A novel T-DNA vector design for selection of transgenic lines with simple transgene integration and stable transgene expression

Plants transformed with Agrobacterium frequently contain T-DNA concatamers with direct-repeat (d/r) or inverted-repeat (i/r) transgene integrations, and these repetitive T-DNA insertions are often associated with transgene silencing. To facilitate the selection of transgenic lines with simple T-DNA insertions, we constructed a binary vector (pSIV) based on the principle of hairpin RNA (hpRNA)-induced gene silencing. The vector is designed so that any transformed cells that contain more than one insertion per locus should generate hpRNA against the selective marker gene, leading to its silencing. These cells should, therefore, be sensitive to the selective agent and less likely to regenerate. Results from Arabidopsis and tobacco transformation showed that pSIV gave considerably fewer transgenic lines with repetitive insertions than did a conventional T-DNA vector (pCON). Furthermore, the transgene was more stably expressed in the pSIV plants than in the pCON plants. Rescue of plant DNA flanking sequences from pSIV plants was significantly more frequent than from pCON plants, suggesting that pSIV is potentially useful for T-DNA tagging. Our results revealed a perfect correlation between the presence of tail-to-tail inverted repeats and transgene silencing, supporting the view that read-through hpRNA transcript derived from i/r T-DNA insertions is a primary inducer of transgene silencing in plants. © CSIRO 2005.

Intranasal immunization with C. muridarum major outer membrane protein (MOMP) and cholera toxin elicits local production of neutralising IgA in the prostate

Successful control of sexually transmitted diseases (STDs) through vaccination will require the development of vaccine strategies that target protective immunity to both the female and male reproductive tracts (MRT). In the male, the immune privileged nature of the male reproductive tract provides a barrier to entry of serum immunoglobulins into the male reproductive ducts, thereby preventing the induction of protective immunity using conventional injectable vaccination techniques. In this study we investigated the potential of intranasal (IN) immunization to elicit anti-chlamydial immunity in BALB/c male mice. Intranasal immunization with Chlamydia muridarum major outer membrane protein (MOMP) admixed with cholera toxin (CT) resulted in high levels of MOMP-specific IgA in prostatic fluids (PF) and MOMP-specific IgA-secreting cells in the prostate. Prostatic fluid IgA inhibited in vitro infection of McCoy cells with C. muridarum. Using RT-PCR we also show that mRNA for the polymeric immunoglobulin receptor (PIgR), which transports IgA across mucosal epithelia, is expressed only in the prostate but not in other regions of the male reproductive ducts upstream of the prostate. These data suggest that using intranasal immunization to target IgA to the prostate may protect males against STDs while at the same time maintaining the state of immune privilege within the MRT.

Book review: ‘Cornea. Color Atlas & Synopsis of Clinical Ophthalmology. Wills Eye Institute’, by C.J. Rapuano. Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia 2012

This book is one in a series of seven atlases covering the ophthalmic sub-specialties: cornea, retina, glaucoma, oculoplastics, neuro-ophthalmology, uveitis and paediatrics. The author of Cornea and editor of the series is Christopher Rapuano, Attending Surgeon and Director of the Cornea Service at Wills Eye Hospital in Philadelphia, Pennsylvania, USA. In the introduction to the book, Rapuano states ‘The goal of this series is to provide an up-to-date clinical overview of the major areas of ophthalmology for students, residents and practitioners in all the healthcare professions’...

The first x-ray diffraction peak of water as a function of temperature in the range 4 – 63 °C

X-ray diffraction structure functions for water flowing in a 1.5 mm diameter siphon in the temperature range 4 – 63 °C were obtained using a 20 keV beam at the Australian Synchrotron. These functions were compared with structure functions obtained at the Advanced Light Source for a 0.5 mm thick sample of water in the temperature range 1 – 77 °C irradiated with an 11 keV beam. The two sets of structure functions are similar, but there are subtle differences in the shape and relative position of the two functions suggesting a possible differences between the structure of bulk and siphon water. In addition, the first structural peak (Q0) for water in a siphon, showed evidence of a step-wise increase in Q0 with increasing temperature rather than a smoothly varying increase. More experiments are required to investigate this apparent difference.

