An extracellular signal-regulated kinase 2 survival pathway mediates resistance of human mesothelioma cells to asbestos-induced injury

We hypothesized that normal human mesothelial cells acquire resistance to asbestos-induced toxicity via induction of one or more epidermal growth factor receptor (EGFR) - linked survival pathways (phosphoinositol-3-kinase/AKT/ mammalian target of rapamycin and extracellular signal - regulated kinase [ERK] 1/2) during simian virus 40 (SV40) transformation and carcinogenesis. Both isolated HKNM-2 mesothelial cells and a telomerase-immortalized mesothelial line (LP9/TERT-1) were more sensitive to crocidolite asbestos toxicity than an SV40 Tag-immortalized mesothelial line (MET5A) and malignant mesothelioma cell lines (HMESO and PPM Mill). Whereas increases in phosphorylation of AKT (pAKT) were observed in MET5A cells in response to asbestos, LP9/TERT-1 cells exhibited dose-related decreases in pAKT levels. Pretreatment with an EGFR phosphorylation or mitogen-activated protein kinase kinase 1/2 inhibitor abrogated asbestos-induced phosphorylated ERK (pERK) 1/2 levels in both LP9/TERT-1 and MET5A cells as well as increases in pAKT levels in MET5A cells. Transient transfection of small interfering RNAs targeting ERK1, ERK2, or AKT revealed that ERK1/2 pathways were involved in cell death by asbestos in both cell lines. Asbestos-resistant HMESO or PPM Mill cells with high endogenous levels of ERKs or AKT did not show dose-responsive increases in pERK1/ERK1, pERK2/ERK2, or pAKT/AKT levels by asbestos. However, small hairpin ERK2 stable cell lines created from both malignant mesothelioma lines were more sensitive to asbestos toxicity than shERK1 and shControl lines, and exhibited unique, tumor-specific changes in endogenous cell death - related gene expression. Our results suggest that EGFR phosphorylation is causally linkedto pERK and pAKT activation by asbestos in normal and SV40 Tag - immortalized human mesothelial cells. They also indicate that ERK2 plays a role in modulating asbestos toxicity by regulating genes critical to cell injury and survival that are differentially expressed in human mesotheliomas.

Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA

Many examples of extreme virus resistance and posttranscriptional gene silencing of endogenous or reporter genes have been described in transgenic plants containing sense or antisense transgenes. In these cases of either cosuppression or antisense suppression, there appears to be induction of a surveillance system within the plant that specifically degrades both the transgene and target RNAs. We show that transforming plants with virus or reporter gene constructs that produce RNAs capable of duplex formation confer virus immunity or gene silencing on the plants. This was accomplished by using transcripts from one sense gene and one antisense gene colocated in the plant genome, a single transcript that has self-complementarity, or sense and antisense transcripts from genes brought together by crossing. A model is presented that is consistent with our data and those of other workers, describing the processes of induction and execution of posttranscriptional gene silencing.

Production of transgenic rice with rice ragged stunt virus synthetic resistance genes

Rice ragged stunt virus (RRSV) is an important pathogen of rice affecting its cultivation in South and South East Asia. An approach based on pathogen derived resistance (PDR) was used to produce RRSV resistant rice cultivars. Sequences from the coding region of RRSV genome segments 7 and 10 (non-structural genes), and 5, 8 and 9 (structural genes) were placed in sense or antisense orientation behind the plant expression promoters CaMV35S, RolC, Ubil, Actl and RBTV. Rice cultivars Taipei 309 and Chinsurah Boro II were transformed by biolistic and/or Agrobacterium-mediated delivery of one or more of these PDR gene constructs. A large number of transgenic lines were produced from calli derived from mature or immature embryos, co-bombarded with the marker gene hph encoding hygromycin resistance and RRSV PDR genes or co-cultivated with strains having the binary vector containing these two genes. Both Mendelian and non-Mendelian segregations were observed in transgenic progeny, especially with transgenic lines produced by biolistics. Preliminary tests conducted in China on selected transgenic lines indicate that plants with RRSV segment 5 antisense PDR gene confer RRSV resistance.

