Rice ragged stunt oryzavirus genome segments S7 and S10 encode non-structural proteins of M(r) 68 025 (Pns7) and M(r) 32364 (Pns10) : brief report

The nucleotide sequences of genome segments S7 and S10 of a Thai-isolate of rice ragged stunt virus (RRSV) were determined. The 1938 bp S7 sequence contains a single large open reading frame (ORF) spanning nucleotides 20 to 1 843 that is predicted to encode a protein of M(r) 68 025. The 1 162 bp S10 sequence has a major ORF spanning nucleotides 142 to 1 032 that is predicted to encode a protein of M(r) 32364. This S10 ORF is preceded by a small ORF (nt 20-55) which is probably a minicistron. Coupled in vitro transcription-translation from the two major ORFs gave protein products of the expected sizes. However, no protein was visualised from S10 when the small ORF sequence was included. Proteins were expressed in Escherichia coli from the full length ORF of S7 (P7) and from a segment of the S10 ORF (P10) fused to the ORF of glutathione S-transferase (GST). Neither fusion protein was recognised by polyclonal antibodies raised against RRSV particles. Furthermore, polyclonal antibodies raised against GST-P7 fusion protein did not recognise any virion structural polypeptides. These data strongly suggest that the proteins P7 and P10 do not form part of RRSV particle. This is further supported by observed sequence homology (though very weak) of predicted.

A longitudinal study of the impact of chronic psychological stress on health-related quality of life and clinical biomarkers : protocol for the Australian Healthy Aging of Women Study

BACKGROUND: Despite advancements in our understanding of the importance of stress reduction in achieving good health, we still only have limited insight into the impact of stress on cellular function. Recent studies have suggested that exposure to prolonged psychological stress may alter an individual's physiological responses, and contribute to morbidity and mortality. This paper presents an overview of the study protocol we are using to examine the impact of life stressors on lifestyle factors, health-related quality of life and novel and established biomarkers of stress in midlife and older Australian women.The primary aim of this study is to explore the links between chronic psychological stress on both subjective and objective health markers in midlife and older Australian women. The study examines the extent to which exposure frightening, upsetting or stressful events such as natural disasters, illness or death of a relative, miscarriage and relationship conflict is correlated with a variety of objective and subjective health markers.Methods/design: This study is embedded within the longitudinal Healthy Aging of Women's study which has collected data from midlife and older Australian women at 5 yearly intervals since 2001, and uses the Allostastic model of women's health by Groer and colleagues in 2010. The current study expands the focus of the HOW study and will assess the impact of life stressors on quality of life and clinical biomarkers in midlife and older Australian women to explain the impact of chronic psychological stress in women. DISCUSSION: The proposed study hypothesizes that women are at increased risk of exposure to multiple or repeated stressors, some being unique to women, and the frequency and chronicity of stressors increases women's risk of adverse health outcomes. This study aims to further our understanding of the relationships between stressful life experiences, perceived quality of life, stress biomarkers, chronic illness, and health status in women.

Correlating flow induced shear stress and chondrocytes activity in micro-porous scaffold using computational fluid dynamics and rapid prototyping

Flow induced shear stress plays an important role in regulating cell growth and distribution in scaffolds. This study sought to correlate wall shear stress and chondrocytes activity for engineering design of micro-porous osteochondral grafts based on the hypothesis that it is possible to capture and discriminate between the transmitted force and cell response at the inner irregularities. Unlike common tissue engineering therapies with perfusion bioreactors in which flow-mediated stress is the controlling parameter, this work assigned the associated stress as a function of porosity to influence in vitro proliferation of chondrocytes. D-optimality criterion was used to accommodate three pore characteristics for appraisal in a mixed level fractional design of experiment (DOE); namely, pore size (4 levels), distribution pattern (2 levels) and density (3 levels). Micro-porous scaffolds (n=12) were fabricated according to the DOE using rapid prototyping of an acrylic-based bio-photopolymer. Computational fluid dynamics (CFD) models were created correspondingly and used on an idealized boundary condition with a Newtonian fluid domain to simulate the dynamic microenvironment inside the pores. In vitro condition was reproduced for the 3D printed constructs seeded by high pellet densities of human chondrocytes and cultured for 72 hours. The results showed that cell proliferation was significantly different in the constructs (p<0.05). Inlet fluid velocity of 3×10-2mms-1 and average shear stress of 5.65×10-2 Pa corresponded with increased cell proliferation for scaffolds with smaller pores in hexagonal pattern and lower densities. Although the analytical solution of a Poiseuille flow inside the pores was found insufficient for the description of the flow profile probably due to the outside flow induced turbulence, it showed that the shear stress would increase with cell growth and decrease with pore size. This correlation demonstrated the basis for determining the relation between the induced stress and chondrocyte activity to optimize microfabrication of engineered cartilaginous constructs.

