Differential expression profiling of components associated with exoskeletal hardening in crustaceans

<p>Abstract</p> <p>Background</p> <p>Exoskeletal hardening in crustaceans can be attributed to mineralization and sclerotization of the organic matrix. Glycoproteins have been implicated in the calcification process of many matrices. Sclerotization, on the other hand, is catalysed by phenoloxidases, which also play a role in melanization and the immunological response in arthropods. Custom cDNA microarrays from <it>Portunus pelagicus </it>were used to identify genes possibly associated with the activation pathways involved in these processes.</p> <p>Results</p> <p>Two genes potentially involved in the recognition of glycosylation, the C-type lectin receptor and the mannose-binding protein, were found to display molt cycle-related differential expression profiles. C-type lectin receptor up-regulation was found to coincide with periods associated with new uncalcified cuticle formation, while the up-regulation of mannose-binding protein occurred only in the post-molt stage, during which calcification takes place, implicating both in the regulation of calcification. Genes presumed to be involved in the phenoloxidase activation pathway that facilitates sclerotization also displayed molt cycle-related differential expression profiles. Members of the serine protease superfamily, trypsin-like and chymotrypsin-like, were up-regulated in the intermolt stage when compared to post-molt, while trypsin-like was also up-regulated in pre-molt compared to ecdysis. Additionally, up-regulation in pre- and intermolt stages was observed by transcripts encoding other phenoloxidase activators including the putative antibacterial protein carcinin-like, and clotting protein precursor-like. Furthermore, hemocyanin, itself with phenoloxidase activity, displayed an identical expression pattern to that of the phenoloxidase activators, i.e. up-regulation in pre- and intermolt.</p> <p>Conclusion</p> <p>Cuticle hardening in crustaceans is a complex process that is precisely timed to occur in the post-molt stage of the molt cycle. We have identified differential expression patterns of several genes that are believed to be involved in biomineralization and sclerotization and propose possible regulatory mechanisms for these processes based on their expression profiles, such as the potential involvement of C-type lectin receptors and mannose binding protein in the regulation of calcification.</p

Gene expression profiling of cuticular proteins across the moult cycle of the crab Portunus pelagicus

Background: Crustaceans represent an attractive model to study biomineralization and cuticle matrix formation, as these events are precisely timed to occur at certain stages of the moult cycle. Moulting, the process by which crustaceans shed their exoskeleton, involves the partial breakdown of the old exoskeleton and the synthesis of a new cuticle. This cuticle is subdivided into layers, some of which become calcified while others remain uncalcified. The cuticle matrix consists of many different proteins that confer the physical properties, such as pliability, of the exoskeleton

Microbial Diversity Profiling of Gut Microbiota of Macropus giganteus Using Three Hypervariable Regions of the Bacterial 16S rRNA

Animal faecal contamination of surface waters poses a human health risk, as they may contain pathogenic bacteria or viruses. Of the numerous animal species residing along surface waterways in Australia, macropod species are a top contributor to wild animals’ faecal pollution load. We characterised the gut microbiota of 30 native Australian Eastern Grey Kangaroos from six geographical regions (five kangaroos from each region) within South East Queensland in order to establish their bacterial diversity and identify potential novel species-specific bacteria for the rapid detection of faecal contamination of surface waters by these animals. Using three hypervariable regions (HVRs) of the 16S rRNA gene (i.e., V1–V3, V3–V4, and V5–V6), for their effectiveness in delineating the gut microbial diversity, faecal samples from each region were pooled and microbial genomic DNA was extracted, sequenced, and analysed. Results indicated that V1-V3 yielded a higher taxa richness due to its larger target region (~480 bp); however, higher levels of unassigned taxa were observed using the V1-V3 region. In contrast, the V3–V4 HVR (~569 bp) attained a higher likelihood of a taxonomic hit identity to the bacterial species level, with a 5-fold decrease in unassigned taxa. There were distinct dissimilarities in beta diversity between the regions, with the V1-V3 region displaying the highest number of unique taxa (n = 42), followed by V3–V4 (n = 11) and V5–V6 (n = 8). Variations in the gut microbial diversity profiles of kangaroos from different regions were also observed, which indicates that environmental factors may impact the microbial development and, thus, the composition of the gut microbiome of these animals

Endotoxin tolerance in abdominal aortic aneurysm macrophages, in vitro: a case–control study