Incorporating anchored learning in a C# Intelligent Tutoring System

Learning programming is known to be difficult. One possible reason why students fail programming is related to the fact that traditional learning in the classroom places more emphasis on lecturing the material instead of applying the material to a real application. For some students, this teaching model may not catch their interest. As a result they may not give their best effort to understand the material given. Seeing how the knowledge can be applied to real life problems can increase student interest in learning. As a consequence, this will increase their effort to learn. Anchored learning that applies knowledge to solve real life problems may be the key to improving student performance. In anchored learning, it is necessary to provide resources that can be accessed by the student as they learn. These resources can be provided by creating an Intelligent Tutoring System (ITS) that can support the student when they need help or experience a problem. Unfortunately, there is no ITS developed for the programming domain that has incorporated anchored learning in its teaching system. Having an ITS that supports anchored learning will not only be able to help the student learn programming effectively but will also make the learning process more enjoyable. This research tries to help students learn C# programming using an anchored learning ITS named CSTutor. Role playing is used in CSTutor to present a real world situation where they develop their skills. A knowledge base using First Order Logic is used to represent the student's code and to give feedback and assistance accordingly.

The politics of open-access publishing : M/C Journal, Public Intellectualism, and Academic Discourses of Legitimacy

This article investigates the discourses of academic legitimacy that surround the production, consumption, and accreditation of online scholarship. Using the web-based media and cultural studies journal (http://journal.media-culture.org.au) as a case study, it examines how online scholarly journals often position themselves as occupying a space between the academic and the popular and as having a functional advantage over print-based media in promoting a spirit of public intellectualism. The current research agenda of both government and academe prioritises academic research that is efficient, self-promoting, and relevant to the public. Yet, although the cost-effectiveness and public-intellectual focus of online scholarship speak to these research priorities, online journals such as M/C Journal have occupied, and continue to occupy, an unstable position in relation to the perceived academic legitimacy of their content. Although some online scholarly journals have achieved a limited form of recognition within a system of accreditation that still privileges print-based scholarship, I argue that this, nevertheless, points to the fact that traditional textual notions of legitimate academic work continue to pervade the research agenda of an academe that increasingly promotes flexible delivery of teaching and online research initiatives.

Design of an integrated platform for V2X wireless communications and positioning supporting C-ITS safety applications

In this paper, an integrated inter-vehicles wireless communications and positioning system supporting alternate positioning techniques is proposed to meet the requirements of safety applications of Cooperative Intelligent Transportation Systems (C-ITS). Recent advances have repeatedly demonstrated that road safety problems can be to a large extent addressed via a range of technologies including wireless communications and positioning in vehicular environments. The novel communication stack utilizing a dedicated frequency spectrum (e.g. at 5.9 GHz band), known as Dedicated Short-Range Communications (DSRC), has been particularly designed for Wireless Access in Vehicular Environments (WAVE) to support safety applications in highly dynamic environments. Global Navigation Satellite Systems (GNSS) is another essential enabler to support safety on rail and roads. Although current vehicle navigation systems such as single frequency Global Positioning System (GPS) receivers can provide route guidance with 5-10 meters (road-level) position accuracy, positioning systems utilized in C-ITS must provide position solutions with lane-level and even in-lane-level accuracies based on the requirements of safety applications. This article reviews the issues and technical approaches that are involved in designing a vehicular safety communications and positioning architecture; it also provides technological solutions to further improve vehicular safety by integrating the DSRC and GNSS-based positioning technologies.

