Optimization of chimeric HIV-1 virus-like particle production in a baculovirus-insect cell expression system

A baculovirus-insect cell expression system potentially provides the means to produce prophylactic HIV-1 virus-like particle (VLP) vaccines inexpensively and in large quantities. However, the system must be optimized to maximize yields and increase process efficiency. In this study, we optimized the production of two novel, chimeric HIV-1 VLP vaccine candidates (GagRT and GagTN) in insect cells. This was done by monitoring the effects of four specific factors on VLP expression: these were insect cell line, cell density, multiplicity of infection (MOI), and infection time. The use of western blots, Gag p24 ELISA, and four-factorial ANOVA allowed the determination of the most favorable conditions for chimeric VLP production, as well as which factors affected VLP expression most significantly. Both VLP vaccine candidates favored similar optimal conditions, demonstrating higher yields of VLPs when produced in the Trichoplusia ni Pro insect cell line, at a cell density of 1 × 106 cells/mL, and an infection time of 96 h post infection. It was found that cell density and infection time were major influencing factors, but that MOI did not affect VLP expression significantly. This work provides a potentially valuable guideline for HIV-1 protein vaccine optimization, as well as for general optimization of a baculovirus-based expression system to produce complex recombinant proteins. © 2009 American Institute of Chemical Engineers.

HIV-1 subtype C Pr55gag virus-like particle vaccine efficiently boosts baboons primed with a matched DNA vaccine

A DNA vaccine expressing human immunodeficiency virus type 1 (HIV-1) southern African subtype C Gag (pTHGag) and a recombinant baculovirus Pr55gag virus-like particle prepared using a subtype C Pr55gag protein (Gag VLP) was tested in a prime-boost inoculation regimen in Chacma baboons. The response of five baboons to Gag peptides in a gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay after three pTHGag immunizations ranged from 100 to 515 spot-forming units (s.f.u.) per 106 peripheral blood mononuclear cells (PBMCs), whilst the response of two baboons to the Gag VLP vaccine ranged from 415 to 465 s.f.u. per 106 PBMCs. An increase in the Gag-specific response to a range of 775-3583 s.f.u. per 106 PBMCs was achieved by boosting with Gag VLPs the five baboons that were primed with pTHGag. No improvement in Gag responses was achieved in this prime-boost inoculation regimen by increasing the number of pTHGag inoculations to six. IFN-γ responses were mapped to several peptides, some of which have been reported to be targeted by PBMCs from HIV-1 subtype C-infected individuals. Gag VLPs, given as a single-modality regimen, induced a predominantly CD8+ T-cell IFN-γ response and interleukin-2 was a major cytokine within a mix of predominantly Th1 cytokines produced by a DNA-VLP prime-boost modality. The prime-boost inoculation regimen induced high serum p24 antibody titres in all baboons, which were several fold above that induced by the individual vaccines. Overall, this study demonstrated that these DNA prime/VLP boost vaccine regimens are highly immunogenic in baboons, inducing high-magnitude and broad multifunctional responses, providing support for the development of these products for clinical trials. © 2008 SGM.

Therapeutic immunisation of rabbits with cottontail rabbit papillomavirus (CRPV) virus-like particles (VLP) induces regression of established papillomas

There is overwhelming evidence that persistent infection with high-risk human papillomaviruses (HR-HPV) is the main risk factor for invasive cancer of the cervix. Due to this global public health burden, two prophylactic HPV L1 virus-like particles (VLP) vaccines have been developed. While these vaccines have demonstrated excellent type-specific prevention of infection by the homologous vaccine types (high and low risk HPV types), no data have been reported on the therapeutic effects in people already infected with the low-risk HPV type. In this study we explored whether regression of CRPV-induced papillomas could be achieved following immunisation of out-bred New Zealand White rabbits with CRPV VLPs. Rabbits immunised with CRPV VLPs had papillomas that were significantly smaller compared to the negative control rabbit group (P ≤ 0.05). This data demonstrates the therapeutic potential of PV VLPs in a well-understood animal model with potential important implications for human therapeutic vaccination for low-risk HPVs. © 2008 Govan et al; licensee BioMed Central Ltd.