Macrophages are implicated in the pathogenesis of abdominal aortic aneurysm (AAA). This study examined the environmentally conditioned responses of AAA macrophages to inflammatory stimuli. Plasma- and blood-derived monocytes were separated from the whole blood of patients with AAA (30-45 mm diameter; = 33) and sex-matched control participants ( = 44). Increased concentrations of pro-inflammatory and pro-oxidant biomarkers were detected in the plasma of AAA patients, consistent with systemic inflammation and oxidative stress. However, in monocyte-derived macrophages, a suppressed cytokine response was observed in AAA compared to the control following stimulation with lipopolysaccharide (LPS) (tumor necrosis factor alpha (TNF-α) 26.9 ± 3.3 vs. 15.5 ± 3.2 ng/mL, < 0.05; IL-6 3.2 ± 0.6 vs. 1.4 ± 0.3 ng/mL, < 0.01). LPS-stimulated production of 8-isoprostane, a biomarker of oxidative stress, was also markedly lower in AAA compared to control participants. These findings are consistent with developed tolerance in human AAA macrophages. As Toll-like receptor 4 (TLR4) has been implicated in tolerance, macrophages were examined for changes in TLR4 expression and distribution. Although TLR4 mRNA and protein expression were unaltered in AAA, cytosolic internalization of receptors and lipid rafts was found. These findings suggest the inflamed, pro-oxidant AAA microenvironment favors macrophages with an endotoxin-tolerant-like phenotype characterized by a diminished capacity to produce pro-inflammatory mediators that enhance the immune response

Molecular insights into land snail neuropeptides through transcriptome and comparative gene analysis

Background: Snails belong to the molluscan class Gastropoda, which inhabit land, freshwater and marine environments. Several land snail species, including Theba pisana, are crop pests of major concern, causing extensive damage to agriculture and horticulture. A deeper understanding of their molecular biology is necessary in order to develop methods to manipulate land snail populations. Results: The present study used in silico gene data mining of T. pisana tissue transcriptomes to predict 24,920 central nervous system (CNS) proteins, 37,661 foot muscle proteins and 40,766 hepatopancreas proteins, which together have 5,236 unique protein functional domains. Neuropeptides, metabolic enzymes and epiphragmin genes dominated expression within the CNS, hepatopancreas and muscle, respectively. Further investigation of the CNS transcriptome demonstrated that it might contain as many as 5,504 genes that encode for proteins destined for extracellular secretion. Neuropeptides form an important class of cell-cell messengers that control or influence various complex metabolic events. A total of 35 full-length neuropeptide genes were abundantly expressed within T. pisana CNS, encoding precursors that release molluscan-type bioactive neuropeptide products. These included achatin, allototropin, conopressin, elevenin, FMRFamide, LFRFamide, LRFNVamide, myomodulins, neurokinin Y, PKYMDT, PXFVamide, sCAPamides and several insulin-like peptides. Liquid chromatography-mass spectrometry of neural ganglia confirmed the presence of many of these neuropeptides. Conclusions: Our results provide the most comprehensive picture of the molecular genes and proteins associated with land snail functioning, including the repertoire of neuropeptides that likely play significant roles in neuroendocrine signalling. This information has the potential to expedite the study of molluscan metabolism and potentially stimulate advances in the biological control of land snail pest species

Moult cycle specific differential gene expression profiling of the crab Portunus pelagicus

Background: Crustacean moulting is a complex process involving many regulatory pathways. A holistic approach to examine differential gene expression profiles of transcripts relevant to the moulting process, across all moult cycle stages, was used in this study. Custom cDNA microarrays were constructed for Portunus pelagicus. The printed arrays contained 5000 transcripts derived from both the whole organism, and from individual organs such as the brain, eyestalk, mandibular organ and Y-organ from all moult cycle stages.Results: A total of 556 clones were sequenced from the cDNA libraries used to construct the arrays. These cDNAs represented 175 singletons and 62 contigs, resulting in 237 unique putative genes. The gene sequences were classified into the following biological functions: cuticular proteins associated with arthropod exoskeletons, farnesoic acid O-methyltransferase (FaMeT), proteins belonging to the hemocyanin gene family, lectins, proteins relevant to lipid metabolism, mitochondrial proteins, muscle related proteins, phenoloxidase activators and ribosomal proteins. Moult cycle-related differential expression patterns were observed for many transcripts. Of particular interest were those relating to the formation and hardening of the exoskeleton, and genes associated with cell respiration and energy metabolism.Conclusions: The expression data presented here provide a chronological depiction of the molecular events associated with the biological changes that occur during the crustacean moult cycle. Tracing the temporal expression patterns of a large variety of transcripts involved in the moult cycle of P. pelagicus can provide a greater understanding of gene function, interaction, and regulation of both known and new genes with respect to the moulting process

