A fission yeast homolog of Int-6, the mammalian oncoprotein and eIF3 subunit, induces drug resistance when overexpressed

Through a screen to identify genes that induce multi-drug resistance when overexpressed, we have identified a fission yeast homolog of Int-6, a component of the human translation initiation factor eIF3. Disruption of the murine Int-6 gene by mouse mammary tumor virus (MMTV) has been implicated previously in tumorigenesis, although the underlying mechanism is not yet understood. Fission yeast Int6 was shown to interact with other presumptive components of eIF3 in vivo, and was present in size fractions consistent with its incorporation into a 43S translation preinitiation complex. Drug resistance induced by Int6 overexpression was dependent on the AP-1 transcription factor Pap1, and was associated with increased abundance of Pap1-responsive mRNAs, but not with Pap1 relocalization. Fission yeast cells lacking the int6 gene grew slowly. This growth retardation could be corrected by the expression of full length Int6 of fission yeast or human origin, or by a C-terminal fragment of the fission yeast protein that also conferred drug resistance, but not by truncated human Int-6 proteins corresponding to the predicted products of MMTV-disrupted murine alleles. Studies in fission yeast may therefore help to explain the ways in which Int-6 function can be perturbed during MMTV-induced mammary tumorigenesis.

Advanced glycation endproducts in horses with insulin-induced laminitis

Advanced glycation endproducts (AGEs) have been implicated in the pathogenesis of cancer, inflammatory conditions and diabetic complications. An interaction of AGEs with their receptor (RAGE) results in increased release of pro-inflammatory cytokines and reactive oxygen species (ROS), causing damage to susceptible tissues. Laminitis, a debilitating foot condition of horses, occurs in association with endocrine dysfunction and the potential involvement of AGE and RAGE in the pathogenesis of the disease has not been previously investigated. Glucose transport in lamellar tissue is thought to be largely insulin-independent (GLUT-1), which may make the lamellae susceptible to protein glycosylation and oxidative stress during periods of increased glucose metabolism. Archived lamellar tissue from horses with insulin-induced laminitis (n=4), normal control horses (n=4) and horses in the developmental stages (6 h, 12 h and 24 h) of the disease (n=12) was assessed for AGE accumulation and the presence of oxidative protein damage and cellular lipid peroxidation. The equine-specific RAGE gene was identified in lamellar tissue, sequenced and is now available on GenBank. Lamellar glucose transporter (GLUT-1 and GLUT-4) gene expression was assessed quantitatively with qRT-PCR in laminitic and control horses and horses in the mid-developmental time-point (24 h) of the disease. Significant AGE accumulation had occurred by the onset of insulin-induced laminitis (48 h) but not at earlier time-points, or in control horses. Evidence of oxidative stress was not found in any group. The equine-specific RAGE gene was not expressed differently in treated and control animals, nor was the insulin-dependent glucose transporter GLUT-4. However, the glucose transporter GLUT-1 was increased in lamellar tissue in the developmental stages of insulin-induced laminitis compared to control horses and the insulin-independent nature of the lamellae may facilitate AGE formation. However, due to the lack of AGE accumulation during disease development and a failure to detect an increase in ROS or upregulation of RAGE, it appears unlikely that oxidative stress and protein glycosylation play a central role in the pathogenesis of acute, insulin-induced laminitis.

Nutritional support in chronic obstructive pulmonary disease: a systematic review and meta-analysis