An investigation into the use of human papillomavirus type 16 virus-like particles as a delivery vector system for foreign proteins: N- and C-terminal fusion of GFP to the L1 and L2 capsid proteins

Development of vaccine strategies against human papillomavirus (HPV), which causes cervical cancer, is a priority. We investigated the use of virus-like particles (VLPs) of the most prevalent type, HPV-16, as carriers of foreign proteins. Green fluorescent protein (GFP) was fused to the N or C terminus of both L1 and L2, with L2 chimeras being co-expressed with native L1. Purified chimaeric VLPs were comparable in size (∼55 nm) to native HPV VLPs. Conformation-specific monoclonal antibodies (Mabs) bound to the VLPs, thereby indicating that they possibly retain their antigenicity. In addition, all of the VLPs encapsidated DNA in the range of 6-8 kb. © 2007 Springer-Verlag.

The experiences of novices in an enhanced graduated driver licensing program in Queensland, Australia

- Young novice drivers - The Queensland graduated driver licensing (GDL) context - Pre-July 2007 (‘Original-GDL’) - Post-July 2007 (‘Enhanced-GDL’) - Experiences of Learners in Queensland’s enhanced-GDL program - Pre-/post-July 2007 comparison - Post-July 2007 only - Implications - Strengths and limitations

Colour and Camp Bring Shakespeare’s As You Like It to Life

As You Like It By Shakespeare. La Boite Theatre Company, Brisbane, February 24. DURING the past three years, La Boite Theatre Company has started each season with a modern adaptation of a Shakespearean favourite. This time, director David Bertold's choice is not a tragedy but As You Like It, a comedy about love's twists and turns in which a strong female figure, Rosalind, takes the leading role...

Living a life with a physical impairment in Taiwan : "It is more than just the impairment"

This thesis is concerned with understanding what it is like to live with a physical impairment in Taiwan. Constructionism was used as the epistemological stance to guide the study and Heideggerian interpretive phenomenology was used as the theoretical perspective. Information was gained through a series of in-depth interviews with seven Taiwanese adults with a physical impairment living in the community. They were recruited from Yunlin and Tainan Counties in Taiwan. Study participants were seen as research partners who had expertise in understanding disability, and the researcher was seen as a learner. Grounded theory principles were used to develop the theory "it is more than just the impairment" from the information provided by the participants. According to their descriptions of how they lived their lives, participants are grouped into three clusters. These are ‘fortress ladies’, ‘social networkers’ and ‘the mind man’. The grounded theory developed portrays their lives, providing a vivid picture of living a life with a physical impairment in Taiwan. The study’s findings contribute to three main areas. First, as an occupational therapist and with my growing understanding of disability learned from the study participants, I recognize the agency of people with an impairment and their expertise in disability. Thus, I argue the need for health professionals to build alliances with them, and suggest ways to achieve such a relationship. Second, I propose the developed conceptual framework is suitable for exploring lived experience in other research areas; I discuss the implications of the subtle interactions between impaired people’s body and mind; I also present three impressive lived experiences provided by study participants as exemplars of the findings, and these form the foundation for discussion. Finally, the development of "it is more than just the impairment" provides a basis from which to theorize disability in a more holistic way.

Identities and transformational experiences for quantitative problem solving : gender comparisons of first-year university science students

Women are underrepresented in science, technology, engineering and mathematics (STEM) areas in university settings; however this may be the result of attitude rather than aptitude. There is widespread agreement that quantitative problem-solving is essential for graduate competence and preparedness in science and other STEM subjects. The research question addresses the identities and transformative experiences (experiential, perception, & motivation) of both male and female university science students in quantitative problem solving. This study used surveys to investigate first-year university students’ (231 females and 198 males) perceptions of their quantitative problem solving. Stata (statistical analysis package version 11) analysed gender differences in quantitative problem solving using descriptive and inferential statistics. Males perceived themselves with a higher mathematics identity than females. Results showed that there was statistical significance (p<0.05) between the genders on 21 of the 30 survey items associated with transformative experiences. Males appeared to have a willingness to be involved in quantitative problem solving outside their science coursework requirements. Positive attitudes towards STEM-type subjects may need to be nurtured in females before arriving in the university setting (e.g., high school or earlier). Females also need equitable STEM education opportunities such as conversations or activities outside school with family and friends to develop more positive attitudes in these fields.