Genomic approach to the study of moulting and cuticle formation in crustaceans

Crustacean moulting is a complex process involving many regulatory pathways. A holistic approach to examine differential gene expression profiles across all moult cycle stages was used in this study. Custom cDNA microarrays were constructed for P. pelagicus. The chips contained 5000 transcripts derived from both the whole organism, and from individual tissues such as the brain, eyestalk, mandibular organ and Y-organ from all moult cycle stages. Thus the arrays were designed to study global gene expression profiles of transcripts relevant to the moulting process, across the entire moult cycle. A total of 556 clones were sequenced from the cDNA libraries used to construct the arrays. These cDNAs represented 217 singlets and 62 contigs, resulting in 279 unique putative genes. The isolated transcripts were classified into the following biological functions: cuticular proteins associated with calcified and/or uncalcified arthropod exoskeletons, FaMeT, proteins belonging to the hemocyanin gene family, lectins, proteins relevant to lipid metabolism, mitochondrial proteins, muscle related proteins, phenoloxidase activators, ribosomal RNA relevant to protein biosynthesis, other sequences which did not fall into these groups, and those transcripts which displayed no significant sequence homology with sequences deposited in the NCBI database. Partial characterisation of the unannotated transcripts was achieved via conceptual domain analysis. Moult cycle-related differential expression profiles were observed for many transcripts. Of particular interest were those relating to the formation and hardening of the exoskeleton, which were shown to be up-regulated at key stages of moulting, and the gene for putative FaMeT which was up-regulated only in intermoult. The moult cycle-related differential expression profiles of genes involved in exoskeletal formation were analysed and described in detail. Additional characterisation was carried out in the form of conceptual domain analysis, uncovering many types of cuticle proteins. Twenty-one distinct transcripts representing crustacean cuticular proteins were isolated. Thirteen contain copies of the cuticle_1 domain, previously isolated only from calcified regions of the crustacean exoskeleton. Four transcripts contained a chitin_bind_4 domain, associated with both the calcified and un-calcified cuticle of crustaceans. Another four transcripts contained an unannotated domain (PfamB_109992) previously isolated from C. pagurus. Additionally cryptocyanin, a haemolymph protein, involved in cuticle synthesis and structural integrity, also displayed moult cycle-related differential expression. Moult stage-specific expression analysis of these transcripts revealed that differential expression occurs both within transcripts containing the same domain and between transcripts containing different domains. Exoskeletal hardening in crustaceans occurs through the mineralisation and sclerotization of the organic matrix. Glycosylation levels of proteins in an organic matrix have been attributed to the regulation of calcification. Two genes potentially involved in the recognition of glycosylation, the C-type lectin receptor and the mannose-binding protein, were found to display moult cyclerelated differential expression profiles. C-type lectin receptor up-regulation was found to coincide with periods associated with new uncalcified cuticle formation, while the up-regulation of mannose-binding protein occurred only in the postmoult stage, during which calcification takes place, implicating both in the regulation of calcification. Genes known to be involved in the phenoloxidase activation pathway, which facilitates sclerotization, also displayed moult cycle-related differential expression profiles. Trypsin and chymotrypsin were up-regulated in the intermoult stage when compared to postmoult, while trypsin was also up-regulated in premoult compared to ecdysis. Furthermore, hemocyanin, itself with phenoloxidase activity, displayed an identical expression pattern to that of the phenoloxidase activators, i.e. up-regulation in pre- and postmoult. FaMeT is the enzyme responsible for the conversion of FA to MF in the final step of MF synthesis. Multiple isoforms of putative FaMeT were isolated from six crustacean species belonging to the families Portunidae, Penaeidae, Scyllaridae and Parastacidae. The portunid crabs Portunus pelagicus and Scylla serrata code for three isoforms. Two isoforms (short and long) were isolated from the penaeid prawns Penaeus monodon and Fenneropenaeus merguiensis, and the scyllarid Thenus orientalis and parastacid Cherax quadricarinatus. Putative FaMeT sequences were also amplified from the genomic DNA of P. pelagicus and compared to the putative FaMeT transcripts expressed. Each putative FaMeT cDNA isoform was represented in the genomic DNA, indicative of a multi-gene family. Various tissues from P. pelagicus were individually screened for putative FaMeT expression using PCR and fragment analysis. Each tissue type expressed all three isoforms of putative FaMeT irrespective of sex or moult stage. Protein domain analysis revealed the presence of a deduced casein kinase II phosphorylation site present in only the long isoform of putative FaMeT

Diagnosis of Preclinical Crohn's Disease: Hurdles, Challenges, and Hopes

LETTER TO THE EDITO