Studies of assisted reproduction in the spotted grass frog Limnodynastes tasmaniensis: ovulation, early development and microinjection (ICSI)

Assisted Reproductive Technologies (ART) offer a wide range of techniques that have the potential to augment efforts to conserve and manage endangered amphibians and improve wild and captive population numbers. Gametes and tissues of species nearing endangered or extinct status can be cryopreserved and stored in gene banks, to provide material that can be utilised in the future as ART methods are refined. The Spotted Grass Frog, Limnodynastes tasmaniensis, is an abundant amphibian species in South-Eastern Australia of the family Myobatrachidae, that is suitable for the development of ART systems that can be applied to the threatened and endangered myobatrachid and other amphibian species native to Australia. The aim of this study was to advance the understanding of ovulation, fertilisation and embryo nic development of Lim. tasmaniensis and in vitro manipulations of reproduction and development for use in the development of advanced ART procedures such as intracytoplasmic spermatozoon injection (ICSI), androgenesis and nuclear transfer. Ovulation in amphibians can be induced by protocols utilising natural or synthetic hormones. All protocols tested on Lim. tasmaniensis in this study required two injections and the most effective protocols continued to require a first injection of pituitary extracts to induce ovulation. The second injection was, however, successfully replaced by synthetic chorionic gonadotrophin at a threshold dosage of 100 iu and halved the number of cane toads required to source the pituitaries. A combination of LHRH and Pimozide offered a less effective protocol, that did not require the use of pituitary extracts, and avoided the risk of pathogen transfer associated with unsterilised pituitary extracts. Unfertilised eggs of Lim. tasmaniensis were exposed to media of various osmolalities to determine media effects on eggs and their surrounding jelly layers that might impact on egg viability and fertilisability. Osmolality had no effect upon the egg diameter, however, rapid swelling of the jelly layers occurred within 15 minutes of exposure to various media treatments and plateaued from 30-90 minutes without further expansion. Swelling of the jelly layers was increased in hypotonic media (2.5% SAR, H2O) and minimised in the isotonic media (100% SAR). The optimal conditions for the culture of Lim. tasmaniensis eggs were identified as a holding media of 100% SAR, followed by a medium change to 2.5% SAR at insemination. This sequence of media minimised the rate of swelling of the jelly layers prior to contact with the spermatozoa, and maximised the activation of spermatozoa and eggs throughout fertilisation and embryonic development. Embryos of Lim. tasmaniensis were cultured at four temperatures (13 C, 17 C, 23 C and 29 C), to determine the effect of temperature on cleavage and embryonic development rates. Embryonic development progressed through a sequence of stages that were not altered by changes in temperature. However cleavage rates were affected by changes in temperature as compared with normal embryonic growth at 23 C. Embryonic development was suspended at the lowest temperature (13 C) while embryonic viability was maintained. A moderate decrease in temperature (17 C) slowed cleavage, while the highest temperature (29 C) increased the cleavage rate, but decreased the embryo survival. Rates of embryonic development can be manipulated by changes in temperature and this method can be used to source blastomeres of a specific size/stage at a predetermined age or halt cleavage at specific stages for embryos or embryo derived cells to be included in ART procedures. This study produced the first report of the application of Intracytoplasmic Spermatozoon Injection (ICSI) in an Australian amphibian. Eggs that were activated by microinjection with a single spermatozoon (n=50) formed more deep, but abnormal, cleavage furrows post-injection (18/50, 36%), than surface changes (12/50, 24%). This result is in contrast to eggs injected without a spermatozoon (n=42), where the majority of eggs displayed limited surface changes (36/42, 86%), and few deep, abnormal furrows (3/42, 7%). Three advanced embryos (3/50, 6%) were produced by ICSI that developed to various stages within the culture system. Technical difficulties were encountered that prevented the generation of any metamorphs from ICSI tadpoles. Nevertheless, when these blocks to ICSI are overcome, the ICSI procedure will be both directly useful as an ART procedure in its own right, and the associated refinement of micromanipulation procedures will assist in the development of other ART procedures in Lim. tasmaniensis. A greater understanding of basic reproductive and developmental biology in Lim. tasmaniensis would greatly facilitate refinement of fertilisation by ICSI. Assisted Reproductive Technologies, in conjunction with gene banks may in the future regenerate extinct amphibian species, and assist in the recovery of declining amphibian populations nationally and worldwide.

