Self-assembling Bioadhesive Inspired by the Fourth Repetitive Sequence of Balanus albicostatus Cement Protein 20 kDa (Balcp-20 k).

Barnacle cement proteins are multi-protein complexes composed of a series of functionally related synergistic proteins that enable barnacles to adhere strongly and consistently to various underwater substrates. There is no post-translational modification of barnacle cement proteins, which provides a possibility for the synthesis of similar adhesive materials. Balcp-20 k has four repetitive sequences with multiple conserved cysteine groups. Whether these repeats are separate functional units and the role of cysteine in adhesion is not clear. In order to investigate the adhesion properties of Balcp-20 k, we amplified and expressed R4 (DHLACNAKHPCWHKHCDCFC)4, which is a quadruple repeat of Balcp-20 k's fourth repetitive sequence, and S0R4 (DHLASNAKHPSWHKHSDSFS)4, all cysteine of R4 replaced by serine. Analysis showed that R4 had a similar structure to Balcp-20 k, and the amyloid fibrils structure formed by self-assembly of R4 played an important role in improving the adhesion strength. The absence of disulfide bonds in S0R4 prevents self-assembly, and the failure of self-assembly after the reduction of disulfide bonds of R4 by DTT indicates that disulfide bonds play an important role in self-assembly. With adhesion and coating analysis, it was found that R4 has good adhesion on different materials surfaces, which is better than Balcp-20 k, while S0R4 has weak adhesion, which is only better than BSA.

Design of RGDS Peptide-Immobilized Self-Assembling ß-Strand Peptide from Barnacle Protein.

We designed three types of RGD-containing barnacle adhesive proteins using self-assembling peptides. In the present study, three types of RGD-containing peptides were synthesized by solid-phase peptide synthesis, and the secondary structures of these peptides were analyzed by CD and FT-IR spectroscopy. The mechanical properties of peptide hydrogels were characterized by a rheometer. We discuss the correlation between the peptide conformation, and cell attachment and cell spreading activity from the viewpoint of developing effective tissue engineering scaffolds. We created a peptide-coated cell culture substrate by coating peptides on a polystyrene plate. They significantly facilitated cell adhesion and spreading compared to a non-coated substrate. When the RGDS sequence was modified at N- or C-terminal of R-Y, it was found that the self-assembling ability was dependent on the strongly affects hydrogel formation and cell adhesion caused by its secondary structure.

Small RNA sequencing of sessile serrated polyps identifies microRNA profile associated with colon cancer.

Sessile serrated adenoma/polyps (SSA/Ps) of the colon account for 20-30% of all colon cancers. Small non-coding RNAs, including microRNAs (miRNAs), may function as oncogenes or tumor suppressor genes involved in cancer development. Small RNA sequencing (RNA-seq) was used to characterize miRNA profiles in SSA/Ps, hyperplastic polyps (HPs), adenomatous polyps and paired uninvolved colon. Our 108 small RNA-seq samples' results were compared to small RNA-seq data from 212 colon cancers from the Cancer Genome Atlas. Twenty-three and six miRNAs were differentially expressed in SSA/Ps compared to paired uninvolved colon and HPs, respectively. Differential expression of MIR31-5p, MIR135B-5p and MIR378A-5p was confirmed by RT-qPCR. SSA/P-specific miRNAs are similarly expressed in colon cancers containing genomic aberrations described in serrated cancers. Correlation of miRNA expression with consensus molecular subtypes suggests more than one subtype is associated with the serrated neoplasia pathway. Canonical pathway analysis suggests many of these miRNAs target growth factor signaling pathways.

DNA Extraction Protocols for Whole-Genome Sequencing in Marine Organisms.

The marine environment harbors a large proportion of the total biodiversity on this planet, including the majority of the earths' different phyla and classes. Studying the genomes of marine organisms can bring interesting insights into genome evolution. Today, almost all marine organismal groups are understudied with respect to their genomes. One potential reason is that extraction of high-quality DNA in sufficient amounts is challenging for many marine species. This is due to high polysaccharide content, polyphenols and other secondary metabolites that will inhibit downstream DNA library preparations. Consequently, protocols developed for vertebrates and plants do not always perform well for invertebrates and algae. In addition, many marine species have large population sizes and, as a consequence, highly variable genomes. Thus, to facilitate the sequence read assembly process during genome sequencing, it is desirable to obtain enough DNA from a single individual, which is a challenge in many species of invertebrates and algae. Here, we present DNA extraction protocols for seven marine species (four invertebrates, two algae, and a marine yeast), optimized to provide sufficient DNA quality and yield for de novo genome sequencing projects.

