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. Results: We have used a custom cDNA microarray chip, developed for the blue swimmer crab Portunus pelagicus, to generate expression profiles of genes involved in exoskeletal formation across the moult cycle. A total of 21 distinct moult-cycle related differentially expressed transcripts representing crustacean cuticular proteins were isolated. Of these, 13 contained copies of the cuticle_1 domain previously isolated from calcified regions of the crustacean exoskeleton, four transcripts contained a chitin_bind_4 domain (RR consensus sequence) associated with both the calcified and un-calcified cuticle of crustaceans, and four transcripts contained an unannotated domain (PfamB_109992) previously isolated from C. pagurus. Additionally, cryptocyanin, a hemolymph protein involved in cuticle synthesis and structural integrity, also displays differential expression related to the moult cycle. Moult stage-specific expression analysis of these transcripts revealed that differential gene expression occurs both among transcripts containing the same domain and among transcripts containing different domains. Conclusion: The large variety of genes associated with cuticle formation, and their differential expression across the crustacean moult cycle, point to the complexity of the processes associated with cuticle formation and hardening. This study provides a molecular entry path into the investigation of the gene networks associated with cuticle formation.

Recovery of the red-finned blue-eye: An endangered fish from springs of the Great Artesian Basin

The red-finned blue-eye (Scaturiginichthys vermeilipinnis) is endemic to a single complex of springs emanating from the Great Artesian Basin, Australia. The species has been recorded as naturally occurring in eight separate very shallow (generally <20 mm) springs, with a combined wetland area of ~0.3 ha. Since its discovery in 1990, five red-finned blue-eye (RFBE) populations have been lost and subsequent colonisation has occurred in two spring wetlands. Current population size is estimated at <3000 individuals. Artesian bores have reduced aquifer pressure, standing water levels and spring-flows in the district. There is evidence of spatial separation within the spring pools where RFBE and the introduced fish gambusia (Gambusia holbrooki) co-occur, although both species are forced together when seasonal extremes affect spring size and water temperature. Gambusia was present in four of the five springs where RFBE populations have been lost. Four out of the five remaining subpopulations of RFBE are Gambusia free. Circumstantial evidence suggests that gambusia is a major threat to red-finned blue-eyes. The impact of Gambusia is probably exacerbated by domestic stock (cattle and sheep), feral goats and pigs that utilise the springs and can negatively affect water quality and flow patterns. Three attempts to translocate RFBE to apparently suitable springs elsewhere within the complex have failed. Opportunities to mitigate threats are discussed, along with directions for future research to improve management of this extremely threatened fish and habitat.

In planta localization and interactions of impatiens necrotic spot tospovirus proteins

Impatiens necrotic spot tospovirus (INSV) is a significant pathogen of ornamentals. The tripartite negative- and ambi-sense RNA genome encodes six proteins that are involved in cytoplasmic replication, movement, assembly, insect transmission and defence. To gain insight into the associations of these viral proteins, we determined their intracellular localization and interactions in living plant cells. Nucleotide sequences encoding the nucleoprotein N, non-structural proteins NSs and NSm, and glycoproteins Gn and Gc of a Kentucky isolate of INSV were amplified by RTPCR, cloned, sequenced and transiently expressed as fusions with autofluorescent proteins in leaf epidermal cells of Nicotiana benthamiana. All proteins accumulated at the cell periphery and co-localized with an endoplasmic reticulum marker. The Gc protein fusion also localized to the nucleus. N and NSm protein self-interactions and an NSm-N interaction were observed by using bimolecular fluorescence complementation. A tospovirus NSm homotypic interaction had not been reported previously.

Pleistocene isolation, secondary introgression and restricted contemporary gene flow in the pig-eye shark, Carcharhinus amboinensis across northern Australia

We examine the structure and phylogeography of the pig-eye shark (Carcharhinus amboinensis) common in shallow coastal environments in northern Australia using two types of genetic markers, two mitochondrial (control region and NADH hydrogenase 4) and two nuclear (microsatellite and Rag 1) DNA. Two populations were defined within northern Australia on the basis of mitochondrial DNA evidence, but this result was not supported by nuclear microsatellite or Rag 1 markers. One possibility for this structure might be sex-specific behaviours such as female philopatry, although we argue it is doubtful that sufficient time has elapsed for any potential signatures from this behaviour to be expressed in nuclear markers. It is more likely that the observed pattern represents ancient populations repeatedly isolated and connected during episodic sea level changes during the Pleistocene epoch, until current day with restricted contemporary gene flow maintaining population genetic structure. Our results show the need for an understanding of both the history and ecology of a species in order to interpret patterns in genetic structure.

Association between feline immunodeficiency virus (FIV) plasma viral RNA load, concentration of acute phase proteins and disease severity

Veterinarians have few tools to predict the rate of disease progression in FIV-infected cats. In contrast, in HIV infection, plasma viral RNA load and acute phase protein concentrations are commonly used as predictors of disease progression. This study evaluated these predictors in cats naturally infected with FIV. In older cats (>5 years), log10 FIV RNA load was higher in the terminal stages of disease compared to the asymptomatic stage. There was a significant association between log10 FIV RNA load and both log10 serum amyloid A concentration and age in unwell FIV-infected cats. This study suggests that viral RNA load and serum amyloid A warrant further investigation as predictors of disease status and prognosis in FIV-infected cats.

Grain sorghum proteomics: integrated approach toward characterization of endosperm storage proteins in kafirin allelic variants

Grain protein composition determines quality traits, such as value for food, feedstock, and biomaterials uses. The major storage proteins in sorghum are the prolamins, known as kafirins. Located primarily on the periphery of the protein bodies surrounding starch, cysteine-rich beta- and gamma-kafirins may limit enzymatic access to internally positioned alpha-kafirins and starch. An integrated approach was used to characterize sorghum with allelic variation at the kafirin loci to determine the effects of this genetic diversity on protein expression. Reversed-phase high performance liquid chromatography and lab-on-a-chip analysis showed reductions in alcohol-soluble protein in beta-kafirin null lines. Gel-based separation and liquid chromatography-tandem mass spectrometry identified a range of redox active proteins affecting storage protein biochemistry. Thioredoxin, involved in the processing of proteins at germination, has reported impacts on grain digestibility and was differentially expressed across genotypes. Thus, redox states of endosperm proteins, of which kafirins are a subset, could affect quality traits in addition to the expression of proteins.

Synthesis of Silica Vesicles with Controlled Entrance Size for High Loading, Sustained Release, and Cellular Delivery of Therapeutical Proteins

A rationally designed two-step synthesis of silica vesicles is developed with the formation of vesicular structure in the first step and fine control over the entrance size by tuning the temperature in the second step. The silica vesicles have a uniform size of ≈50 nm with excellent cellular uptake performance. When the entrance size is equal to the wall thickness, silica vesicles after hydrophobic modification show the highest loading amount (563 mg/g) towards Ribonuclease A with a sustained release behavior. Consequently, the silica vesicles are excellent nano-carriers for cellular delivery applications of therapeutical biomolecules.