Optimization of human papillomavirus type 16 (HPV-16) L1 expression in plants: Comparison of the suitability of different HPV-16 L1 gene variants and different cell-compartment localization

Virus-like particle-based vaccines for high-risk human papillomaviruses (HPVs) appear to have great promise; however, cell culture-derived vaccines will probably be very expensive. The optimization of expression of different codon-optimized versions of the HPV-16 L1 capsid protein gene in plants has been explored by means of transient expression from a novel suite of Agrobacterium tumefaciens binary expression vectors, which allow targeting of recombinant protein to the cytoplasm, endoplasmic reticulum (ER) or chloroplasts. A gene resynthesized to reflect human codon usage expresses better than the native gene, which expresses better than a plant-optimized gene. Moreover, chloroplast localization allows significantly higher levels of accumulation of L1 protein than does cytoplasmic localization, whilst ER retention was least successful. High levels of L1 (>17% total soluble protein) could be produced via transient expression: the protein assembled into higher-order structures visible by electron microscopy, and a concentrated extract was highly immunogenic in mice after subcutaneous injection and elicited high-titre neutralizing antibodies. Transgenic tobacco plants expressing a human codon-optimized gene linked to a chloroplast-targeting signal expressed L1 at levels up to 11% of the total soluble protein. These are the highest levels of HPV L1 expression reported for plants: these results, and the excellent immunogenicity of the product, significantly improve the prospects of making a conventional HPV vaccine by this means. © 2007 SGM.

A deletion and point mutation study of the human papillomavirus type 16 major capsid gene

Recombinant human papillomavirus (HPV) virus-like particles (VLPs) made from the major capsid protein L1 are promising vaccine candidates for use as vaccines against genital and other HPV infections, and particularly against HPV-16. However, HPV-16 genotype variants have different binding affinities for neutralising mouse Mabs raised against HPV-16 L1 VLPs. This paper analyses, using a panel of well-characterised Mabs, the effects on the antigenicity of various C- and N-terminal deletants of HPV-16 L1 made in insect cells via recombinant baculovirus, of an A → T mutation at residue 266 (A266T), and of a C → G mutation at conserved position 428 (C428G). The effects of these changes on assembly of the variant L1s were studied by electron microscopy. Binding of Mab H16:E70 to A266T was reduced by almost half in comparison to wild type L1. Retention of the C-terminal region 428-483 was critical for the binding of conformation-specific Mabs (H16:V5, H16:E70, H16:U4 and H16:9A) whereas deletion of the nuclear localisation signal (NLS) or the C428G mutation or an N-terminal deletion (residues 2-9) did not affect the antigenicity. The N-terminal deletion resulted in a mixed population of 30 and 55 nm VLPs, which differs from the same construct expressed in Escherichia coli, whereas pentamer aggregates resulted from deletion of the 428-465 region or the C428G mutation. The results have implications both for considering use of single-genotype HPV vaccines, and for design of novel second-generation vaccines. © 2006 Elsevier B.V. All rights reserved.