BACKGROUND: The efficacy of nutritional support in the management of malnutrition in chronic obstructive pulmonary disease (COPD) is controversial. Previous meta-analyses, based on only cross-sectional analysis at the end of intervention trials, found no evidence of improved outcomes. OBJECTIVE: The objective was to conduct a meta-analysis of randomized controlled trials (RCTs) to clarify the efficacy of nutritional support in improving intake, anthropometric measures, and grip strength in stable COPD. DESIGN: Literature databases were searched to identify RCTs comparing nutritional support with controls in stable COPD. RESULTS: Thirteen RCTs (n = 439) of nutritional support [dietary advice (1 RCT), oral nutritional supplements (ONS; 11 RCTs), and enteral tube feeding (1 RCT)] with a control comparison were identified. An analysis of the changes induced by nutritional support and those obtained only at the end of the intervention showed significantly greater increases in mean total protein and energy intakes with nutritional support of 14.8 g and 236 kcal daily. Meta-analyses also showed greater mean (±SE) improvements in favor of nutritional support for body weight (1.94 ± 0.26 kg, P < 0.001; 11 studies, n = 308) and grip strength (5.3%, P < 0.050; 4 studies, n = 156), which was not shown by ANOVA at the end of the intervention, largely because of bias associated with baseline imbalance between groups. CONCLUSION: This systematic review and meta-analysis showed that nutritional support, mainly in the form of ONS, improves total intake, anthropometric measures, and grip strength in COPD. These results contrast with the results of previous analyses that were based on only cross-sectional measures at the end of intervention trials.

Effects of enhanced somatosensory information on postural stability in older people and people with Parkinson’s disease

The somatosensory system plays an important role in balance control and age-related changes to this system have been implicated in falls. Parkinson’s disease (PD) is a chronic and progressive disease of the brain, characterized by postural instability and gait disturbance. Previous research has shown that deficiencies in somatosensory feedback may contribute to the poorer postural control demonstrated by PD individuals. However, few studies have comprehensively explored differences in somatosensory function and postural control between PD participants and healthy older individuals. The soles of the feet contain many cutaneous mechanoreceptors that provide important somatosensory information sources for postural control. Different types of insole devices have been developed to enhance this somatosensory information and improve postural stability, but these devices are often too complex and expensive to integrate into daily life. Textured insoles provide a more passive intervention that may be an inexpensive and accessible means to enhance the somatosensory input from the plantar surface of the feet. However, to date, there has been little work conducted to test the efficacy of enhanced somatosensory input induced by textured insoles in both healthy and PD populations during standing and walking. Therefore, the aims of this thesis were to determine: 1) whether textured insole surfaces can improve postural stability by enhancing somatosensory information in younger and older adults, 2) the differences between healthy older participants and PD participants for measures of physiological function and postural stability during standing and walking, 3) how changes in somatosensory information affect postural stability in both groups during standing and walking; and 4), whether textured insoles can improve postural stability in both groups during standing and walking. To address these aims, Study 1 recruited seven older individuals and ten healthy young controls to investigate the effects of two textured insole surfaces on postural stability while performing standing balance tests on a force plate. Participants were tested under three insole surface conditions: 1) barefoot; 2) standing on a hard textured insole surface; and 3), standing on a soft textured insole surface. Measurements derived from the centre of pressure displacement included the range of anterior-posterior and medial-lateral displacement, path length and the 90% confidence elliptical area (C90 area). Results of study 1 revealed a significant Group*Surface*Insole interaction for the four measures. Both textured insole surfaces reduced postural sway for the older group, especially in the eyes closed condition on the foam surface. However, participants reported that the soft textured insole surface was more comfortable and, hence, the soft textured insoles were adopted for Studies 2 and 3. For Study 2, 20 healthy older adults (controls) and 20 participants with Parkinson’s disease were recruited. Participants were evaluated using a series of physiological assessments that included touch sensitivity, vibratory perception, and pain and temperature threshold detection. Furthermore, nerve function and somatosensory evoked potentials tests were utilized to provide detailed information regarding peripheral nerve function for these participants. Standing balance and walking were assessed on different surfaces using a force plate and the 3D Vicon motion analysis system, respectively. Data derived from the force plate included the range of anterior-posterior and medial-lateral sway, while measures of stride length, stride period, cadence, double support time, stance phase, velocity and stride timing variability were reported for the walking assessment. The results of this study demonstrated that the PD group had decrements in somatosensory function compared to the healthy older control group. For electrodiagnosis, PD participants had poorer nerve function than controls, as evidenced by slower nerve conduction velocities and longer latencies in sural nerve and prolonged latency in the P37 somatosensory evoked potential. Furthermore, the PD group displayed more postural sway in both the anterior-posterior and medial-lateral directions relative to controls and these differences were increased when standing on a foam surface. With respect to the gait assessment, the PD group took shorter strides and had a reduced stride period compared with the control group. Furthermore, the PD group spent more time in the stance phase and had increased cadence and stride timing variability than the controls. Compared with walking on the firm surface, the two groups demonstrated different gait adaptations while walking on the uneven surface. Controls increased their stride length and stride period and decreased their cadence, which resulted in a consistent walking velocity on both surfaces. Conversely, while the PD patients also increased their stride period and decreased their cadence and stance period on the uneven surface, they did not increase their stride length and, hence walked slower on the uneven surface. In the PD group, there was a strong positive association between decreased somatosensory function and decreased clinical balance, as assessed by the Tinetti test. Poorer somatosensory function was also strongly positively correlated with the temporospatial gait parameters, especially shorter stride length. Study 3 evaluated the effects of manipulating the somatosensory information from the plantar surface of the feet using textured insoles in the same populations assessed in Study 2. For this study, participants performed the standing and walking balance tests under three footwear conditions: 1) barefoot; 2) with smooth insoles; and 3), with textured insoles. Standing balance and walking were evaluated using a force plate and a Vicon motion analysis system and the data were analysed in the same way outlined for Study 2. The findings showed that the smooth and textured insoles caused different effects on postural control during both the standing and walking trials. Both insoles decreased medial-lateral sway to the same level on the firm surface. The greatest benefits were observed in the PD group while wearing the textured insole. When standing under a more challenging condition on the foam surface with eyes closed, only the textured insole decreased medial-lateral sway in the PD group. With respect to the gait trials, both insoles increased walking velocity, stride length and stride time and decreased cadence, but these changes were more pronounced for the textured insoles. The effects of the textured insoles were evident under challenging conditions in the PD group and increased walking velocity and stride length, while decreasing cadence. Textured insoles were also effective in reducing the time spent in the double support and stance phases of the gait cycle and did not increase stride timing variability, as was the case for the smooth insoles for the PD group. The results of this study suggest that textured insoles, such as those evaluated in this research, may provide a low-cost means of improving postural stability in high-risk groups, such as people with PD, which may act as an important intervention to prevent falls.

