Project description:MicroRNAs (miRNAs) are 18-24 nucleotide autonomous regulatory RNA molecules found in all eukaryotes. They are involved in the regulation of a multitude of genetic and biological pathways through post transcriptional gene silencing and/or translational repression. Previous data has suggested a slow evolutionary rate for the saltwater crocodile (Crocodylus porosus) over the past several million years when compared to its closest extant relatives, the birds. Understanding genome regulation, adaptive capabilities and physiological features in the saltwater crocodile in the context of relatively slow genomic change thus holds significant potential for the investigation of genomics, evolution and adaptive studies. Utilizing eleven different tissue types and sixteen small RNA libraries, we report a catalog of 644 miRNAs in the saltwater crocodile with > 78% of miRNAs being potentially novel to crocodilians. We also predicted and identified targets for the miRNAs as well as analyzed the relationship of the miRNA repertoire to transposable elements (TEs) in the saltwater crocodile that showed an increased association of DNA transposons with miRNA biogenesis when compared to retrotransposons. Phylogenetic analysis of C. porosus miRNA expectedly revealed highest number of miRNAs in sister crocodilian clades of the American Alligator and the Indian Gharial. This work reports the first comprehensive analysis of miRNAs in Crocodylus porosus for and addresses the potential impacts of miRNAs in regulating the genome in the saltwater crocodile as well as supporting the role of TEs as a source for miRNAs, adding to the increasing evidence that TEs play a significant role in the evolution of gene regulation.
Project description:Identification and characterization of microRNAs (miRNAs) and their transposable element origins in the saltwater crocodile, Crocodylus porosus
Project description:Crocodiles are remarkable animals that have the ability to endure extremely harsh conditions and can survive up to a 100 years while being exposed to noxious agents that are detrimental to Homo sapiens. Besides their immunity, we postulate that the microbial gut flora of crocodiles may produce substances with protective effects. In this study, we isolated and characterized selected bacteria colonizing the gastrointestinal tract of Crocodylusporosus and demonstrated their inhibitory effects against three different cancerous cell lineages. Using liquid chromatography-mass spectrometry, several molecules were identified. For the first time, we report partial analyses of crocodile's gut bacterial molecules.
Project description:BackgroundCancer remains a global threat resulting in significant morbidity and mortality despite advances in therapeutic interventions, suggesting urgency for identification of anticancer agents. Crocodiles thrive in polluted habitat, feed on germ-infested meat, are exposed to carcinogenic heavy metals, are the very few species to survive the catastrophic Cretaceous-Paleogene extinction event, yet have a prolonged lifespan and rarely been reported to develop cancer. Therefore, we hypothesised that animals living in polluted environments such as crocodiles possess anticancer molecules/mechanisms.MethodsCrocodylus porosus was procured, blood collected, dissected and lysates prepared from internal organs. Organ lysates and sera were tested for growth inhibition, cytotoxic effects and cell survival against HeLa, PC3 and MCF7 cells and subjected to liquid chromatography mass spectrometry. RNA transcriptome analysis and differential gene analysis were performed using Galaxy Bioinformatics.ResultsSera exhibited potent growth inhibition and cytotoxic effects against cancer cells. 80 molecules were detected from C. porosus and 19 molecules were putatively identified. Additionally, more than 100 potential anticancer peptides were identified from sera using bioinformatics based on peptide amino acid composition, binary profile, dipeptide composition and pseudo-amino acid composition. Following transcriptome analysis, 14 genes in treated HeLa cells, 51 genes in treated MCF7 cells and 2 genes in treated PC3 cells, were found to be expressed, compared with untreated controls.ConclusionAnimals residing in polluted milieus are an unexploited source for prospective pharmaceutical drugs, and could lead to identification of novel antitumour compound(s) and/or further understanding of the mechanisms of cancer resistance.
Project description:West Nile virus, Kunjin strain (WNVKUN) is endemic in Northern Australia, but rarely causes clinical disease in humans and horses. Recently, WNVKUN genomic material was detected in cutaneous lesions of farmed saltwater crocodiles (Crocodylus porosus), but live virus could not be isolated, begging the question of the pathogenesis of these lesions. Crocodile hatchlings were experimentally infected with either 105 (n = 10) or 104 (n = 11) TCID50-doses of WNVKUN and each group co-housed with six uninfected hatchlings in a mosquito-free facility. Seven hatchlings were mock-infected and housed separately. Each crocodile was rotationally examined and blood-sampled every third day over a 3-week period. Eleven animals, including three crocodiles developing typical skin lesions, were culled and sampled 21 days post-infection (dpi). The remaining hatchlings were blood-sampled fortnightly until experimental endpoint 87 dpi. All hatchlings remained free of overt clinical disease, apart from skin lesions, throughout the experiment. Viremia was detected by qRT-PCR in infected animals during 2-17 dpi and in-contact animals 11-21 dpi, indicating horizontal mosquito-independent transmission. Detection of viral genome in tank-water as well as oral and cloacal swabs, collected on multiple days, suggests that shedding into pen-water and subsequent mucosal infection is the most likely route. All inoculated animals and some in-contact animals developed virus-neutralizing antibodies detectable from 17 dpi. Virus-neutralizing antibody titers continued to increase in exposed animals until the experimental endpoint, suggestive of persisting viral antigen. However, no viral antigen was detected by immunohistochemistry in any tissue sample, including from skin and intestine. While this study confirmed that infection of saltwater crocodiles with WNVKUN was associated with the formation of skin lesions, we were unable to elucidate the pathogenesis of these lesions or the nidus of viral persistence. Our results nevertheless suggest that prevention of WNVKUN infection and induction of skin lesions in farmed crocodiles may require management of both mosquito-borne and water-borne viral transmission in addition to vaccination strategies.
Project description:Saltwater crocodilepox virus (SwCRV), belonging to the genus Crocodylidpoxvirus, are large DNA viruses posing an economic risk to Australian saltwater crocodile (Crocodylus porosus) farms by extending production times. Although poxvirus-like particles and sequences have been confirmed, their infection dynamics, inter-farm genetic variability and evolutionary relationships remain largely unknown. In this study, a poxvirus infection dynamics study was conducted on two C. porosus farms. One farm (Farm 2) showed twice the infection rate, and more concerningly, an increase in the number of early- to late-stage poxvirus lesions as crocodiles approached harvest size, reflecting the extended production periods observed on this farm. To determine if there was a genetic basis for this difference, 14 complete SwCRV genomes were isolated from lesions sourced from five Australian farms. They encompassed all the conserved genes when compared to the two previously reported SwCRV genomes and fell within three major clades. Farm 2's SwCRV sequences were distributed across all three clades, highlighting the likely mode of inter-farm transmission. Twenty-four recombination events were detected, with one recombination event resulting in consistent fragmentation of the P4c gene in the majority of the Farm 2 SwCRV isolates. Further investigation into the evolution of poxvirus infection in farmed crocodiles may offer valuable insights in evolution of this viral family and afford the opportunity to obtain crucial information into natural viral selection processes in an in vivo setting.