Maize streak virus: An old and complex 'emerging' pathogen

Maize streak virus (MSV; Genus Mastrevirus, Family Geminiviridae) occurs throughout Africa, where it causes what is probably the most serious viral crop disease on the continent. It is obligately transmitted by as many as six leafhopper species in the Genus Cicadulina, but mainly by C. mbila Naudé and C. storeyi. In addition to maize, it can infect over 80 other species in the Family Poaceae. Whereas 11 strains of MSV are currently known, only the MSV-A strain is known to cause economically significant streak disease in maize. Severe maize streak disease (MSD) manifests as pronounced, continuous parallel chlorotic streaks on leaves, with severe stunting of the affected plant and, usuallly, a failure to produce complete cobs or seed. Natural resistance to MSV in maize, and/or maize infections caused by non-maize-adapted MSV strains, can result in narrow, interrupted streaks and no obvious yield losses. MSV epidemiology is primarily governed by environmental influences on its vector species, resulting in erratic epidemics every 3-10 years. Even in epidemic years, disease incidences can vary from a few infected plants per field, with little associated yield loss, to 100% infection rates and complete yield loss. Taxonomy: The only virus species known to cause MSD is MSV, the type member of the Genus Mastrevirus in the Family Geminiviridae. In addition to the MSV-A strain, which causes the most severe form of streak disease in maize, 10 other MSV strains (MSV-B to MSV-K) are known to infect barley, wheat, oats, rye, sugarcane, millet and many wild, mostly annual, grass species. Seven other mastrevirus species, many with host and geographical ranges partially overlapping those of MSV, appear to infect primarily perennial grasses. Physical properties: MSV and all related grass mastreviruses have single-component, circular, single-stranded DNA genomes of approximately 2700 bases, encapsidated in 22 × 38-nm geminate particles comprising two incomplete T = 1 icosahedra, with 22 pentameric capsomers composed of a single 32-kDa capsid protein. Particles are generally stable in buffers of pH 4-8. Disease symptoms: In infected maize plants, streak disease initially manifests as minute, pale, circular spots on the lowest exposed portion of the youngest leaves. The only leaves that develop symptoms are those formed after infection, with older leaves remaining healthy. As the disease progresses, newer leaves emerge containing streaks up to several millimetres in length along the leaf veins, with primary veins being less affected than secondary or tertiary veins. The streaks are often fused laterally, appearing as narrow, broken, chlorotic stripes, which may extend over the entire length of severely affected leaves. Lesion colour generally varies from white to yellow, with some virus strains causing red pigmentation on maize leaves and abnormal shoot and flower bunching in grasses. Reduced photosynthesis and increased respiration usually lead to a reduction in leaf length and plant height; thus, maize plants infected at an early stage become severely stunted, producing undersized, misshapen cobs or giving no yield at all. Yield loss in susceptible maize is directly related to the time of infection: Infected seedlings produce no yield or are killed, whereas plants infected at later times are proportionately less affected. Disease control: Disease avoidance can be practised by only planting maize during the early season when viral inoculum loads are lowest. Leafhopper vectors can also be controlled with insecticides such as carbofuran. However, the development and use of streak-resistant cultivars is probably the most effective and economically viable means of preventing streak epidemics. Naturally occurring tolerance to MSV (meaning that, although plants become systemically infected, they do not suffer serious yield losses) has been found, which has primarily been attributed to a single gene, msv-1. However, other MSV resistance genes also exist and improved resistance has been achieved by concentrating these within individual maiz genotypes. Whereas true MSV immunity (meaning that plants cannot be symptomatically infected by the virus) has been achieved in lines that include multiple small-effect resistance genes together with msv-1, it has proven difficult to transfer this immunity into commercial maize genotypes. An alternative resistance strategy using genetic engineering is currently being investigated in South Africa. Useful websites: 〈http://www.mcb.uct.ac.za/MSV/mastrevirus.htm〉; 〈http://www. danforthcenter.org/iltab/geminiviridae/geminiaccess/mastrevirus/Mastrevirus. htm〉. © 2009 Blackwell Publishing Ltd.

The porcine circovirus type 1 capsid gene promoter improves antigen expression and immunogenicity in a HIV-1 plasmid vaccine

Background. One of the promising avenues for development of vaccines against Human immunodeficiency virus type 1 (HIV-1) and other human pathogens is the use of plasmid-based DNA vaccines. However, relatively large doses of plasmid must be injected for a relatively weak response. We investigated whether genome elements from Porcine circovirus type 1 (PCV-1), an apathogenic small ssDNA-containing virus, had useful expression-enhancing properties that could allow dose-sparing in a plasmid vaccine. Results. The linearised PCV-1 genome inserted 5' of the CMV promoter in the well-characterised HIV-1 plasmid vaccine pTHgrttnC increased expression of the polyantigen up to 2-fold, and elicited 3-fold higher CTL responses in mice at 10-fold lower doses than unmodified pTHgrttnC. The PCV-1 capsid gene promoter (Pcap) alone was equally effective. Enhancing activity was traced to a putative composite host transcription factor binding site and a "Conserved Late Element" transcription-enhancing sequence previously unidentified in circoviruses. Conclusions. We identified a novel PCV-1 genome-derived enhancer sequence that significantly increased antigen expression from plasmids in in vitro assays, and improved immunogenicity in mice of the HIV-1 subtype C vaccine plasmid, pTHgrttnC. This should allow significant dose sparing of, or increased responses to, this and other plasmid-based vaccines. We also report investigations of the potential of other circovirus-derived sequences to be similarly used. © 2011 Tanzer et al; licensee BioMed Central Ltd.