Combination of MEK-ERK inhibitor and hyaluronic acid has a synergistic effect on anti-hypertrophic and pro-chondrogenic activities in osteoarthritis treatment

We hypothesised that a potentially disease-modifying osteoarthritis (OA) drug such as hyaluronic acid (HA) given in combination with anti-inflammatory signalling agents such as mitogen-activated protein kinase kinase–extracellular signal-regulated kinase (MEK-ERK) signalling inhibitor (U0126) could result in additive or synergistic effects on preventing the degeneration of articular cartilage. Chondrocyte differentiation and hypertrophy were evaluated using human OA primary cells treated with either HA or U0126, or the combination of HA + U0126. Cartilage degeneration in menisectomy (MSX) induced rat OA model was investigated by intra-articular delivery of either HA or U0126, or the combination of HA + U0126. Histology, immunostaining, RT-qPCR, Western blotting and zymography were performed to assess the expression of cartilage matrix proteins and hypertrophic markers. Phosphorylated ERK (pERK)1/2-positive chondrocytes were significantly higher in OA samples compared with those in healthy control suggesting the pathological role of that pathway in OA. It was noted that HA + U0126 significantly reduced the levels of pERK, chondrocyte hypertrophic markers (COL10 and RUNX2) and degenerative markers (ADAMTs5 and MMP-13), however, increased the levels of chondrogenic markers (COL2) compared to untreated or the application of HA or U0126 alone. In agreement with the results in vitro, intra-articular delivery of HA + U0126 showed significant therapeutic improvement of cartilage in rat MSX OA model compared with untreated or the application of HA or U0126 alone. Our study suggests that the combination of HA and MEK-ERK inhibition has a synergistic effect on preventing cartilage degeneration.