Evolutionary and biogeographical patterns of barnacles from deep-sea hydrothermal vents.

The characterization of evolutionary and biogeographical patterns is of fundamental importance to identify factors driving biodiversity. Due to their widespread but discontinuous distribution, deep-sea hydrothermal vent barnacles represent an excellent model for testing biogeographical hypotheses regarding the origin, dispersal and diversity of modern vent fauna. Here, we characterize the global genetic diversity of vent barnacles to infer their time of radiation, place of origin, mode of dispersal and diversification. Our approach was to target a suite of multiple loci in samples representing seven of the eight described genera. We also performed restriction-site associated DNA sequencing on individuals from each species. Phylogenetic inferences and topology hypothesis tests indicate that vent barnacles have colonized deep-sea hydrothermal vents at least twice in history. Consistent with preliminary estimates, we find a likely radiation of barnacles in vent ecosystems during the Cenozoic. Our analyses suggest that the western Pacific was the place of origin of the major vent barnacle lineage, followed by circumglobal colonization eastwards through the Southern Hemisphere during the Neogene. The inferred time of radiation rejects the classic hypotheses of antiquity of vent taxa. The timing and the mode of origin, radiation and dispersal are consistent with recent inferences made for other deep-sea taxa, including nonvent species, and are correlated with the occurrence of major geological events and mass extinctions. Thus, we suggest that the geological processes and dispersal mechanisms discussed here can explain the current distribution patterns of many other marine taxa and have played an important role shaping deep-sea faunal diversity. These results also constitute the critical baseline data with which to assess potential effects of anthropogenic disturbances on deep-sea ecosystems.

Molecular phylogeny of the acorn barnacle family Tetraclitidae (Cirripedia: Balanomorpha: Tetraclitoidea): validity of shell morphology and arthropodal characteristics in the systematics of Tetraclitid barnacles.

Shell structure is a crucial aspect of barnacle systematics. Within Tetraclitidae, the diametric and monometric growth patterns and number of rows of parietal tubes in the shells are key characteristics used to infer evolutionary trends. We used molecular analysis based on seven genes (mitochondrial COI, 16S and 12S rRNA, and nuclear EF1, RPII, H3, and 18S rRNA) to test two traditional phylogenetic hypothesis: (1) Tetraclitid barnacles are divided into two major lineages, which are distinguished according to monometric and diametric shell growth patterns, and (2) the evolutionary trend in shell parietal development began with a solid shell, which developed into a single tubiferous shell, which then developed into multitubiferous shells. The results indicated that Tetraclitinae and Newmanellinae are not monophyletic, but that Austrobalaninae and Tetraclitellinae are. The phylogram based on the genetic data suggested that Bathylasmatidae is nested within the Tetraclitidae, forming a sister relationship with the Austrobalaninae and Tetraclitinae/Newmanellinae clade. Within the Tetraclitinae/Newmanellinae clade, the genera Tetraclita (multitubiferous shell), Tesseropora (single tubiferous shell), and Yamaguchiella (multitubiferous shell) are polyphyletic. The results suggested that shell morphology and growth patterns do not reflect the evolutionary history of Tetraclitidae, whereas the arthropodal characteristics are informative.

Genealogical approaches to the temporal origins of the Central American gap: Speciation and divergence in Pacific Chthamalus (Sessilia: Chthamalidae)