The Yd2 resistance to barley yellow dwarf virus is effective in barley plants but not in their leaf protoplasts

The Yd2 gene for “resistance” to barley yellow dwarf virus (BYDV) has been widely used in barley (Hordeum vulgare). We have tested Australian isolates of BYDV of varying severity against barley genotypes with and without the Yd2 gene and report here a positive relationship between symptoms and virus levels determined by ELISA. Cultivar Shannon is the result of backcrossing the resistant line CI 3208 to cultivar Proctor, a susceptible line. It appears to be intermediate in reaction to BYDV between Proctor and CI 3208, although it carries the major gene, Yd2. Unlike the whole plant studies, no significant differences were observed with regard to the ability of protoplasts derived from these various genotypes to support BYDV replication. It is therefore demonstrated for the first time that the Yd2 gene is not among the small number of resistance genes which are effective against virus replication in isolated protoplasts.

Agrobacterium tumefaciens-mediated transformation of an elite Australian barley cultivar with virus resistance and reporter genes

Efficient transformation of barley cv. Schooner was achieved using Agrobacterium delivery, hygromycin or bialaphos selection and embryogenic callus. Using this system, transgenic plants were generated that contained either the green fluorescent protein gene, or transgenes derived from barley yellow dwarf (BYDV) and cereal yellow dwarf (CYDV) viruses. Many of these plants contained 1-3 transgene copies that were inherited in a simple Mendelian manner. Some plants containing BYDV and/or CYDV derived transgenes showed reduced virus symptoms and rates of viral replication when challenged with the appropriate virus. The ability to transform Schooner is a significant advance for the Australian barley industry, as this elite malting variety is, and has for the last 15 years been, the most widely grown barley variety in eastern Australia.

Virus resistance and gene silencing : killing the messenger

On occasion, virus-derived transgenes in plants can be poorly expressed and yet provide excellent virus resistance, and transgene constructs designed to supplement the expression of endogenous genes can have the effect of co-suppressing themselves and the endogenous genes. These two phenomena appear to result from the same post-transcriptional silencing mechanism, which operates by targeted-RNA degradation. Recent research into RNA-mediated virus resistance and co-suppression has provided insights into the interactions between plant viruses and their hosts, and spawned several models to explain the phenomenon.

A rapid and simple method of assaying plants transformed with hygromycin or PPT resistance genes

A very simple leaf assay is described that rapidly and reliably identifies transgenic plants expressing the hygromycin resistance gene, hph or the phosphinothricin resistance gene, bar. Leaf tips were cut from plants propagated either in the glasshouse or in tissue culture and the cut surface embedded in solid medium containing the appropriate selective agent. Non-transgenic barley or rice leaf tips had noticeable symptoms of either bleaching or necrosis after three days on the medium and were completely bleached or necrotic after one week. Transgenic leaf tips remained green and healthy over this period. This gave unambiguous discrimination between transgenic and non-transgenic plants. The leaf assay was also effective for dicot plants tested (tobacco and peas).

The human cost of change : tales from the campus about personal change fatigue, resistance, and resilience

For the last decade, one question has haunted me: what helps people to cope with large-scale organisational change in their workplace? This study explores the construct of personal change resilience, and its potential for identifying solutions to the problems of change fatigue and change resistance. The thesis has emerged from the fields of change management, leadership, training, mentoring, evaluation, management and trust within the context of higher education in Australia at the beginning of the twenty-first century. In this thesis I present a theoretical model of the factors to consider in increasing peoples’ personal change resilience as they navigate large-scale organisational change at work, thereby closing a gap in the literature on the construct of change resilience. The model presented is based on both the literature in the realms of business and education, and on the findings of the research. In this thesis, an autoethnographic case study of two Australian university projects is presented as one narrative, resulting in a methodological step forward in the use of multiple research participants’ stories in the development of a single narrative. The findings describe the experiences of workers in higher education and emphasise the importance of considerate management in the achievement of positive experiences of organisational change. This research makes a significant contribution to new knowledge in three ways. First, it closes a gap in the literature in the realm of change management around personal change resilience as a solution to the problem of change fatigue by presenting models of both change failure and personal change resilience. Second, it is methodologically innovative in the use of personae to tell the stories of multiple participants in one coherent tale presented as a work of ethnographic fiction seen through an autoethnographic lens. By doing so, it develops a methodology for giving a voice to those to whom change is done in the workplace. Third, it provides a perspective on organisational change management from the view of the actual workers affected by change, thereby adding to the literature that currently exists, which is based on the views of those with responsibility for leading or managing change rather than those it affects. This thesis is intended as a practical starting point for conversations by actual change managers in higher education, and it is written in such a way as to help them see how theory can be applied in real life, and how empowering and enabling the actual working staff members, and engaging with them in a considerate way before, during and even after the change process, can help to make them resilient enough to cope with the change, rather than leaving them burned out or disengaged and no longer a well-functioning member of the institution. This thesis shows how considerately managed large-scale organisational change can result in positive outcomes for both the organisation and the individuals who work in it.