Local stress-strain distribution and load transfer across cartilage matrix at micro-scale using combined microscopy-based finite element method

Articular cartilage is the load-bearing tissue that consists of proteoglycan macromolecules entrapped between collagen fibrils in a three-dimensional architecture. To date, the drudgery of searching for mathematical models to represent the biomechanics of such a system continues without providing a fitting description of its functional response to load at micro-scale level. We believe that the major complication arose when cartilage was first envisaged as a multiphasic model with distinguishable components and that quantifying those and searching for the laws that govern their interaction is inadequate. To the thesis of this paper, cartilage as a bulk is as much continuum as is the response of its components to the external stimuli. For this reason, we framed the fundamental question as to what would be the mechano-structural functionality of such a system in the total absence of one of its key constituents-proteoglycans. To answer this, hydrated normal and proteoglycan depleted samples were tested under confined compression while finite element models were reproduced, for the first time, based on the structural microarchitecture of the cross-sectional profile of the matrices. These micro-porous in silico models served as virtual transducers to produce an internal noninvasive probing mechanism beyond experimental capabilities to render the matrices micromechanics and several others properties like permeability, orientation etc. The results demonstrated that load transfer was closely related to the microarchitecture of the hyperelastic models that represent solid skeleton stress and fluid response based on the state of the collagen network with and without the swollen proteoglycans. In other words, the stress gradient during deformation was a function of the structural pattern of the network and acted in concert with the position-dependent compositional state of the matrix. This reveals that the interaction between indistinguishable components in real cartilage is superimposed by its microarchitectural state which directly influences macromechanical behavior.

Foot massage and physiological stress in people with dementia : a randomized controlled trial

Background There is increasing interest in using complementary and alternative treatments to manage behavioural and psychological symptoms of dementia such as agitation, aggression and depressed mood. Objective To compare the effect of foot massage (intervention) and quiet presence (control) on agitation and mood in people with dementia. Design A randomised controlled trial using a within-subjects, crossover design. Settings Five long-term care facilities in Brisbane, Australia. The primary outcome was the Cohen-Mansfield Agitation Inventory (CMAI) and the secondary outcome was the Observed Emotion Rating Scale (OERS). The screening and data collection research assistants, families, and care staff were blinded to participant allocation. Participants Participants of the study were 55 long-term care residents aged 74–103 years (mean age 86.5), with moderate to severe dementia and a history of agitated behaviour according to the Pittsburgh Agitation Scale. A computer-program randomised participants to 10-min foot massage (intervention) or quiet presence (control), every weekday for 3 weeks. Results A carry-over effect was identified in the data, and so the data was treated as a parallel groups RCT. The mean total CMAI increased in both groups (reflecting an increase in agitation) with this increase greater in the quiet presence group than the foot massage group (p=0.03). There was a trend towards a difference on OERS General Alertness, with a positive change in alertness for participants in the foot massage group (indicating reduced alertness) and a negative change for participants in the quiet presence group (indicating increased alertness) (F(1,51)=3.88, p=0.05, partial ή2=0.07). Conclusions The findings highlight the need for further research on the specific conditions under which massage might promote relaxation and improve mood for people with dementia. The unfamiliar research assistants and variations in usual activity may have contributed to the increase in agitation and this needs further research.

Stress, lifestyle and quality of life in midlife and older Australian women : results from the stress and the health of women study

Background Chronic psychological stress may pose a serious threat to health, although the mechanisms are not fully understood. This study examines the impact of stress on modifiable lifestyle factors, depressive symptoms, health-related quality of life (HRQOL) and chronic illness in older Australian women. Methods Cross-sectional data were collected from a random sample of 181 older adults aged 60-70 years from rural and urban areas of South-East Queensland, Australia. We used structural equation modelling to examine associations between stress, modifiable lifestyle factors, HRQoL, and chronic illness. Findings Parameter estimates show that older women who reported life stressors where they felt helpless and feared for their life (high magnitude stressors) also reported higher body mass index (p = 0.03) and more chronic illness (p <0.01). In contrast, duration of exposure to life stressors was associated with higher depressive symptom scores (CES-D, p = 0.02) and sleep disturbance scores (p <0.01). Conclusions Our findings support the link between traumatic personal histories (exposure to high magnitude stressors) and unhealthy lifestyle factors. Findings highlight the need for more research on how stress reduction healthy lifestyle and positive coping strategies can be used to reduce the effects of high magnitude stress on health-related quality of life and chronic illness.