A large section of the tropical Eastern Pacific coastline is nearly devoid of reef or consolidated habitat, and is known as the Central American Gap as it is associated with a biogeographic transition in fish and invertebrate species. We analyze phylogeographic data for intertidal barnacles (Chthamalus) to identify relevant temporal patterns that describe the origins of this biogeographic transition (the Mexican-Panamic Transition Zone). These contrasts of populations on either side of the transition zone include two pairs of closely related species (C. panamensis and C. hedgecocki; C. southwardorum and a Southern form of C. southwardorum), as well as gene flow data within one species (C. panamensis) that currently is found on both sides of the boundary between provinces. Using sequence data from a prior phylogenetic study, we used traditional (net nucleotide divergence) measures as well as coalescent analyses that incorporate the isolation-migration model to identify the likely time of separation between Northern and Southern taxa in two species pairs. A total of 67 individuals were sequenced at two mitochondrial (cytochrome c oxidase I, 16S) and one nuclear (elongation factor 1-alpha) gene regions. Our analyses indicate that the regional isolation of these intertidal barnacles occurred approximately 315-400kya, with subsequent expansion of C. panamensis from the Southern region into the North much more recently. There are insufficient survey data to conclusively document the absence of species from this group within the Central American Gap region near the Gulf of Tehuantepec. However, appropriate habitat is quite sparse in this region and other environmental factors, including upwelling and water temperature, are likely to be associated with isolation of many species in the Mexican and Panamic provinces sensu stricto. Some taxa may maintain gene flow across this region, but very few genetic studies have been completed on such taxa. Until further work is done, distinguishing between prior hypotheses of a faunal gap, or a faunal transition zone, is somewhat speculative. Additional taxonomic revision will be necessary in Chthamalus but is beyond the scope of this paper.

La taxonomía del complejo de especies de cirripedios (Chthamalus) se ha confundido en la literatura desde hace casi 30 años, por lo tanto analizamos datos de su filogeografía para identificar modelos temporales relevantes que describan los orígenes de la zona de transición entre las provincias Mexicana y Panameña. Estos contrastes de poblaciones a ambos lados de la zona de transición incluyen a dos pares de especies estrechamente relacionadas, así como datos de flujo de genes dentro de una especie que actualmente es encontrada en ambos lados del límite entre provincias. Usando datos de secuencia de un estudio previo de filogenética, usamos medidas tradicionales, así como análisis de coalescencia que incorporan el modelo de migración y aislamiento para identificar el tiempo probable de la separación entre los taxa del norte y del sur en dos pares de especies. Nuestros análisis indican que el aislamiento regional de estos ciripedios ocurrió aproximadamente hace 315-400 mil años, con una extensión subsecuente de Chthamalus panamensis de la región del sur hacia el norte mucho más reciente. No hay datos suficientes para documentar conclusivamente la ausencia de especies de este grupo dentro de la región de Centro América cerca del Golfo de Tehuantepec. Sin embargo, el hábitat apropiado es bastante escaso en esta región y otros factores ambientales, incluyendo corrientes y temperatura acuática, probablemente están relacionados con el aislamiento de muchas especies en estas provincias. Algunos taxa pueden mantener el flujo de genes a través de esta región, pero muy pocos estudios genéticos han sido realizados en tales taxa. Hasta que no se desarrollen trabajos adicionales, la distinción entre hipótesis previas de un gap faunal o de una zona de transición faunal es algo especulativo.

Regional variation in the spatial scale of selection at MPI* and GPI* in the acorn barnacle Semibalanus balanoides (Crustacea).

Elucidating the ecological processes by which adaptive genetic polymorphism is maintained in heterogeneous environments requires knowledge on the spatial scale at which alternate habitats affect genotype-specific fitness. The general objective of this study was to document patterns of temporal and spatial variation of genetic polymorphism in the acorn barnacle (Semibalanus balanoides) at MPI* and GPI* allozyme loci. A total of 7261 barnacles were sampled in the intertidal at various locations north and south of the Miramichi estuary, New Brunswick, Canada. The results of this study supported the hypothesis that both MPI* and GPI* are under the effect of strong directional selection south of the Miramichi, whereas neutrality cannot be ruled out at sampling sites located north of the estuary. Comparisons between this study and previous ones also question the generality of current hypotheses regarding ecological processes that are responsible for maintaining polymorphism at MPI* and GPI* in the acorn barnacle.

Unraveling the evolutionary radiation of the thoracican barnacles using molecular and morphological evidence: a comparison of several divergence time estimation approaches.