Examining preservice teachers’ applied learning experiences in the Teacher Education Done Differently (TEDD) project

Reviews have criticised universities for not embedding sufficient praxis for preparing preservice teachers for the profession. The Teacher Education Done Differently (TEDD) project explored praxis development for preservice teachers within existing university coursework. This mixed-method investigation involved an analysis of multiple case studies with preservice teacher involvement in university programs, namely: Ed Start for practicum I (n=26), III (n=23), and IV (n=12); Move It Use It (Health and Physical Education program; n=38), Studies of Society and its Environment (SOSE, n=24), and Science in Schools (n=38). The project included preservice teachers teaching primary students at the campus site in gifted education (the B-GR8 program, n=22). The percentage range for preservice teacher agreement of their praxis development leading up to practicum I, III, and IV was between 91-100% with a high mean score range (4.26-5.00). Other university units had similar findings except for SOSE (i.e., percentage range: 10-86%; M range: 2.33-4.00; SD range: 0.55-1.32). Qualitative data presented an understanding of the praxis development leading to the conclusion that additional applied learning experiences as lead-up days for field experiences and as avenues for exploring the teaching of specific subject areas presented opportunities for enhancing praxis.

Australian new graduate experiences during their transition program in a rural/regional acute setting

The transition process from student to Registered Nurse has been recognised as an important yet challenging time for newly graduated nurses. Knowledge about this experience from the nurse’s perspective, particularly in a rural setting, is limited. This paper reports the findings of a qualitative study of the experiences of newly graduated nurses working in a rural acute care facility in New South Wales. The study examined, from the perspective of the new nurse, the orientation and support which can help to facilitate the transition from student to registered nurse. Four themes emerged which were being supported, being challenged, reflections on being a new graduate, and reflections on a rural new graduate program. These findings contribute to what is know about the transition of new graduates in a rural facility and have implications for program improvements, specifically within the rural acute care environment. The findings are also relevant to students considering rural employment on graduation and for the recruitment and retention of New Graduate Registered Nurses in rural areas.

Negotiating : experiences of community nurses when contracting with clients

A community nurse is required to have excellent interpersonal, teaching, collaborative and clinical skills in order to develop effective individualised client care contracts. Using a descriptive qualitative design data was collected from two focus groups of fourteen community nurses to explore the issues surrounding negotiating and contracting client care contracts from the perspective of community nurses. Thematic analysis revealed three themes: ‘assessment of needs’, ‘education towards enablement’, and ‘negotiation’. ‘Assessment of needs’ identified that community nurses assess both the client’s requirements for health care as well as the ability of the nurse to provide that care. ‘Education towards enablement’ described that education of the client is a common strategy used by community nurses to establish realistic goals of health care as part of developing an ongoing care plan. The final theme, ‘negotiation’, involved an informed agreement between the client and the community nurse which forms the origin of the care contract that will direct the partnership between the client and the nurse. Of importance for community nurses is that development of successful person-centred care contracts requires skillful negotiation of care that strikes the balance between the needs of the client and the ability of the nurse to meet those needs.

The politics of gang research in New Zealand

Like many other Western jurisdictions over the past sixty years, New Zealand has had to contend with episodes of moral panic regarding the activities of youth gangs. The most recent episode occurred in 2005-2007 and was spurred by a perceived escalation in inter-gang conflict and violence in the Counties Manukau areas within greater Auckland, New Zealand. This particular episode was unique in the New Zealand context for the level of attention given to youth gangs by the government and policy makers. This paper reports on the authors’ experiences of carrying out research on the youth gang situation inCounties Manukauas part of an inter-agency project to develop a response to gang-related violence. Particular attention is paid to the ways in which government officials attempted to mould the research process and findings to suit an already emerging policy framework, predicated on supporting ‘business as usual’, at the expense of research participants calls for great autonomy to develop and delivery appropriate youth services to their communities.