Differential Regulation of Clusterin Isoforms by the Androgen Receptor

Clusterin (CLU) was initially reported as an androgen-repressed gene which is now shown to be an androgen-regulated ATP-independent cytoprotective molecular chaperone. CLU binds to a wide variety of client proteins to potently inhibit stress-induced protein aggregation and chaperone or stabilise conformations of proteins at times of cell stress. CLU is an enigmatic protein, being ascribed both pro- and anti-apoptotic roles. Recent evidence has shown that both secreted (sCLU) and nuclear (nCLU) isoforms can be produced, and that protein function is dependent on the sub-cellular localisation. We and others have shown that sCLU is cytoprotective, while nCLU is pro-apoptotic. It now seems likely that the apparently dichotomous functions of CLU result from the expression of different but related CLU isoforms and splice variants, and that cell survival depends in part on the relative expression of pro- versus anti-apoptotic CLU proteins. In cancer cells, increased sCLU expression is associated with increased resistance to apoptotic triggers and treatment resistance. CLU is a stress-induced protein upregulated after apoptotic triggers like androgen ablation and chemotherapy. Treatment strategies targeting stress-associated increases in sCLU expression enhance treatment-induced apoptosis and delay the emergence of androgen independence. Differential regulation of CLU isoforms and splice variants by androgens may be a pathway whereby cancer cells develop treatment resistance and evade apoptosis.

Cellular stress triggers the human topoisomerase I damage response independently of DNA damage in a p53 controlled manner

The 'human topoisomerase I (htopoI) damage response' was reported to be triggered by various kinds of DNA lesions. Also, a high and persistent level of htopoI cleavage complexes correlated with apoptosis. In the present study, we demonstrate that DNA damage-independent induction of cell death using colcemid and tumor necrosis factor is also accompanied by a strong htopoI response that correlates with the onset of apoptotic hallmarks. Consequently, these results suggest that htopoI cleavage complex formation may be caused by signaling pathways independent of the kind of cellular stress. Thus, protein interactions or signaling cascades induced by DNA damage or cellular stress might lead to the formation of stabilized cleavage complexes rather than the DNA lesion itself. Finally, we show that p53 not only plays a key role in the regulation of the htopoI response to UV-C irradiation but also to treatment with colcemid.

The role of the glucocorticoids in developing resilience to stress and addiction

There is emerging evidence that individuals have the capacity to learn to be resilient by developing protective mechanisms that prevent them from the maladaptive effects of stress that can contribute to addiction.The emerging field of the neuroscience of resilience is beginning to uncover the circuits and molecules that protect against stress-related neuropsychiatric diseases, such as addiction. Glucocorticoids (GCs) are important regulators of basal and stress-related homeostasis in all higher organisms and influence a wide array of genes in almost every organ and tissue. GCs, therefore, are ideally situated to either promote or prevent adaptation to stress. In this review, we will focus on the role of GCs in the hypothalamic-pituitary adrenocortical axis and extra-hypothalamic regions in regulating basal and chronic stress responses. GCs interact with a large number of neurotransmitter and neuropeptide systems that are associated with the development of addiction. Additionally, the review will focus on the orexinergic and cholinergic pathways and highlight their role in stress and addiction. GCs play a key role in promoting the development of resilience or susceptibility and represent important pharmacotherapeutic targets that can reduce the impact of a maladapted stress system for the treatment of stress-induced addiction.