The Thoracica includes the ordinary barnacles found along the sea shore and is the most diverse and well-studied superorder of Cirripedia. However, although the literature abounds with scenarios explaining the evolution of these barnacles, very few studies have attempted to test these hypotheses in a phylogenetic context. The few attempts at phylogenetic analyses have suffered from a lack of phylogenetic signal and small numbers of taxa. We collected DNA sequences from the nuclear 18S, 28S, and histone H3 genes and the mitochondrial 12S and 16S genes (4,871 bp total) and data for 37 adult and 53 larval morphological characters from 43 taxa representing all the extant thoracican suborders (except the monospecific Brachylepadomorpha). Four Rhizocephala (highly modified parasitic barnacles) taxa and a Rhizocephala + Acrothoracica (burrowing barnacles) hypothetical ancestor were used as the outgroup for the molecular and morphological analyses, respectively. We analyzed these data separately and combined using maximum likelihood (ML) under "hill-climbing" and genetic algorithm heuristic searches, maximum parsimony procedures, and Bayesian inference coupled with Markov chain Monte Carlo techniques under mixed and homogeneous models of nucleotide substitution. The resulting phylogenetic trees answered key questions in barnacle evolution. The four-plated Iblomorpha were shown as the most primitive thoracican, and the plateless Heteralepadomorpha were placed as the sister group of the Lepadomorpha. These relationships suggest for the first time in an invertebrate that exoskeleton biomineralization may have evolved from phosphatic to calcitic. Sessilia (nonpedunculate) barnacles were depicted as monophyletic and appear to have evolved from a stalked (pedunculate) multiplated (5+) scalpelloidlike ancestor rather than a five-plated lepadomorphan ancestor. The Balanomorpha (symmetric sessile barnacles) appear to have the following relationship: (Chthamaloidea(Coronuloidea(Tetraclitoidea, Balanoidea))). Thoracican divergence times were estimated under ML-based local clock, Bayesian, and penalized likelihood approaches using an 18S data set and three calibration points: Heteralepadomorpha = 530 million years ago (MYA), Scalpellomorpha = 340 MYA, and Verrucomorpha = 120 MYA. Estimated dates varied considerably within and between approaches depending on the calibration point. Highly parameterized local clock models that assume independent rates (r > or = 15) for confamilial or congeneric species generated the most congruent estimates among calibrations and agreed more closely with the barnacle fossil record. Reasonable estimates were also obtained under the Bayesian procedure of Kishino et al. (2001, Mol. Biol. Evol. 18:352-361) but using multiple calibrations. Most of the dates estimated under the Bayesian procedure of Aris-Brosou and Yang (2002, Syst. Biol. 51:703-714) and the penalized likelihood method using single and/or multiple calibrations were inconsistent among calibrations and did not fit the fossil record.

4-n-Nonylphenol and 17-beta estradiol may induce common DNA effects in developing barnacle larvae.

There is a growing concern over the potential effects of environmental endocrine disrupters on both human and wildlife populations. However, to date, minimal research has been conducted to determine the effect of estrogens and xenoestrogens at the DNA level. In this study, we used the random amplified polymorphic DNA (RAPD) assay to evaluate the effects on the genomic DNA of barnacle larvae that had been exposed to 17beta-estradiol (E2) and low concentrations of 4-n-nonylphenol (NP). DNA effects include DNA damage as well as mutations and possibly other effects at the DNA level that can be induced by chemical or physical agents that directly and/or indirectly interact with genomic DNA. Not only did exposure to NP and E2 induce changes in RAPD profiles in the exposed barnacle larvae when compared to control patterns, but also, and more importantly, there were similarities in the RAPD modifications in the exposed populations that had been treated to either chemical. We propose that NP and E2 induced some common DNA effects in barnacle larvae and that these specific modifications in RAPD patterns may arise as a consequence of hot spot DNA damage (e.g. DNA adducts) and/or mutations (point mutations or genomic rearrangements). This could help to explain how xenoestrogens mimic the effects produced by natural estrogens. In conclusion, in the field of endocrine disruption, the study of DNA effects induced by estrogens and/or xenoestrogens warrants further investigation. Indeed, changes at the DNA levcl may be the precursors of some of the numerous effects reported at higher levels of biological organisation such as the feminization of males, developmental abnormalities, and infertility.

PCR-cloning of cadmium-inducible peptides in the barnacle, Megabalanus volcano.