Investigating knowledge transfer through transnational programs between Indonesian and Australian universities : A conceptual framework

Globalization stimulated demand for transnational higher education. However, little is known about how transnational programs can facilitate knowledge transfer between the partner universities—something that is often implied in the partnership arrangement. For the purpose of this paper transnational programs will be limited to dual degree programs between Indonesian and Australian universities, which, in recent years have been rising. Nevertheless, the discussion and synthesis may have value to other transnational programs. Situating the knowledge transfer in the wider context of globalization of higher education and the current reforms in Indonesian education, the paper acknowledges the tension between the need to generate revenue and the ideals of facilitating knowledge transfer and capacity development for Indonesian universities. Drawing on a detailed review of research findings in the business and education sector literature, this paper presents a conceptual framework that may be used to analyze knowledge transfer between Indonesian and Australian universities. Such investigations delineate the significant drivers underlying transnational programs for supporting knowledge transfer. The proposed framework incorporates the types of knowledge, knowledge transfer mechanisms, and the processes. It also considers how the inter-university antecedents may affect the potential for knowledge transfer between the universities. A discussion of how the framework may be used to implemented knowledge transfer concludes this paper.

Reduced expression of retinoblastoma gene product (pRB) and high expression of p53 are associated with poor prognosis in ovarian cancer

Paraffin sections (n = 168, 27 benign, 16 low malignant potential [LMP] and 125 malignant tumours) from epithelial ovarian tumours were evaluated immunohistochemically for expression of retinoblastoma gene product (pRB) and p53 protein, and the relationship among pRB, p53 and cyclin-dependent kinase inhibitor 2 (CDKN2) gene product p16INK4A (p16) was analysed, following our previous study of p16. Forty-one percent of the benign, 50% of the LMP and most (71%) of the malignant tumours showed high pRB expression. High expression of pRB (>50% pRB-positive cells) significantly correlated with non-mucinous histological subtypes. Reduced pRB expression, substage and residual disease were significant predictors for poor prognosis in stage I patients. All the benign and most of the LMP (81%) tumours were in either the p53-negative or low p53-positive category, but nearly half of the malignant tumours had high p53 expression. High p53 accumulation was found in non-mucinous, high grade and late stage tumours. For well-differentiated carcinomas, high p53 expression was a predictor of poor prognosis. However, even though high p53 expression was not associated with histological subtype, stage or the presence of residual disease, high p53 expression was not an independent predictor when all clinical parameters were combined. For all ovarian cancers, a close correlation was found between high p53 and high p16 expression. The relationship between the expression of pRB and p16 depended on tumour stage. In stage I tumours, high pRB was associated with low p16 reactivity. On the other hand, most advanced tumours showed both high pRB and high p16 reactivity. Int. J. Cancer 74:407–415, 1997. © 1997 Wiley-Liss, Inc.

Increased expression of cyclin-dependent kinase inhibitor 2 (CDKN2A) gene product P16INK4A in ovarian cancer is associated with progression and unfavourable prognosis

Paraffin sections from 190 epithelial ovarian tumours, including 159 malignant and 31 benign epithelial tumours, were analysed immunohistochemically for expression of cyclin-dependent kinase inhibitor 2 (CDKN2A) gene product p16INK4A (p16). Most benign tumours showed no p16 expression in the tumour cells, whereas only 11% of malignant cancers were p16 negative. A high proportion of p16-positive tumour cells was associated with advanced stage and grade, and with poor prognosis in cancer patients. For FIGO stage 1 tumours, a high proportion of p16-positive tumour cells was associated with poorer survival, suggesting that accumulation of p16 is an early event of ovarian tumorigenesis. In contrast to tumour cells, high expression of p16 in the surrounding stromal cells was not associated with the stage and grade, but was associated with longer survival. When all parameters were combined in multivariate analysis, high p16 expression in stromal cells was not an independent predictor for survival, indicating that low p16 expression in stromal cells is associated with other markers of tumour progression. High expression of p16 survival in the stromal cells of tumours from long-term survivors suggests that tumour growth is limited to some extent by factors associated with p16 expression in the matrix.

