MKK3 Was Involved in Larval Settlement of the Barnacle Amphibalanus amphitrite through Activating the Kinase Activity of p38MAPK

The p38 mitogen-activated protein kinase (p38MAPK) plays a key role in larval settlement of the barnacle Amphibalanus amphitrite. To study the signaling pathway associated with p38MAPK during larval settlement, we sought to identify the upstream kinase of p38MAPK. Three MKKs (MKK3, MKK4 and MKK7) and three MAPKs (p38MAPK, ERK and JNK) in A. amphitrite were cloned and recombinantly expressed in E. coli. Through kinase assays, we found that MKK3, but not MKK4 or MKK7, phosphorylated p38MAPK. Furthermore, MKK3 activity was specific to p38MAPK, as it did not phosphorylate ERK or JNK. To further investigate the functional relationship between MKK3 and p38MAPK in vivo, we studied the localization of phospho-MKK3 (pMKK3) and MKK3 by immunostaining. Consistent with the patterns of p38MAPK and phospho-p38MAPK (pp38MAPK), pMKK3 and MKK3 mainly localized to the antennules of the cyprids. Western blot analysis revealed that pMKK3 levels, like pp38MAPK levels, were elevated at cyprid stage, compared to nauplii and juvenile stages. Moreover, pMKK3 levels increased after treatment with adult barnacle crude extracts, suggesting that MKK3 might mediate the stimulatory effects of adult barnacle extracts on the p38MAPK pathway.     

A Recurrent PDGFRB Mutation Causes Familial Infantile Myofibromatosis

Infantile myofibromatosis (IM) is the most common benign fibrous tumor of soft tissues affecting young children. By using whole-exome sequencing, RNA sequencing, and targeted sequencing, we investigated germline and tumor DNA in individuals from four distinct families with the familial form of IM and in five simplex IM cases with no previous family history of this disease. We identified a germline mutation c.1681C>T (p.Arg561Cys) in platelet-derived growth factor receptor β (PDGFRB) in all 11 affected individuals with familial IM, although none of the five individuals with nonfamilial IM had mutations in this gene. We further identified a second heterozygous mutation in PDGFRB in two myofibromas from one of the affected familial cases, indicative of a potential second hit in this gene in the tumor. PDGFR-β promotes growth of mesenchymal cells, including blood vessels and smooth muscles, which are affected in IM. Our findings indicate p.Arg561Cys substitution in PDGFR-β as a cause of the dominant form of this disease. They provide a rationale for further investigations of this specific mutation and gene to assess the benefits of targeted therapies against PDGFR-β in aggressive life-threatening familial forms of the disease.     

Nitric oxide inhibits larval settlement in Amphibalanus amphitrite cyprids by repressing muscle locomotion and molting

Nitric oxide (NO) is a universal signaling molecule and plays a negative role in the metamorphosis of many biphasic organisms. Recently, the NO/cGMP (cyclic guanosine monophosphate) signaling pathway was reported to repress larval settlement in the barnacle Amphibalanus amphitrite. To understand the underlying molecular mechanism, we analyzed changes in the proteome of A. amphitrite cyprids in response to different concentrations of the NO donor sodium nitroprusside (SNP; 62.5, 250, and 1000 μM) using a label‐free proteomics method. Compared with the control, the expression of 106 proteins differed in all three treatments. These differentially expressed proteins were assigned to 13 pathways based on KEGG pathway enrichment analysis. SNP treatment stimulated the expression of heat shock proteins and arginine kinase, which are functionally related to NO synthases, increased the expression levels of glutathione transferases for detoxification, and activated the iron‐mediated fatty acid degradation pathway and the citrate cycle through ferritin. Moreover, NO repressed the level of myosins and cuticular proteins, which indicated that NO might inhibit larval settlement in A. amphitrite by modulating the process of muscle locomotion and molting.     

Trophic responsiveness of purified postnatal and adult rat retinal ganglion cells