A 340 bp DNA fragment was amplified from barnacle (Megabalanus volcano) cDNA by polymerase chain reaction using primers designed based on the amino acid sequences of barnacle cadmium-inducible peptides CdlP1 and CdlP2. The whole sequence was determined by rapid amplification of cDNA ends method. The cDNA contained an open reading frame encoding 71 amino acid residues and the sequences for CdlP1 and CdlP2 were found to be located in the center of this coding region. Although CdlP1 and CdlP2 had been detected only in the cadmium-exposed barnacles, their mRNA was present both in cadmium-exposed barnacles and in unexposed barnacles. These results suggest that posttranslational proteolytic processing may be induced in the presence of cadmium.

Differential patterns of spatial divergence in microsatellite and allozyme alleles: further evidence for locus-specific selection in the acorn barnacle, Semibalanus balanoides?

We compared patterns of genetic structure at potentially selected (two allozyme loci) and neutral molecular markers (six microsatellite loci) in the acorn barnacle, Semibalanus balanoides from the Gulf of St. Lawrence. Our results confirmed the presence of a geographical shift in alleles MPI and GPI near the Miramichi River. In contrast, no significant patterns of population differentiation among samples located north and south of the river mouth were detected for four of six microsatellite loci. However, analysis of molecular variance (amova) at individual loci revealed that a significant proportion of the total variance in allele frequencies was partitioned among samples located north and south of the river for both the allozyme and the other two microsatellite loci. The two most common alleles at these microsatellites showed frequencies that were highly correlated (r = 0.65-0.74, P < 0.05) with those of the MPI*2 allele, perhaps because of either physical linkage or epistasis. The two allozyme loci were significantly correlated in barnacles located north of the Miramichi River (r = 0.86, P < 0.05). Overall, our results supported the hypothesis that the broad scale pattern of allozyme allelic shifts is maintained by selection. They also indicated that microsatellites may not always behave in a neutral way and must be used cautiously, especially when evidence for genetic structuring relies on only a few assayed loci.

Novel barnacle underwater adhesive protein is a charged amino acid-rich protein constituted by a Cys-rich repetitive sequence.

Barnacle cement is an underwater adhesive that is used for permanent settlement, and is an insoluble protein complex. A method for rendering soluble the cement of Megabalanus rosa has been developed, and three major proteins have been identified in a previous study. To survey the M. rosa cement proteins in a lower molecular mass range, the cement proteins were separated by reversed-phase HPLC and a previously unidentified protein named 20 kDa M. rosa cement protein (Mrcp-20k) was found. Mrcp-20k cDNA was cloned to reveal its primary structure. This cDNA was 902 bp long and encoded a 202 amino acid-long open reading frame, including 19 amino acids of the signal sequence. The molecular mass in the disulphide form was calculated to be 20357 Da and the isoelectric point of the mature polypeptide was 4.72. Mrcp-20k was characterized by an abundance of Cys residues and charged amino acids. The most common amino acid was Cys (17.5%), with Asp (11.5%), Glu (10.4%) and His (10.4%) following in order of magnitude. The alignment of the Cys residues indicated the primary structure of this protein to consist of six degenerated repeats, each about 30 residues long. Mrcp-20k has no intermolecular disulphide bonds and no free thiol groups of Cys in the insoluble cement complex. Abundant Cys is thought to play a role in maintaining the topology of charged amino acids on the molecular surface by intramolecular disulphide-bond formation. The possible function of abundant charged amino acids, including the interaction with a variety of surface metals on the substratum, is discussed.

Cloning and expression of the engrailed.a gene of the barnacle Sacculina carcini.

Cirripedia (barnacles) constitute a crustacean monophyletic taxon which is very well defined by several synapomorphies. In particular, all cirripedes are composed of six thoracic segments, but are devoid of any complete abdominal segment. This body plan is preserved in the adult in non-parasitic groups, while the parasitic rhizocephalan cirripedes completely lose arthropodian segmentation at the adult stage. These traits make them a particularly favourable model for studying the formation and maintenance of segmental identity. For the above reasons, it seemed worthwhile to look at the segmentation gene engrailed in a cirripede. A complete engrailed.a cDNA was isolated from larvae of the rhizocephalan cirripede Sacculina carcini. Its expression was monitored during larval development by use of the monoclonal antibody MAb4D9 directed against the Drosophila homologous proteins. The Sacculina engrailed.a gene is expressed during the second and third larval stages in stripes within a posterior area corresponding to the presumptive trunk segments. Surprisingly, these stripes appear in a posterior to anterior sequence. Six engrailed.a stripes characterize the thoracic segments of the cirripedean ground plan.