Prognostic significance of IGF and ECM induced signalling proteins in breast cancer patients

Breast cancer is a leading contributor to the burden of disease in Australia. Fortunately, the recent introduction of diverse therapeutic strategies have improved the survival outcome for many women. Despite this, the clinical management of breast cancer remains problematic as not all approaches are sufficiently sophisticated to take into account the heterogeneity of this disease and are unable to predict disease progression, in particular, metastasis. As such, women with good prognostic outcomes are exposed to the side effects of therapies without added benefit. Furthermore, women with aggressive disease for whom these advanced treatments would deliver benefit cannot be distinguished and opportunities for more intensive or novel treatment are lost. This study is designed to identify novel factors associated with disease progression, and the potential to inform disease prognosis. Frequently overlooked, yet common mediators of disease are the interactions that take place between the insulin-like growth factor (IGF) system and the extracellular matrix (ECM). Our laboratory has previously demonstrated that multiprotein insulin-like growth factor-I (IGF-I): insulin-like growth factor binding protein (IGFBP): vitronectin (VN) complexes stimulate migration of breast cancer cells in vitro, via the cooperative involvement of the insulin-like growth factor type I receptor (IGF-IR) and VN-binding integrins. However, the effects of IGF and ECM protein interactions on the dissemination and progression of breast cancer in vivo are unknown. It was hypothesised that interactions between proteins required for IGF induced signalling events and those within the ECM contribute to breast cancer metastasis and are prognostic and predictive indicators of patient outcome. To address this hypothesis, semiquantitative immunohistochemistry (IHC) analyses were performed to compare the extracellular and subcellular distribution of IGF and ECM induced signalling proteins between matched normal, primary cancer, and metastatic cancer among archival formalin-fixed paraffin-embedded (FFPE) breast tissue samples collected from women attending the Princess Alexandra Hospital, Brisbane. Multivariate Cox proportional hazards (PH) regression survival models in conjunction with a modified „purposeful selection of covariates. method were applied to determine the prognostic potential of these proteins. This study provides the first in-depth, compartmentalised analysis of the distribution of IGF and ECM induced signalling proteins. As protein function and protein localisation are closely correlated, these findings provide novel insights into IGF signalling and ECM protein function during breast cancer development and progression. Distinct IGF signalling and ECM protein immunoreactivity was observed in the stroma and/or in subcellular locations in normal breast, primary cancer and metastatic cancer tissues. Analysis of the presence and location of stratifin (SFN) suggested a causal relationship in ECM remodelling events during breast cancer development and progression. The results of this study have also suggested that fibronectin (FN) and ¥â1 integrin are important for the formation of invadopodia and epithelial-to-mesenchymal transition (EMT) events. Our data also highlighted the importance of the temporal and spatial distribution of IGF induced signalling proteins in breast cancer metastasis; in particular, SFN, enhancer-of-split and hairy-related protein 2 (SHARP-2), total-akt/protein kinase B 1 (Total-AKT1), phosphorylated-akt/protein kinase B (P-AKT), extracellular signal-related kinase-1 and extracellular signal-related kinase-2 (ERK1/2) and phosphorylated-extracellular signal-related kinase-1 and extracellular signal-related kinase-2 (P-ERK1/2). Multivariate survival models were created from the immunohistochemical data. These models were found to fit well with these data with very high statistical confidence. Numerous prognostic confounding effects and effect modifications were identified among elements of the ECM and IGF signalling cascade and corroborate the survival models. This finding provides further evidence for the prognostic potential of IGF and ECM induced signalling proteins. In addition, the adjusted measures of associations obtained in this study have strengthened the validity and utility of the resulting models. The findings from this study provide insights into the biological interactions that occur during the development of breast tissue and contribute to disease progression. Importantly, these multivariate survival models could provide important prognostic and predictive indicators that assist the clinical management of breast disease, namely in the early identification of cancers with a propensity to metastasise, and/or recur following adjuvant therapy. The outcomes of this study further inform the development of new therapeutics to aid patient recovery. The findings from this study have widespread clinical application in the diagnosis of disease and prognosis of disease progression, and inform the most appropriate clinical management of individuals with breast cancer.

