Fresh and cultured buccal cells as a source of mRNA and protein for molecular analysis

We developed a method for obtaining viable buccal cells from mouthwash samples for use as a source of mRNA and protein. Immunofluorescent analysis showed that most cells were derived from nonkeratinized parabasal epithelia, with a minor proportion of proliferative cells. Gene expression was detected in buccal cells using reverse transcription PCR, Western blot analysis, and immunofluorescence. Using a keratinocyte-specific medium, buccal cells could be cultured on Matrigel™-coated permeable filters for up to 2 weeks while maintaining the expression of some epithelial-specific markers, including cytokeratin 13, cytokeratin 10, transferrin receptor, and β-integrin. The basal marker cytokeratin 14 and Ki67, an indicator of cellular proliferation, were detected in a few cells. We show that buccal cells can be obtained from a noninvasive procedure for use as a source of material for biochemical analyses. A population of the buccal cells can be maintained in culture for up to 2 weeks using keratinocyte-specific medium in combination with extracellular matrix.

Molecular analysis of Australian Newcastle disease virus isolates

The results presented demonstrate that outbreaks of virulent Newcastle disease in Australia arose from the emergence of virulent virus from an endemic avirulent quasispecies. Environmental selection pressures, or industrial practices, led to the emergence of this species and its dominance in several distinct ourbreaks from 1998-2002.

Molecular analysis of the human copper uptake protein hCTR1

This study examined the role of the high-affinity copper uptake protein hCTR1 in cellular copper homeostasis and found hCTR1 was internalized in response to raised copper levels. The work from this thesis supports a model in which the regulation of hCTR1 is partially or wholly dependent upon internal copper levels.

Detection of Candida spp. in the vagina of a cohort of nulliparous pregnant women by culture and molecular methods: is there an association between maternal vaginal and infant oral colonisation?

BACKGROUND: Most studies describing vaginal Candida spp. in pregnancy focus on symptomatic vaginitis, rather than asymptomatic colonisation, and solely utilise microbiological culture. The extent to which asymptomatic vaginal carriage may represent a reservoir for infant oral colonisation has been highly debated. MATERIALS AND METHODS: This study formed part of the Candida and Staphylococcus Transmission Longitudinal Evaluation (CASTLE) study, in Melbourne, Australia, from 2009 to 2011 and used culture and molecular methods to examine vaginal swabs collected late in the third trimester of pregnancy for Candida spp. Oral swabs from infants were also examined using culture methods. RESULTS: Overall, 80 of 356 (22%) women were positive for Candida spp; the majority being Candida albicans (83%). Candida glabrata and other Candida spp. were also identified, but in much lower numbers. Molecular analysis identified numerous positive samples not detected by culture, including 13 cases of C. albicans. In addition, some positive samples only recorded to genus level by culture were accurately identified as either C. albicans or C. glabrata following molecular analyses. Eighteen infants recorded positive Candida spp. cultures, predominantly C. albicans. However, there were only four (25%) mother/infant dyads where C. albicans was detected. CONCLUSIONS: This study provides valuable data on asymptomatic colonisation rates of Candida spp. within an asymptomatic population of women late in pregnancy. The utilisation of molecular methods improved the rate of detection and provided a more accurate means for identification of non-albicans Candida spp. The low mother/infant colonisation rate suggests that non-maternal sources are likely involved in determining infant oral colonisation status.

Hematopoietic perturbation in zebrafish expressing a tel-jak2a fusion

Objective
Various TEL-JAK2 fusions have been identified in patients with lymphoblastic and myeloid leukemias that result in constitutive activation of the JAK2 kinase domain. Such fusions can mediate factor-independent growth of hematopoietic cell lines and induction of malignancy in mouse models.
Materials and methods
To assess whether zebrafish could be utilized as a suitable model for the study of myeloid oncogenesis, we generated a zebrafish tel-jak2a fusion oncoprotein based on that seen in a case of chronic myeloid leukemia. This was transiently expressed in zebrafish embryos under the control of the spi1 promoter, which is strongly active in myeloid precursors.
Results
Visual, histological, and molecular analysis revealed disruption of normal embryonic hematopoiesis, including perturbation of the myeloid and erythroid lineages.
Conclusion
These results indicate that the zebrafish tel-jak2a oncoprotein is functional, and suggest that this organism will be useful for the experimental study of myeloid malignancy.