Biological markers of stress in pediatric acute burn injury

BACKGROUND Burns and their associated wound care procedures evoke significant stress and anxiety, particularly for children. Little is known about the body's physiological stress reactions throughout the stages of re-epithelialization following an acute burn injury. Previously, serum and urinary cortisol have been used to measure stress in burn patients, however these measures are not suitable for a pediatric burn outpatient setting. AIM To assess the sensitivity of salivary cortisol and sAA in detecting stress during acute burn wound care procedures and to investigate the body's physiological stress reactions throughout burn re-epithelialization. METHODS Seventy-seven participants aged four to thirteen years who presented with an acute burn injury to the burn center at the Royal Children's Hospital, Brisbane, Australia, were recruited between August 2011 and August 2012. RESULTS Both biomarkers were responsive to the stress of burn wound care procedures. sAA levels were on average 50.2U/ml higher (p<0.001) at 10min post-dressing removal compared to baseline levels. Salivary cortisol levels showed a blunted effect with average levels at ten minutes post dressing removal decreasing by 0.54nmol/L (p<0.001) compared to baseline levels. sAA levels were associated with pain (p=0.021), no medication (p=0.047) and Child Trauma Screening Questionnaire scores at three months post re-epithelialization (p=0.008). Similarly, salivary cortisol was associated with no medication (p<0.001), pain scores (p=0.045) and total body surface area of the burn (p=0.010). CONCLUSION Factors which support the use of sAA over salivary cortisol to assess stress during morning acute burn wound care procedures include; sensitivity, morning clinic times relative to cortisol's diurnal peaks, and relative cost.

The IL-6 response to Chlamydia from primary reproductive epithelial cells is highly variable and may be involved in differential susceptibility to the immunopathological consequences of chlamydial infection

Background Chlamydia trachomatis infection results in reproductive damage in some women. The process and factors involved in this immunopathology are not well understood. This study aimed to investigate the role of primary human cellular responses to chlamydial stress response proteases and chlamydial infection to further identify the immune processes involved in serious disease sequelae. Results Laboratory cell cultures and primary human reproductive epithelial cultures produced IL-6 in response to chlamydial stress response proteases (CtHtrA and CtTsp), UV inactivated Chlamydia, and live Chlamydia. The magnitude of the IL-6 response varied considerably (up to 1000 pg ml-1) across different primary human reproductive cultures. Thus different levels of IL-6 production by reproductive epithelia may be a determinant in disease outcome. Interestingly, co-culture models with either THP-1 cells or autologous primary human PBMC generally resulted in increased levels of IL-6, except in the case of live Chlamydia where the level of IL-6 was decreased compared to the epithelial cell culture only, suggesting this pathway may be able to be modulated by live Chlamydia. PBMC responses to the stress response proteases (CtTsp and CtHtrA) did not significantly vary for the different participant cohorts. Therefore, these proteases may possess conserved innate PAMPs. MAP kinases appeared to be involved in this IL-6 induction from human cells. Finally, we also demonstrated that IL-6 was induced by these proteins and Chlamydia from mouse primary reproductive cell cultures (BALB/C mice) and mouse laboratory cell models. Conclusions We have demonstrated that IL-6 may be a key factor for the chlamydial disease outcome in humans, given that primary human reproductive epithelial cell culture showed considerable variation in IL-6 response to Chlamydia or chlamydial proteins, and that the presence of live Chlamydia (but not UV killed) during co-culture resulted in a reduced IL-6 response suggesting this response may be moderated by the presence of the organism.

Targeting nuclear factor-kappa B to overcome resistance to chemotherapy

Intrinsic or acquired resistance to chemotherapeutic agents is a common phenomenon and a major challenge in the treatment of cancer patients. Chemoresistance is defined by a complex network of factors including multi-drug resistance proteins, reduced cellular uptake of the drug, enhanced DNA repair, intracellular drug inactivation, and evasion of apoptosis. Pre-clinical models have demonstrated that many chemotherapy drugs, such as platinum-based agents, antracyclines, and taxanes, promote the activation of the NF-κB pathway. NF-κB is a key transcription factor, playing a role in the development and progression of cancer and chemoresistance through the activation of a multitude of mediators including anti-apoptotic genes. Consequently, NF-κB has emerged as a promising anti-cancer target. Here, we describe the role of NF-κB in cancer and in the development of resistance, particularly cisplatin. Additionally, the potential benefits and disadvantages of targeting NF-κB signaling by pharmacological intervention will be addressed.