Improved treatment of nicotine addiction and emerging pulmonary drug delivery

Nicotine addiction remains the leading cause of death and disease in developed and developing nations and a major cause of mortality around the world. Currently, nicotine replacement therapies (NRTs), bupropion, and varenicline are approved by the regulatory agencies as first-line treatments for nicotine addiction. Emerging evidence indicates that varenicline and bupropion have some therapeutic limitations for treating nicotine addiction with oral route of administration. Thus, continued investigation of innovative drug delivery for nicotine addiction remains a critical priority. This review will discuss some novel strategies and future directions for pulmonary drug delivery, an emerging route of administration for smoking cessation. It is anticipated that the advancement of knowledge on pulmonary drug delivery will provide better management for nicotine addiction and other addictive disorders.

DRD2/ANKK1 Taq1A (rs 1800497 C>T) genotypes are associated with susceptibility to second generation antipsychotic-induced akathisia

Rationale Although the advent of atypical, second-generation antipsychotics (SGAs) has resulted in reduced likelihood of akathisia, this adverse effect remains a problem. Extrapyramidal adverse effects are associated with increased drug occupancy of dopamine 2 receptors (DRD2). The A1 allele of the DRD2/ANKK1,rs1800497, is associated with decreased striatal DRD2 density. Objectives The aim of this study was to identify whether the A1(T) allele of the DRD2/ANKK1 was associated with akathisia (measured with the Barnes Akathisia Rating Scale) in a clinical sample of 234 patients treated with antipsychotics. Results Definite akathisia (a score≥ 2 for the global clinical assessment of akathisia) was significantly less common in subjects prescribed SGAs (16.8 %) than those prescribed FGAs (47.6%), p<0.0001. Overall, 24.1% of A1+ (A1A2/A1A1) patients treated with SGAs had akathisia compared to 10.8% of A1- (A2A2) patients. A1+ (A1A2/A1A1) patients administered SGAs also had higher global clinical assessment of akathisia scores than A1- subjects (p=0.01). SGAs maintained their advantage over FGAs regarding akathisia even in A1+ patients treated with SGAs. Conclusions These results strongly suggest that A1+ variants of the DRD2/ANKK1 Taq1A allele confer risk for akathisia in patients treated with SGAs and may explain inconsistencies across prior studies comparing FGAs and SGAs.

Nutritional support and functional capacity in chronic obstructive pulmonary disease: A systematic review and meta-analysis

Currently there is confusion about the value of using nutritional support to treat malnutrition and improve functional outcomes in chronic obstructive pulmonary disease (COPD). This systematic review and meta-analysis of randomised controlled trials (RCTs) aimed to clarify the effectiveness of nutritional support in improving functional outcomes in COPD. A systematic review identified 12 RCTs (n = 448) in stable COPD patients investigating the effects of nutritional support [dietary advice (1 RCT), oral nutritional supplements (ONS; 10 RCTs), enteral tube feeding (1 RCT)] versus control on functional outcomes. Meta-analysis of the changes induced by intervention found that whilst respiratory function (FEV(1,) lung capacity, blood gases) was unresponsive to nutritional support, both inspiratory and expiratory muscle strength (PI max +3.86 SE 1.89 cm H(2) O, P = 0.041; PE max +11.85 SE 5.54 cm H(2) O, P = 0.032) and handgrip strength (+1.35 SE 0.69 kg, P = 0.05) were significantly improved, and associated with weight gains of ≥ 2 kg. Nutritional support produced significant improvements in quality of life in some trials, although meta-analysis was not possible. It also led to improved exercise performance and enhancement of exercise rehabilitation programmes. This systematic review and meta-analysis demonstrates that nutritional support in COPD results in significant improvements in a number of clinically relevant functional outcomes, complementing a previous review showing improvements in nutritional intake and weight.