Comparative distribution of a putative egg-laying hormone in neural and reproductive tissues of four Decapoda crustaceans

Evidence for the presence of a putative egg-laying (ELH) hormone has been previously described in the black tiger shrimp, Penaeus monodon, so a further investigation was carried out to detect its presence in a range of Decapoda crustaceans prior to a full molecular analysis. The crustaceans were represented by the Australian fresh water yabbie, Cherax destructor, the Australian southern rock lobster, Jasus edwardsii, the snow crab, Chionoecetes opilio, and the blue swimmer crab, Portunus pelagicus. Female cerebral ganglia, ventral nerve cords and gonads were investigated in a comparative study of the distribution of the immunoreactive hormone using immunoenzyme and immunofluorescence techniques. Immunoreactivity was detected in all tissues of interest, and the distribution patterns showed similarity within the four species, as well as that of P. monodon reported in the earlier study. There were minor variations. These data indicate that a putative ELH-like neuropeptide is widespread in crustaceans, and supports its previous identification in a range of molluscs and other invertebrates. Elucidation of the molecular structure of the peptide hormone and its encoding gene, as well as its involvement in spawning behaviour of crustaceans, is now fully under investigation.

Comparative genomics and transcriptomics of lactation

Lactation is an important characteristic of mammalian reproduction sometimes referred to as the quintessence of mammals. Comparative genomics and transcriptomics experiments are allowing a more in-depth molecular analysis of the evolution of lactation throughout the mammalian kingdom and these recent results are reviewed here. Milk cell and mammary gland gene expression analysis with sequencing methodology have started to reveal conserved or specific milk protein and components of the lactation system of monotreme, marsupial and eutherian lineages. These experiments have confirmed the ancient origin of the complex lactation system and provided useful insight into the function of specific milk proteins in the control of the lactation programme or the role of milk in the regulation of growth and development of the young beyond simple nutritive aspects.

Effects of alterations of primer-binding site sequences on human immunodeficiency virus type 1 replication

The human immunodeficiency virus type 1 genomic RNA primer-binding site (PBS) sequence comprises 18 nucleotides which are complementary to those at the 3' end of the replication initiation primer tRNA(3Lys). To investigate the role of the PBS in viral replication, we either deleted the original wild-type PBS (complementary to tRNA(3Lys) or replaced it with DNA sequences complementary to either tRNA(1,2Lys) or tRNA(Phe). Transfection of COS cells with such molecular constructs yielded similar levels of viral progeny that were indistinguishable with regard to viral proteins and tRNA content. Virus particles derived from PBS-deleted molecular clones were noninfectious for MT-4, Jurkat, and CEM-T4 cells. However, infectious viruses were derived from constructs in which the PBS had been altered to sequences complementary to either tRNA(1,2Lys) or tRNA(Phe), although mutated forms showed significant lags in replication efficiency in comparison with wild types. Molecular analysis of reverse-transcribed DNA in cells infected by the mutated viruses indicated that both tRNA(1,2Lys) and tRNA(Phe) could function as primers for reverse transcription during the early stages of infection. Sequencing of full-length proviral DNA, obtained 6 days after infection, revealed the mutated PBS, indicating that a complete cycle of reverse transcription had occurred. During subsequent rounds of infection, reversion of the mutated PBS to wild-type sequences was observed, accompanied by increased production of viral gene products. Reversion to wild-type PBS sequences was confirmed both by specific PCR analysis, using distinct primer pairs, and by direct sequencing of amplified segments. We also performed endogenous in vitro reverse transcription experiments in which synthesis of minus-strand strong-stop viral DNA was primed from a synthetic RNA template containing a PBS complementary to various tRNA isoacceptors. These results showed that tRNA(3Lys) was a much more efficient primer of such reactions than either tRNA(1,2Lys) or tRNA(Phe).