Modeling heat and mass transfer process during convection drying of fruit

Fruit drying is a process of removing moisture to preserve fruits by preventing microbial spoilage. It increases shelf life, reduce weight and volume thus minimize packing, storage, and transportation cost and enable storage of food under ambient environment. But, it is a complex process which involves combination of heat and mass transfer and physical property change and shrinkage of the material. In this background, the aim of this paper to develop a mathematical model to simulate coupled heat and mass transfer during convective drying of fruit. This model can be used predict the temperature and moisture distribution inside the fruits during drying. Two models were developed considering shrinkage dependent and temperature dependent moisture diffusivity and the results were compared. The governing equations of heat and mass transfer are solved and a parametric study has been done with Comsol Multiphysics 4.3. The predicted results were validated with experimental data.

Using social media to facilitate knowledge transfer in complex engineering environments : a primer for educators

While highly cohesive groups are potentially advantageous they are also often correlated with the emergence of knowledge and information silos based around those same functional or occupational clusters. Consequently, an essential challenge for engineering organisations wishing to overcome informational silos is to implement mechanisms that facilitate, encourage and sustain interactions between otherwise disconnected groups. This paper acts as a primer for those seeking to gain an understanding of the design, functionality and utility of a suite of software tools generically termed social media technologies in the context of optimising the management of tacit engineering knowledge. Underpinned by knowledge management theory and using detailed case examples, this paper explores how social media technologies achieve such goals, allowing for the transfer of knowledge by tapping into the tacit and explicit knowledge of disparate groups in complex engineering environments.

Stromal upregulation of lateral epithelial adhesions : gene expression analysis of signalling pathways in prostate epithelium

BACKGROUND: Stromal signalling increases the lateral cell adhesions of prostate epithelial cells grown in 3D culture. The aim of this study was to use microarray analysis to identify significant epithelial signalling pathways and genes in this process. METHODS: Microarray analysis was used to identify genes that were differentially expressed when epithelial cells were grown in 3D Matrigel culture with stromal co-culture compared to without stroma. Two culture models were employed: primary epithelial cells (ten samples) and an epithelial cell line (three experiments). A separate microarray analysis was performed on each model system and then compared to identify tissue-relevant genes in a cell line model. RESULTS: TGF beta signalling was significantly ranked for both model systems and in both models the TGF beta signalling gene SOX4 was significantly down regulated. Analysis of all differentially expressed genes to identify genes that were common to both models found several morphology related gene clusters; actin binding (DIAPH2, FHOD3, ABLIM1, TMOD4, MYH10), GTPase activator activity (BCR, MYH10), cytoskeleton (MAP2, MYH10, TMOD4, FHOD3), protein binding (ITGA6, CD44), proteinaceous extracellular matrix (NID2, CILP2), ion channel/ ion transporter activity (CACNA1C, CACNB2, KCNH2, SLC8A1, SLC39A9) and genes associated with developmental pathways (POFUT1, FZD2, HOXA5, IRX2, FGF11, SOX4, SMARCC1). CONCLUSIONS: In 3D prostate cultures, stromal cells increase lateral epithelial cell adhesions. We show that this morphological effect is associated with gene expression changes to TGF beta signalling, cytoskeleton and anion activity.