Resistance to exercise-induced weight loss : compensatory behavioral adaptations

In many interventions that are based on an exercise program intended to induce weight loss, the mean weight loss observed is modest and sometimes far less than what the individual expected. The individual responses are also widely variable, with some individuals losing a substantial amount of weight, others maintaining weight, and a few actually gaining weight. The media have focused on the subpopulation that loses little weight, contributing to a public perception that exercise has limited utility to cause weight loss. The purpose of the symposium was to present recent, novel data that help explain how compensatory behaviors contribute to a wide discrepancy in exercise-induced weight loss. The presentations provide evidence that some individuals adopt compensatory behaviors, that is, increased energy intake and/or reduced activity, that offset the exercise energy expenditure and limit weight loss. The challenge for both scientists and clinicians is to develop effective tools to identify which individuals are susceptible to such behaviors and to develop strategies to minimize their effect.

Effects of scaffold architecture on mechanical characteristics and osteoblast response to static and perfusion bioreactor cultures

Tissue engineering focuses on the repair and regeneration of tissues through the use of biodegradable scaffold systems that structurally support regions of injury whilst recruiting and/or stimulating cell populations to rebuild the target tissue. Within bone tissue engineering, the effects of scaffold architecture on cellular response have not been conclusively characterized in a controlled-density environment. We present a theoretical and practical assessment of the effects of polycaprolactone (PCL) scaffold architectural modifications on mechanical and flow characteristics as well as MC3T3-E1 preosteoblast cellular response in an in vitro static plate and custom-designed perfusion bioreactor model. Four scaffold architectures were contrasted, which varied in inter-layer lay-down angle and offset between layers, whilst maintaining a structural porosity of 60 ± 5%. We established that as layer angle was decreased (90° vs. 60°) and offset was introduced (0 vs. 0.5 between layers), structural stiffness, yield stress, strength, pore size and permeability decreased, whilst computational fluid dynamics-modeled wall shear stress was increased. Most significant effects were noted with layer offset. Seeding efficiencies in static culture were also dramatically increased due to offset (~45% to ~86%), with static culture exhibiting a much higher seeding efficiency than perfusion culture. Scaffold architecture had minimal effect on cell response in static culture. However, architecture influenced osteogenic differentiation in perfusion culture, likely by modifying the microfluidic environment.

Mechanical stimulation of the pro-angiogenic capacity of human fracture haematoma: Involvement of VEGF mechano-regulation

Compromised angiogenesis appears to be a major limitation in various suboptimal bone healing situations. Appropriate mechanical stimuli support blood vessel formation in vivo and improve healing outcomes. However, the mechanisms responsible for this association are unclear. To address this question, the paracrine angiogenic potential of early human fracture haematoma and its responsiveness to mechanical loading, as well as angiogenic growth factors involved, were investigated in vitro. Human haematomas were collected from healthy patients undergoing surgery within 72. h after bone fracture. The haematomas were embedded in a fibrin matrix, and cultured in a bioreactor resembling the in vivo conditions of the early phase of bone healing (20 compression, 1. Hz) over 3. days. Conditioned medium (CM) from the bioreactor was then analyzed. The matrices were also incubated in fresh medium for a further 24. h to evaluate the persistence of the effects. Growth factor (GF) concentrations were measured in the CM by ELISAs. In vitro tube formation assays were conducted on Matrigel with the HMEC-1 cell line, with or without inhibition of vascular endothelial growth factor receptor 2 (VEGFR2). Cell numbers were quantified using an MTS test. In vitro endothelial tube formation was enhanced by CM from haematomas, compared to fibrin controls. The angiogenesis regulators, vascular endothelial growth factor (VEGF) and transforming growth factor β1 (TGF-β1), were released into the haematoma CM, but not angiopoietins 1 or 2 (Ang1, 2), basic fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF). Mechanical stimulation of haematomas, but not fibrin controls, further increased the induction of tube formation by their CM. The mechanically stimulated haematoma matrices retained their elevated pro-angiogenic capacity for 24. h. The pro-angiogenic effect was cancelled by inhibition of VEGFR2 signalling. VEGF concentrations in CM tended to be elevated by mechanical stimulation; this was significant in haematomas from younger, but not from older patients. Other GFs were not mechanically regulated. In conclusion, the paracrine pro-angiogenic capacity of early human haematomas is enhanced by mechanical stimulation. This effect lasts even after removing the mechanical stimulus and appears to be VEGFR2-dependent.