Creatine pretreatment prevents birth asphyxia–induced injury of the newborn spiny mouse kidney

Background:
Acute kidney injury (AKI) is a major complication for infants following an asphyxic insult at birth. We aimed to determine if kidney structure and function were affected in an animal model of birth asphyxia and if maternal dietary creatine supplementation could provide an energy reserve to the fetal kidney, maintaining cellular respiration during asphyxia and preventing AKI.

Methods:
Pregnant spiny mice were maintained on normal chow or chow supplemented with creatine from day 20 gestation. On day 38 (term ~39 d), pups were delivered by cesarean section (c-section) or subjected to intrauterine asphyxia. Twenty-four hours after insult, kidneys were collected for histological or molecular analysis. Urine and plasma were also collected for biochemical analysis.

Results:
AKI was evident at 24 h after birth asphyxia, with a higher incidence of shrunken glomeruli (P < 0.02), disturbance to tubular arrangement, tubular dilatation, a twofold increase (P < 0.02) in expression of Ngal (early marker of kidney injury), and decreased expression of the podocyte differentiation marker nephrin. Maternal creatine supplementation prevented the glomerular and tubular abnormalities observed in the kidney at 24 h and the increased expression of Ngal.

Conclusion:
Maternal creatine supplementation may prove useful in ameliorating kidney injury associated with birth asphyxia.

Evaluating environmental, demographic and genetic effects on population-level survival in an island endemic

The population dynamics of island species are considered particularly sensitive to variation in environmental, demographic and/or genetic processes. However, few studies have attempted to evaluate the relative importance of these processes for key vital rates in island endemics. We integrated the results of long-term capture–mark–recapture analysis, prey surveys, habitat quality assessments and molecular analysis to determine the causes of variation in the survival rates of Komodo dragons Varanus komodoensis at 10 sites on four islands in Komodo National Park, Indonesia. Using open population capture–mark–recapture methods, we ranked competing models that considered environmental, ecological, genetic and demographic effects on site-specific Komodo dragon survival rates. Site-specific survival rates ranged from 0.49 (95% CI: 0.33–0.68) to 0.92 (0.79–0.97) in the 10 study sites. The three highest-ranked models (i.e. ΔQAICc < 2) explained ∼70% of variation in Komodo dragon survival rates and identified interactions between inbreeding coefficients, prey biomass density and habitat quality as important explanatory variables. There was evidence of additive effects from ecological and genetic (e.g. inbreeding) processes affecting Komodo dragon survival rates. Our results indicate that maintaining high ungulate prey biomass and habitat quality would enhance the persistence of Komodo dragon populations. Assisted gene flow may also increase the genetic and demographic viability of the smaller Komodo dragon populations.

Manual on application of molecular tools in aquaculture and inland fisheries management. Part I : laboratory protocols, data management and analysis