Central neurons lose the ability for axonal re-growth during development and typically do not regenerate their axons following axotomy once they become mature unless given a growth-permissive environment i.e. peripheral nerve graft. In the present study, the growth responsiveness of purified retinal ganglion cells (RGCs) at different ages to neurotrophic factors and Schwann cell (SC)-secreted factors were examined directly. The purity of adult RGCs was 97% as assessed by retrograde labelling with 4,6-diamidino-2-phenylindole. The stability of cultures were demonstrated by long-term survival (30 days) in medium contained brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and forskolin (F) (BCF). RGCs from postnatal (P) (P0, P4, P8, P21) and adult (P90) rats showed decreasing levels of survival and neuritogenesis when grown in BCF. In contrast, the opposite was observed in SC-conditioned medium (CM)-treated P0-P8 RGCs which were increasingly responsive. SCCM induced maximal neurite outgrowth in P8 RGCs via the activation of extracellular regulated kinase 1/2 (Erk1/2). Inhibition of mitogen-activated protein kinase-Erk1/2 signaling using an Erk1/2-specific inhibitor (UO126) abolished SCCM-induced Erk1/2 phosphorylation and neuritogenesis completely. Although both SCCM and BCF failed to sustain the same levels of growth in P21 or P90 cultures as observed in P8 cultures, SCCM promoted higher survival and neuritogenesis than BCF-treated adult RGCs. This study is the first report of adult rat RGC purification and demonstrates that mature RGCs need multiple factors for survival and neurite outgrowth.     

Exploring the regulatory role of nitric oxide (NO) and the NO-p38MAPK/cGMP pathway in larval settlement of the bryozoan Bugula neritina

The bryozoan Bugula neritina is a cosmopolitan marine fouling species that causes major fouling problems in sub-tropical waters. Settlement of B. neritina larvae can be triggered without an obvious external cue. Here, the negative regulatory role of nitric oxide (NO) during larval settlement of B. neritina was demonstrated to be mediated by cyclic guanosine monophosphate (cGMP). Although the regulatory role of the NO-p38 MAPK signaling axis in larval settlement was not evident, inhibition of nitric oxide synthase (NOS) led to the deactivation of p38 MAPK. Exclusive localization of NO and NO signaling components in sensory-related organs of the larvae is consistent with its signal transduction function in metamorphosis. Overall, this study provides new insights into the regulatory roles of the NO-p38MAPK/cGMP pathway in B. neritina settlement.     

Inter- and Intraspecific Variations of Bacterial Communities Associated with Marine Sponges from San Juan Island,Washington