Gene expression profile of primary prostate epithelial and stromal cells in response to sulforaphane or iberin exposure

BACKGROUND: Broccoli consumption has been associated with a reduced risk of prostate cancer. Isothiocyanates (ITCs) derived from glucosinolates that accumulate in broccoli are dietary compounds that may mediate these health effects. Sulforaphane (SF, 4-methylsulphinylbutyl ITC) derives from heading broccoli (calabrese) and iberin (IB, 3-methylsulphinypropyl ITC) from sprouting broccoli. While there are many studies regarding the biological activity of SF, mainly undertaken with cancerous cells, there are few studies associated with IB. METHODS: Primary epithelial and stromal cells were derived from benign prostatic hyperplasia tissue. Affymetrix U133 Plus 2.0 whole genome arrays were used to compare global gene expression between these cells, and to quantify changes in gene expression following exposure to physiologically appropriate concentrations of SF and IB. Ontology and pathway analyses were used to interpret results. Changes in expression of a subset of genes were confirmed by real-time RT-PCR. RESULTS: Global gene expression profiling identified epithelial and stromal-specific gene expression profiles. SF induced more changes in epithelial cells, whereas IB was more effective in stromal cells. Although IB and SF induced different changes in gene expression in both epithelial and stromal cells, these were associated with similar pathways, such as cell cycle and detoxification. Both ITCs increased expression of PLAGL1, a tumor suppressor gene, in stromal cells and suppressed expression of the putative tumor promoting genes IFITM1, CSPG2, and VIM in epithelial cells. CONCLUSION: These data suggest that IB and SF both alter genes associated with cancer prevention, and IB should be investigated further as a potential chemopreventative agent.

The phylogeny of Ureaplasma urealyticum based on the mba gene fragment

Sequencing of mba gene fragments of reference strains of Ureaplasma urealyticum serovars 1, 3, 6, 14, in addition to 33 clinical U. urealyticum isolates is reported. A phylogenetic tree deduced from an alignment of these sequences clearly demonstrates two major clusters (confidence limit 100%), which equate to the parvo and T960 biovars, and five types which we have designated mba 1, 3, 6, 8 and X. These relationships are supported by bootstrap analysis. Polymorphisms within the mba fragment of types mba 1, 3, and 6 were used to define nine subtypes (mba 1a, 1b, 3a, 3b, 3c, 3d, 3e, 6a, and 6b) thus facilitating high resolution typing of U. urealyticum. Inclusion of the reference strains for serovars 1, 3, 6, and 8 in the mba typing scheme showed that the results of this analysis are broadly consistent with currently accepted serotyping. In addition a ure gene fragment from nine of the clinical isolates was amplified and sequenced. Comparisons of the sequences clearly distinguished the two biovars of U. urealyticum; however this fragment was invariant within the parvo biovar. This study has shown that the sequence of the mba can reveal the fine details of the relationships between U. urealyticum isolates and also supports the significant evolutionary gap between the two biovars.

Transnational higher education programs for facilitating inter-university knowledge transfer : University of Indonesia’s experience

This paper explores the impacts and extent of knowledge transfer (KT) in an undergraduate engineering transnational program with an Australian university partner at the University of Indonesia (UI) using an inter-university KT conceptual framework (Sutrisno, Lisana, & Pillay 2012). For the purpose of this paper, the opportunity for KT in curriculum design is examined. Given the explicit nature of curriculum knowledge, assessing each partner’s curriculum was pivotal in allowing UI to enrich its own curriculum. The KT mechanism of face-to-face contact between Indonesian and Australian academics led to not only transfer of knowledge related to the curriculum of the undergraduate program but also to other cooperation beyond the transnational program in the form of joint research and joint supervision of post-graduate theses. Positive inter-university dynamics, such as trust and willingness to work together between the partners were underpinned by the presence of key actors from both sides at the earlier stages of the partnership. Retrospectively exploring the KT process in the UI’s transnational programs with its Australian partner suggests that there have been both structured and unstructured mechanisms, highlighting the ubiquitous and unbounded nature of KT between universities. While initially successful in facilitating KT, due to rapid succession of persons in charge of the program and the increasing focus on revenue generation, the useful lessons and practices unfortunately are being lost. Although the intention to use the transnational program for KT was always implied, it gradually was overlooked by newer staff members. Based on UI’s experience as the first provider of transnational program in Indonesia and other similar cases in China, seemingly transnational programs driven by short-term immediate financial return are unsuccessful in facilitating KT due to sensitivities to unfavourable economic situation. Those that remain operational and contribute to knowledge exchange between the partners apparently have genuine long-term engagement objective.