Does DE need a C? A proposal for a DE curriculum

The vision of a digital earth (DE) is continuously evolving, and the next-generation infrastructures, platforms and applications are being implemented. In this article, we attempt to initiate a debate within the DE and with affine communities about 'why' a digital earth curriculum (DEC) is needed, 'how' it should be developed, and 'what' it could look like. It is impossible to do justice to the Herculean effort of DEC development without extensive consultations with the broader community. We propose a frame for the debate (what, why, and how of a DEC) and a rationale for and elements of a curriculum for educating the coming generations of digital natives and indicate possible realizations. We particularly argue that a DEC is not a déjà vu of classical research and training agendas of geographic information science, remote sensing, and similar fields by emphasizing its unique characteristics.

Electron capture of tetracyanoethylene oxide in the gas phase. Rearrangement of the parent radical anion to form [(NC)(3)C](-). A joint experimental and ab initio study

Capture of an electron by tetracyanoethylene oxide can initiate a number of decomposition pathways. One of these decompositions yields [(NC)3C]− as the ionic product. Ab initio calculations (at the B3LYP/6-31+G∗ level of theory) indicate that the formation of [(NC)3C]− is initiated by capture of an electron into the LUMO of tetracyanoethylene oxide to yield the anion radical [(NC)2C–O–C(CN)2]−· that undergoes internal nucleophilic substitution to form intermediate [(NC)3C–OCCN]−·. This intermediate dissociates to form [(NC)3C]− (m/z 90) as the ionic product. The radical (NC)3C· has an electron affinity of 4.0 eV (385 kJ mol−1). Ab initio calculations show that [(NC)3C]− is trigonal planar with the negative charge mainly on the nitrogens. A pictorial representation of this structure is the resonance structure formed from three degenerate contributing structures (NC)2–CCN−. The other product of the reaction is nominally (NCCO)·, but there is no definitive experimental evidence to indicate whether this radical survives intact, or decomposes to NC· and CO. The overall process [(NC)2C–O–C(CN)2]−· → [(NC)3C]− + (NCCO)· is calculated to be endothermic by 21 kJ mol−1 with an overall barrier of 268 kJ mol−1.

The Credit Act Advisory System (CAAS) : conversion from an expert system prototype to a C++ commercial system

CAAS is a rule-based expert system, which provides advice on the Victorial Credit Act 1984. It is currently in commercial use, and has been developed in conjunction with a law firm. It uses an object-oriented hybrid reasoning approach. The system was initially prototyped using the expert system shell NExpert Object, and was then converted into the C++ language. In this paper we describe the advantages that this methodology has, for both commercial and research development.

Conversion of linear to rhombic C-4 in the gas phase: A joint experimental and theoretical study

Charge reversal (CR) and neutralization reionization (NR) experiments carried out on a 4-sector mass spectrometer demonstrate that isotopically labeled, linear C-4 anion rearranges upon collisional oxidation. The cations and neutrals formed in these experiments exhibit differing degrees of isotopic scrambling in their fragmentation patterns, indicative of (at least) partial isomerization of both states. Theoretical studies, employing the CCSD(T)/aug-cc-pVDZ//B3LYP/6-31G(d) level of theory, favor conversion to the rhombic C-4 isomer on both cationic and neutral potential-energy surfaces with the rhombic structures predicted to be slightly more stable than the linear forms in each case. The combination of experiment with theory indicates that the elusive rhombic C-4 is formed as a cation and as a neutral following charge stripping of linear C-4(-)

Epidermal growth factor promotes MDA-MB-231 breast cancer cell migration through a phosphatidylinositol 3'-kinase and phospholipase C-dependent mechanism

Epidermal growth factor receptor (EGFR) levels predict a poor outcome in human breast cancer and are most commonly associated with proliferative effects of epidermal growth factor (EGF), with little emphasis placed on motogenic responses to EGF. We found that MDA-MB-231 human breast cancer cells elicited a potent chemotactic response despite their complete lack of a proliferative response to EGF. Antagonists of EGFR ligation, the EGFR kinase, phosphatidylinositol 3'-kinase, and phospholipase C, but not the mitogen- activated protein kinases (extracellular signal-regulated protein kinase 1 and 2), blocked MDA-MB-231 chemotaxis. These findings suggest that EGF may influence human breast cancer progression via migratory pathways, the signaling for which appears to be dissociated, at least in part, from the proliferative pathways.