This study attempted to assess whether conspecific or congeneric sponges around San Juan Island, Washington, harbor specific bacterial communities. We used a combination of culture-independent DNA fingerprinting techniques (terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis [DGGE]) and culture-dependent approaches. The results indicated that the bacterial communities in the water column consisted of more diverse bacterial ribotypes than and were drastically different from those associated with the sponges. High levels of similarity in sponge-associated bacterial communities were found only in Myxilla incrustans and Haliclona rufescens, while the bacterial communities in Halichondria panicea varied substantially among sites. Certain terminal restriction fragments or DGGE bands were consistently obtained for different individuals of M. incrustans and H. rufescens collected from different sites, suggesting that there are stable or even specific associations of certain bacteria in these two sponges. However, no specific bacterial associations were found for H. panicea or for any one sponge genus. Sequencing of nine DGGE bands resulted in recovery of seven sequences that best matched the sequences of uncultured Proteobacteria. Three of these sequences fell into the sponge-specific sequence clusters previously suggested. An uncultured alphaproteobacterium and a culturable Bacillus sp. were found exclusively in all M. incrustans sponges, while an uncultured gammaproteobacterium was unique to H. rufescens. In contrast, the cultivation approach indicated that sponges contained a large proportion of Firmicutes, especially Bacillus, and revealed large variations in the culturable bacterial communities associated with congeneric and conspecific sponges. This study revealed sponge species-specific but not genus- or site-specific associations between sponges and bacterial communities and emphasized the importance of using a combination of techniques for studying microbial communities.Marine sponges (phylum Porifera) harbor a remarkable array of microorganisms, including bacteria (51, 54), unicellular algae (50), cyanobacteria (45, 48), dinoflagellates (14), zoochlorellae (58), and members of the domain Archaea (33). Of these microorganisms, bacteria are the most dominant group of microbial associates in sponges and can account for up to 40 to 50% of a sponge''s biomass (17). The density of bacteria can be up to 10⁸ to 10¹⁰ bacteria per g (wet weight) of sponge (18). The great abundance of bacteria in sponges caused workers to coin the term “bacteriosponges” (35) and has attracted much research interest in the role and specificity of the sponge-bacterium association.There are generally two pathways by which sponges may acquire their bacterial associates. The first pathway is filter feeding and selective retention of bacteria (44). Bacteria in the surrounding seawater can be captured by sponges when sponges filter the food particles out of the water column. The bacteria that resist digestion by sponge choanocytes and archaeocytes can survive and live inside the sponges. Sponge-associated bacterial communities acquired via this pathway are therefore heavily influenced by the type of bacteria in the water column. The second pathway is vertical transmission of bacterial associates from adult sponges to their progeny (10, 40, 49). Bacteria acquired through this pathway may exhibit specificity for genera or species of sponges due to coevolution (for a review, see reference 44).Sponge-bacterium symbioses are often mutualistic; while bacteria may benefit from the favorable nutritional conditions in sponges (18, 44), some bacterial associates may help their hosts eliminate metabolic waste (4), stabilize the sponge skeleton (36), and defend against pathogens, predators, or competitors via the production of bioactive secondary metabolites (5, 21, 39, 47). In addition, some cyanobacterial symbionts are a source of nutrients for their hosts because of their photosynthetic and nitrogen-fixing abilities (3, 57).There is a large body of knowledge concerning the possible roles of bacterial associates in sponges, but whether sponges harbor specific bacterial communities deserves more detailed study. Many previous investigations demonstrated that associated bacterial communities in certain species of sponges were highly similar and consistently different from the bacterial communities in the ambient environment (6, 22, 46). For instance, Hentschel et al. (16) showed that there were uniform microbial communities in the marine sponges Aplysina aerophoba, Rhopaloeides odorabile, and Theonella swinhoei from different geographic regions that were distinct from those in the water column or in sediments. In addition, Friedrich et al. (12) demonstrated that the composition of sponge-associated bacterial communities was resistant to environmental perturbations resulting from transplantation to different habitats. These studies suggest that there is a stable, specific, and perhaps mutualistic relationship between the two types of organisms (44). However, some bacterium-sponge symbioses do not appear to be consistent. For instance, Wichels et al. (56) demonstrated that the bacterial communities associated with the North Sea sponge Halichondria panicea varied substantially over time. Qian et al. (34) and Lee et al. (25) showed that the congeneric Callyspongia and Mycale sponges from different biogeographic regions had different bacterial associates. Although the discrepancies may be attributed to the methods employed in different studies, whether consistent sponge-bacterium associations occur in different species or genera of sponges remains unclear. Furthermore, most of the previous studies focused on only one sponge species or involved a few sponges from geographically separated regions. So far, there has been no large-scale study comparing the bacterial communities associated with sponges of different genera and species.In this study, we compared the bacterial communities associated with marine sponges around San Juan Island, Washington, in order to investigate the specificity of congeneric and conspecific sponge-associated bacterial communities. Most previous studies have relied on only one method to assess the bacterial communities, limiting the resolution of community assessments. In addition, different studies have used different approaches to address the same question, creating uncertainties and making generalizations difficult. Here, we employed both culture-independent and -dependent approaches to compensate for the limitations of different methods and to obtain a more reliable assessment of the associated bacterial communities. We used two DNA fingerprinting techniques, terminal restriction fragment length polymorphism (TRFLP) (27) and denaturing gradient gel electrophoresis (DGGE) (11). TRFLP is an effective, sensitive, high-throughput technique that differentiates bacterial community structures, while DGGE allows subsequent identification of bacteria of interest by excision and sequencing of specific bands. Both techniques have been successfully and widely used for characterization of bacterial communities in marine samples (13, 26, 28, 43, 56). In addition, bacteria associated with sponges were isolated using cultivation methods and identified by comparative analysis of 16S rRNA gene sequences. Phylogenetic affiliations of the isolates were determined and compared for different samples in order to study the specificity of the sponge-associated culturable bacterial communities.     

Nontoxic piperamides and their synthetic analogues as novel antifouling reagents

Bioassay-guided isolation of an acetone extract from a terrestrial plant Piper betle produced four known piperamides with potent antifouling (AF) activities, as evidenced by inhibition of settlement of barnacle cypris larvae. The AF activities of the four piperamides and 15 synthesized analogues were compared and their structure–activity relationships were probed. Among the compounds, piperoleine B and 1-[1-oxo-7-(3′,4′-methylenedioxyphenyl)-6E-heptenyl]-piperidine (MPHP) showed strong activity against settlement of cyprids of the barnacle Balanus amphitrite, having EC₅₀ values of 1.1?±?0.3 and 0.5?±?0.2?μg?ml^?1, respectively. No toxicity against zebra fish was observed following incubation with these two compounds. Besides being non-toxic, 91% of piperoleine B-treated cyprids and 84% of MPHP-treated cyprids at a concentration of 100?μM completed normal metamorphosis in recovery bioassays, indicating that the anti-settlement effect of these two compounds was reversible. Hydrolysis and photolysis experiments indicated that MPHP could be decomposed in the marine environment. It is concluded that piperamides are promising compounds for use in marine AF coatings.