacrylate (HEMA). The hydroxyl groups of HEMA polymer (PHEMA) were then converted to carboxyl groups for coupling of chitosan (CS) to impart the SS surface with both antifouling and antibacterial properties. The surface-functionalized SS reduced bovine serum albumin adsorption, bacterial adhesion, and exhibited antibacterial efficacy against Escherichia coli (E. coli). The effectiveness of barnacle cement as an initiator anchor was compared to that of dopamine, a marine mussel inspired biomimetic anchor previously used in surface-initiated polymerization. The results indicate that the barnacle cement is a stable and effective anchor for functional surface coatings and polymer brushes.

The nanoscale morphology and protein secondary structure of barnacle adhesive plaques were characterized using atomic force microscopy (AFM), far-UV circular dichroism (CD) spectroscopy, transmission Fourier transform infrared (FTIR) spectroscopy, and Thioflavin T (ThT) staining. Both primary cement (original cement laid down by the barnacle) and secondary cement (cement used for reattachment) from the barnacle Balanus amphitrite (= Amphibalanus amphitrite) were analyzed. Results showed that both cements consisted largely of nanofibrillar matrices having similar composition. Of particular significance, the combined results indicate that the nanofibrillar structures are consistent with amyloid, with globular protein components also identified in the cement. Potential properties, functions, and formation mechanisms of the amyloid-like nanofibrils within the adhesive interface are discussed. Our results highlight an emerging trend in structural biology showing that amyloid, historically associated with disease, also has functional roles.

SUMMARYRising levels of atmospheric CO 2 lead to acidification of the ocean and alter seawater carbonate chemistry, which can negatively impact calcifying organisms, including mollusks. In estuaries, exposure to elevated CO 2 levels often co-occurs with other stressors, such as reduced salinity, which enhances the acidification trend, affects ion and acid-base regulation of estuarine calcifiers and modifies their response to ocean acidification. We studied the interactive effects of salinity and partial pressure of CO 2 (P CO2 ) on biomineralization and energy homeostasis in juveniles of the eastern oyster, Crassostrea virginica, a common estuarine bivalve. Juveniles were exposed for 11weeks to one of two environmentally relevant salinities (30 or 15PSU) either at current atmospheric P CO2 (~400atm, normocapnia) or P CO2 projected by moderate IPCC scenarios for the year 2100 (~700-800atm, hypercapnia). Exposure of the juvenile oysters to elevated P CO2 and/or low salinity led to a significant increase in mortality, reduction of tissue energy stores (glycogen and lipid) and negative soft tissue growth, indicating energy deficiency. Interestingly, tissue ATP levels were not affected by exposure to changing salinity and P CO2, suggesting that juvenile oysters maintain their cellular energy status at the expense of lipid and glycogen stores. At the same time, no compensatory upregulation of carbonic anhydrase activity was found under the conditions of low salinity and high P CO2 . Metabolic profiling using magnetic resonance spectroscopy revealed altered metabolite status following low salinity exposure; specifically, acetate levels were lower in hypercapnic than in normocapnic individuals at low salinity. Combined exposure to hypercapnia and low salinity negatively affected mechanical properties of shells of the juveniles, resulting in reduced hardness and fracture resistance. Thus, our data suggest that the combined effects of elevated P CO2 and fluctuating salinity may jeopardize the survival of eastern oysters because of weakening of their shells and increased energy consumption.

SUMMARYEnzymes and biochemical mechanisms essential to survival are under extreme selective pressure and are highly conserved through evolutionary time. We applied this evolutionary concept to barnacle cement polymerization, a process critical to barnacle fitness that involves aggregation and cross-linking of proteins. The biochemical mechanisms of cement polymerization remain largely unknown. We hypothesized that this process is biochemically similar to blood clotting, a critical physiological response that is also based on aggregation and cross-linking of proteins. Like key elements of vertebrate and invertebrate blood clotting, barnacle cement polymerization was shown to involve proteolytic activation of enzymes and structural precursors, transglutaminase cross-linking and assembly of fibrous proteins. Proteolytic activation of structural proteins maximizes the potential for bonding interactions with other proteins and with the surface. Transglutaminase cross-linking reinforces cement integrity. Remarkably, epitopes and sequences homologous to bovine trypsin and human transglutaminase were identified in barnacle cement with tandem mass spectrometry and/or western blotting. Akin to blood clotting, the peptides generated during proteolytic activation functioned as signal molecules, linking a molecular level event (protein aggregation) to a behavioral response (barnacle larval settlement). Our results draw attention to a highly conserved protein polymerization mechanism and shed light on a long-standing biochemical puzzle. We suggest that barnacle cement polymerization is a specialized form of wound healing. The polymerization mechanism common between barnacle cement and blood may be a theme for many marine animal glues. Supplementary material available online at