Pro-matrix metalloproteinase-2 transfection increases orthotopic primary growth and experimental metastasis of MDA-MB-231 human breast cancer cells in nude mice

The ability to activate pro-matrix metalloproteinase (pro-MMP)-2 via membrane type-MMP is a hallmark of human breast cancer cell lines that show increased invasiveness, suggesting that MMP-2 contributes to human breast cancer progression. To investigate this, we have stably transfected pro-MMP-2 into the human breast cancer cell line MDA-MB-231, which lacks MMP-2 expression but does express its cell surface activator, membrane type 1-MMP. Multiple clones were derived and shown to produce pro-MMP-2 and to activate it in response to concanavalin A. In vitro analysis showed that the pro-MMP-2-transfected clones exhibited an increased invasive potential in Boyden chamber and Matrigel outgrowth assays, compared with the parental cells or those transfected with vector only. When inoculated into the mammary fat pad of nude mice, each of the MMP-2-tranfected clones grew faster than each of the vector controls tested. After intracardiac inoculation into nude mice, pro-MMP-2-transfected clones showed a significant increase in the incidence of metastasis to brain, liver, bone, and kidney compared with the vector control clones but not lung. Increased tumor burden was seen in the primary site and in lung metastases, and a trend toward increased burden was seen in bone, however, no change was seen in brain, liver, or kidney. This data supports a role for MMP-2 in breast cancer progression, both in the growth of primary tumors and in their spread to distant organs. MMP-2 may be a useful target for breast cancer therapy when refinement of MMP inhibitors provides for MMP-specific agents.

Expression of c-ets-1 mRNA is associated with an invasive, EMT-derived phenotype in breast carcinoma cell lines

We have previously observed in vitro that some stromal proteinases (MMP- 2, MT1-MMP) were expressed or activated by invasive carcinoma cell lines exhibiting mesenchymal features, presumably acquired through an epithelial to mesenchymal transition (EMT). To examine the potential contribution of c- ets-1 to this phenotype, we have compared here the expression of c-ets-1 with invasiveness in vitro and expression of vimentin, E-cadherin, uPA, MMP-1 and MMP-3 in a panel of human breast cancer cell lines. Our results clearly demonstrate an association between c-ets-1 expression and the invasive, EMT- derived phenotype, which is typified by the expression of vimentin and the lack of E-cadherin. While absent from the two non-invasive, vimentin-negative cell lines, c-ets-1 was abundantly expressed in all the four vimentin- positive lines. However, we could not find a clear quantitative or qualitative relationship between the expression of c-ets-1 and the three proteinases known to he regulated by c-ets-1, except that when they were expressed, it was only in the invasive c-ets-1-positive lines. UPA mRNAs were found in three of the four vimentin-positive lines, MMP-1 in two of the four, and MMP-3 could not be detected in any of the cell lines. Intriguingly, MDA- MB-435 cells, which exhibit the highest metastatic potential of these cell lines in nude mice, expressed vimentin and c-ets-1, but lacked expression of these three proteinases, at least under the culture conditions employed. Taken together, our results show that c-ets-1 expression is associated with an invasive, EMT-derived phenotype in breast cancer cells, although it is apparently not sufficient to ensure the expression of uPA, MMP-1 or MMP-3, in the vimentin-positive cells. Such proteases regulation is undoubtedly qualified by the cellular context. This study therefore advances our understanding of the molecular regulation of invasiveness in EMT-associated carcinoma progression, and suggests that c-ets-1 may contribute to the invasive phenotype in carcinoma cells.