Cyclic mechanical strain guides capillary-like morphology in vitro

Introduction Stretching of tissue stimulates angiogenesis but increased motion at a fracture site hinders revascularisation. In vitro studies have indicated that mechanical stimuli promote angiogenic responses in endothelial cells, but can either inhibit or enhance responses when applied directly to angiogenesis assays. We anticipated that cyclic tension applied during endothelial network assembly would increase vascular structure formation up to a certain threshold. Methods Fibroblast/HUVEC co-cultures were subjected to cyclic equibiaxial strain (1 Hz; 6 h/day; 7 days) using the FlexerCell FX-4000T system and limiting rings for simultaneous application of multiple strain magnitudes (0–13%). Cells were labelled using anti-PECAM-1, and image analysis provided measures of endothelial network length and numbers of junctions. Results Cyclic stretching had no significant effect on the total length of endothelial networks (P > 0.2) but resulted in a strain-dependent decrease in branching and localised alignments of endothelial structures, which were in turn aligned with the supporting fibroblastic construct. Conclusion The organisation of endothelial networks under cyclic strain is dominated by structural adaptation to the supporting construct. It may be that, in fracture healing, the formation and integrity of the granulation tissue and callus is ultimately critical in revascularisation and its failure under severe strain conditions.

A simulation-based large deflection and inelastic analysis of steel frames under fire

This paper presents an accurate and robust geometric and material nonlinear formulation to predict structural behaviour of unprotected steel members at elevated temperatures. A fire analysis including large displacement effects for frame structures is presented. This finite element formulation of beam-column elements is based on the plastic hinge approach to model the elasto-plastic strain-hardening material behaviour. The Newton-Raphson method allowing for the thermal-time dependent effect was employed for the solution of the non-linear governing equations for large deflection in thermal history. A combined incremental and total formulation for determining member resistance is employed in this nonlinear solution procedure for the efficient modeling of nonlinear effects. Degradation of material strength with increasing temperature is simulated by a set of temperature-stress-strain curves according to both ECCS and BS5950 Part 8, which implicitly allows for creep deformation. The effects of uniform or non-uniform temperature distribution over the section of the structural steel member are also considered. Several numerical and experimental verifications are presented.

Molecular mechanisms of cisplatin resistance in lung cancer

Cisplatin is one of the most potent anticancer agents, displaying significant clinical activity against a variety of solid tumours. To date, cisplatin-based combination treatment remains the most effective systemic chemotherapy for non-small cell lung cancer (NSCLC) patients. Unfortunately, the outcome of cisplatin therapy in NSCLC has reached a plateau due to the development of both intrinsic and acquired resistance that have become a major obstacle in the use of cisplatin in the clinical setting. The molecular mechanisms that underlie chemoresistance are largely unknown. Mechanisms of acquired resistance to cisplatin include reduced intracellular accumulation of the drug, enhanced drug inactivation by metallothionine and glutathione, increased repair activity of DNA damage, and altered expression of oncogenes and regulatory proteins. Cisplatin-induced cytotoxicity is mediated through the induction of apoptosis and cell cycle arrest as a result of cisplatin-DNA adduct formation, which in turn, activates multiple signaling pathways and mediators. These include p53, Bcl-2 family, caspases, cyclins, CDKs, MAPK and PI3K/Akt. Increased expression of anti-apoptotic genes and mutations in the intrinsic apoptotic pathway may also contribute to the inability of cells to detect DNA damage or to induce apoptosis. This chapter will provide an insight into the mechanisms involved in cisplatin resistance and a better understanding of the molecular basis of the cellular response to cisplatin-based chemotherapy in lung cancer.