The role of the Eph and ephrin proteins in prostate cancer

Prostate cancer is the most commonly diagnosed malignancy and the second leading cause of cancer related deaths in Australian men. Treatment in the early stages of the disease involves surgery, radiation and/or hormone therapy. However, in late stages of the disease these treatments are no longer effective and only palliative care is available. Therefore, there is a focus on exploration of novel therapies to increase survival and treatment efficacy. Advanced prostate cancer is characterised by bone or other distant metastasis. Spreading of the primary tumour to a secondary location is a complex process requiring an initial loss in cell-cell adhesion followed by increased cell migration and invasion. One gene family that has been known to affect cell-to-cell contact in other model systems are the Eph receptor tyrosine kinases. They are the largest family of receptor tyrosine kinases made up of 14 vertebrate Eph receptors that bind to nine cell membrane bound ephrin ligands. Eph-ephrin interaction is crucial in regulating cell behaviour in developmental processes and it is now thought that the underlying mechanisms involved in development may also be involved in cancer. Aberrant expression has been reported in many human malignancies including prostate cancer. Furthermore, expression has been linked with metastasis and poor prognosis in other tumour models. This study explores the potential role of the Eph receptor family in prostate cancer, in particular the roles of EphA2, EphA3 and ephrin-A5. Gene expression profiles were established for the Eph family in a series of prostate cancer cell lines using quantitative real time RT-PCR. A smaller subset of the most prominently expressed genes was chosen to screen a cohort of clinical samples. Elevated levels of EphA2, EphA3 and their ligands, ephrin-A1 and ephrin-A5 were observed in individual cell lines. Interestingly high EphA3 expression was observed in the androgen responsive cell lines while EphA2 was more prominent in the androgen independent cell lines. However, studies using 5-dihydrotestosterone suggest that EphA3 expression in not regulated by androgen. Cells expressing EphA2 showed a greater ability for migration and invasion while cells expressing EphA3 showed poor migration and invasion. Forced expression of EphA2 in the LNCaP cell line resulted in a more invasive phenotype while forced expression of EphA3 in the PC-3 cell line suggests a possible negative effect for EphA3 on cell migration and invasion. Cell signalling studies show activation of EphA2 decreases activity of proteins thought to be involved in pathways regulating cell movement including Akt, Src and FAK. Changes to the activation status of Rho family members, including RhoA and Rac1, associated with reorganisation of the actin cytoskeleton, an important part of cell migration was also observed. As a result, activation of EphA2 in PC-3 cells resulted in a less invasive phenotype. A novel finding in this study was the discovery of a combination of two EphA2 Mabs able to activate EphA2. Preliminary results show a potential for this antibody combination to reduce prostate cancer invasion in vitro. A unique aspect of Eph-ephrin interaction is the resulting bi-directional signalling that occurs through both the receptor and ligand. In this study a potential role for ephrin-A5 mediated signalling in prostate cancer was observed. LNCaP cells express high levels of EphA3 and its high affinity ligand ephrin-A5. In stripe assays, used to study guidance cues, LNCaP cells show strong attraction/migration to EphA3-Fc stripes but not ephrin-A5-Fc stripes suggesting ephrin-A5 mediated reverse cell signalling is involved. Knockdown of ephrin-A5 using shRNA resulted in a decrease in attraction/migration to EphA3-Fc stripes. Furthermore a reduction in proliferation was also observed in vitro. A subcutaneous xenograft model using ephrin-A5 shRNA cells versus controls showed a decrease in tumour formation. This study demonstrates a difference in EphA2 and EphA3 function in prostate cancer migration/invasion and a potential role for ephrin-A5 in prostate cancer cell adhesion and growth.

Posttraumatic stress disorder and traumatic stress : from bench to bedside, from war to disaster

War is a tragic event and its mental health consequences can be profound. Recent studies indicate substantial rates of posttraumatic stress disorder and other behavioral alterations because of war exposure. Understanding the psychological, behavioral, and neurobiological mechanism of mental health and behavioral changes related to war exposure is critical to helping those in need of care. Substantial work to encourage bench to bedside to community knowledge and communication is a core component of addressing this world health need.

PTSD and traumatic stress : from gene to community and bench to bedside

Individuals and communities are exposed to traumatic events, those that are accidents or naturally occurring and those that are intentional or human made. Although resilience is the expected response, for some, posttraumatic stress disorder may be the outcome. Brain models of PTSD require understanding the phenomenology of the disorder and the brain “break down” that occurs. Among several models, importantly, is the perspective that PTSD is a “forgetting” disorder. Other elements in the onset and triggers of PTSD can identify further models to examine at the bench. New studies of the 5-HT2A receptor, the glucocorticoid receptor, p11, mitochondrial genes and cannabinoids are bringing new perspectives to understanding brain function in PTSD. Effective treatments indicate areas for bench research on the mechanisms of the disorder.