The aim of this manual is to provide a comprehensive practical tool for the generation and analysis of genetic data for subsequent application in aquatic resources management in relation to genetic stock identification in inland fisheries and aquaculture. The material only covers general background on genetics in relation to aquaculture and fisheries resource management, the techniques and relevant methods of data analysis that are commonly used to address questions relating to genetic resource characterisation and population genetic analyses. No attempt is made to include applications of genetic improvement techniques e.g. selective breeding or producing genetically modified organisms (GMOs). The manual includes two ‘stand-alone’ parts, of which this is the second volume: Part 1 – Conceptual basis of population genetic approaches: will provide a basic foundation on genetics in general, and concepts of population genetics. Issues on the choices of molecular markers and project design are also discussed. Part 2 – Laboratory protocols, data management and analysis: will provide step-by-step protocols of the most commonly used molecular genetic techniques utilised in population genetics and systematic studies. In addition, a brief discussion and explanation of how these data are managed and analysed is also included. This manual is expected to enable NACA member country personnel to be trained to undertake molecular genetic studies in their own institutions, and as such is aimed at middle and higher level technical grades. The manual can also provide useful teaching material for specialised advanced level university courses in the region and postgraduate students. The manual has gone through two development/improvement stages. The initial material was tested at a regional workshop and at the second stage feedback from participants was used to improve the contents.

Development of molecular techniques for the genetic analysis of abalone populations

Three DNA techniques: random amplified polymorphic DNA (RAPD), minisatellite, and microsatellite analyses, were developed for use in abalone population genetic structure studies. The techniques were assessed using sample sets of blacklip and greenlip abalone. The study identifies a potential for the application of these DNA markers in abalone fisheries management, but microsatellites are the recommended method for future studies.

Molecular characterization and analysis of a truncated serotonin receptor gene expressed in neural and reproductive tissues of abalone

In molluscs, the neurotransmitter serotonin (5-HT) has been linked to a variety of biological roles including gamete maturation and spawning. The possible involvement of 5-HT in abalone gamete release was demonstrated by a dose-dependent increase in Haliotis rubra gonad contractile bioactivity following 5-HT stimulation. Physiological functions associated with 5-HT, are mediated through binding to 5-HT receptors. A cDNA encoding a putative 5-HT receptor consisting of 359 amino acids was isolated from the tropical abalone H. asinina, termed 5-HT_{1 ha}. The 5-HT_{1 ha} shares G-protein-coupled receptor motifs with metazoan 5-HT receptors, including predicted transmembrane domains, active sites for protein kinase action, and N-linked glycosylation sites. However, the third intracellular loop of 5-HT_{1 ha} is relatively short, and only six transmembrane domains are predicted, implying a truncated receptor. Phylogenetic analysis with known 5-HT receptor genes suggests that 5-HT_{1 ha} belongs to the type 1 5-HT receptor family. Expression analysis by RT-PCR showed that 5-HT_{1 ha}  mRNA was present in all tissues examined, including the neural ganglia and gonad tissues. Immunocytochemistry revealed the presence of 5-HT_{1 ha} specifically within the soma of neuronal cells located in the outer cortex of both cerebral and pleuropedal ganglia. In ovarian and testicular tissues, 5-HT_{1 ha} immunoreactivity was observed in epithelial cells of the outer capsule and connective tissue of the trabeculae to which the gamete follicles adhere. Whether this receptor transcript is translated to a functional protein needs to be verified, but if so, it could play a role in reproduction.

Molecular identification of marine yeast and its spectroscopic analysis establishes unsaturated fatty acid accumulation

Marine microbes are competent organisms, some of which can accumulate large amounts of lipids. A yeast strain, Rhodotorula mucilaginosa AMCQ8A was isolated from the marine water of the Queenscliff region, Victoria, Australia. The yeast isolate was identified by sequencing 18s rDNA genes. Scanning electron microscopy images revealed scars on the surface of the yeast cells. The Fourier transform infrared spectroscopy microspectroscopy studies demonstrated the presence of unsaturated fatty acids by differential microscopic analysis. The sharp band at 1745 cm^-1 was represented by ν(C=O) stretches of ester functional groups from lipids and fats, and therefore indicated the presence of total lipids produced by the cells. Over 65% of the fatty acids from the yeast strain were analyzed as C¹⁶ and C^18:1 with omega-3 content from about 6% to 7%. Thus, this marine-derived yeast could be a potential source of lipids, including omega-3 fatty acids. 2012, The Society for Biotechnology, Japan. All rights reserved.