Positive experiences for participants in suicide bereavement groups : a grounded theory model of elements that contribute to the process of adjusting to the loss

Grounded Theory was used to examine the experiences of 13 participants who had attended psycho-educational support groups for those bereaved by suicide. Results demonstrated core and central categories which fit well with group therapeutic factors developed by Yalom (1995) and emphasised the importance of universality, imparting information and instilling hope, catharsis and self-disclosure, and broader meaning making processes surrounding acceptance or adjustment. Participants were commonly engaged in a lengthy process of oscillating between loss oriented and restoration focused reappraisals. The functional experience of the group comprised feeling normal within the group, providing a sense of permission to feel and to express emotions and thoughts and to bestow meaning. Structural variables of information and guidance and different perspectives on the suicide and bereavement were gained from other participants, the facilitators, group content and process. Personal changes, including in relationships and in their sense of self, assisted participants to develop an altered and more positive personal narrative.

Generation and characterisation of Cisplatin-resistant non-small cell lung cancer cell lines displaying a stem-like signature

Introduction: Inherent and acquired cisplatin resistance reduces the effectiveness of this agent in the management of non-small cell lung cancer (NSCLC). Understanding the molecular mechanisms underlying this process may result in the development of novel agents to enhance the sensitivity of cisplatin. Methods: An isogenic model of cisplatin resistance was generated in a panel of NSCLC cell lines (A549, SKMES-1, MOR, H460). Over a period of twelve months, cisplatin resistant (CisR) cell lines were derived from original, age-matched parent cells (PT) and subsequently characterized. Proliferation (MTT) and clonogenic survival assays (crystal violet) were carried out between PT and CisR cells. Cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis. A panel of cancer stem cell and pluripotent markers was examined in addition to the EMT proteins, c-Met and β-catenin. Cisplatin-DNA adduct formation, DNA damage (γH2AX) and cellular platinum uptake (ICP-MS) was also assessed. Results: Characterisation studies demonstrated a decreased proliferative capacity of lung tumour cells in response to cisplatin, increased resistance to cisplatin-induced cell death, accumulation of resistant cells in the G0/G1 phase of the cell cycle and enhanced clonogenic survival ability. Moreover, resistant cells displayed a putative stem-like signature with increased expression of CD133+/CD44+cells and increased ALDH activity relative to their corresponding parental cells. The stem cell markers, Nanog, Oct-4 and SOX-2, were significantly upregulated as were the EMT markers, c-Met and β-catenin. While resistant sublines demonstrated decreased uptake of cisplatin in response to treatment, reduced cisplatin-GpG DNA adduct formation and significantly decreased γH2AX foci were observed compared to parental cell lines. Conclusion: Our results identified cisplatin resistant subpopulations of NSCLC cells with a putative stem-like signature, providing a further understanding of the cellular events associated with the cisplatin resistance phenotype in lung cancer. © 2013 Barr et al.

Differentiating first episode substance induced and primary psychotic disorders with concurrent substance use in young people

Objective: Substance use is common in first-episode psychosis, and complicates the accurate diagnosis and treatment of the disorder. The differentiation of substance-induced psychotic disorders (SIPD) from primary psychotic disorders (PPD) is particularly challenging. This cross-sectional study compares the clinical, substance use and functional characteristics of substance using first episode psychosis patients diagnosed with a SIPD and PPD. Method: Participants were 61 young people (15-24 years) admitted to a psychiatric inpatient service with first episode psychosis, reporting substance use in the past month. Diagnosis was determined using the Psychiatric Research Interview for DSM-IV Substance and Mental disorders (PRISM-IV). Measures of clinical (severity of psychotic symptoms, level of insight, history of trauma), substance use (frequency/quantity, severity) and social and occupational functioning were also administered. Results: The PRISM-IV differentially diagnosed 56% of first episode patients with a SIPD and 44% with a PPD. Those with a SIPD had higher rates of substance use and disorders, higher levels of insight, were more likely to have a forensic and trauma history and had more severe hostility and anxious symptoms than those with a PPD. Logistic regression analysis indicated a family history of psychosis, trauma history and current cannabis dependence were the strongest predictors of a SIPD. Almost 80% of diagnostic predictions of a SIPD were accurate using this model. Conclusions: This clinical profile of SIPD could help to facilitate the accurate diagnosis and treatment of SIPD versus PPD in young people with first episode psychosis admitted to an inpatient psychiatric service.