Combined expression of KLK4, KLK5, KLK6, and KLK7 by ovarian cancer cells leads to decreased adhesion and paclitaxel-induced chemoresistance.

OBJECTIVE: Chemoresistance is a critical feature of advanced ovarian cancer with only 30% of patients surviving longer than 5 years. We have previously shown that four kallikrein-related (KLK) peptidases, KLK4, KLK5, KLK6 and KLK7 (KLK4-7), are implicated in peritoneal invasion and tumour growth, but underlying mechanisms were not identified. We also reported that KLK7 overexpression confers chemoresistance to paclitaxel, and cell survival via integrins. In this study, we further explored the functional consequenses of overexpression of all four KLKs (KLK4-7) simultaneously in the ovarian cancer cell line, OV-MZ-6, and its impact on integrin expression and signalling, cell adhesion and survival as contributors to chemoresistance and metastatic progression. METHODS: Quantitative gene and protein expression analyses, confocal microscopy, cell adhesion and chemosensitivity assays were performed. RESULTS: Expression of α5β1/αvβ3 integrins was downregulated upon combined stable KLK4-7 overexpression in OV-MZ-6 cells. Accordingly, the adhesion of these cells to vitronectin and fibronectin, the extracellular matrix binding proteins of α5β1/αvβ3 integrins and two predominant proteins of the peritoneal matrix, was decreased. KLK4-7-transfected cells were more resistant to paclitaxel (10-100 nmol/L: 38-54%), but not to carboplatin, which was associated with decreased apoptotic stimuli. However, the KLK4-7-induced paclitaxel resistance was not blocked by the MEK1/2 inhibitor, U0126. CONCLUSIONS: This study demonstrates that combined KLK4-7 expression by ovarian cancer cells promotes reduced integrin expression with consequently less cell-matrix attachment, and insensitivity to paclitaxel mediated by complex integrin and MAPK independent interactions, indicative of a malignant phenotype and disease progression suggesting a role for these KLKs in this process.

Histological and morphometric lesions in the pre-clinical, developmental phase of insulin-induced laminitis in Standardbred horses

Lamellar pathology in experimentally-induced equine laminitis associated with euglycaemic hyperinsulinaemia is substantial by the acute, clinical phase (∼48 h post-induction). However, lamellar pathology of the developmental, pre-clinical phase requires evaluation. The aim of this study was to analyse lamellar lesions both qualitatively and quantitatively, 6, 12 and 24 h after the commencement of hyperinsulinaemia. Histological and histomorphometrical analyses of lamellar pathology at each time-point included assessment of lamellar length and width, epidermal cell proliferation and death, basement membrane (BM) pathology and leucocyte infiltration. Archived lamellar tissue from control horses and those with acute, insulin-induced laminitis (48 h) was also assessed for cellular proliferative activity by counting the number of cells showing positive nuclear immuno labelling for TPX2. Decreased secondary epidermal lamellar (SEL) width and increased histomorphological evidence of SEL epidermal basal (and supra-basal) cell death occurred early in disease progression (6 h). Increased cellular proliferation in SELs, infiltration of the dermis with small numbers of leucocytes and BM damage occurred later (24 and 48 h). Some lesions, such as narrowing of the SELs, were progressive over this time period (6–48 h). Cellular pathology preceded leucocyte infiltration and BM pathology, indicating that the latter changes may be secondary or downstream events in hyperinsulinaemic laminitis.

Metabolomic analysis characterizes tissue specific indomethacin-induced metabolic perturbations of rats

In this study, the promising metabolomic approach integrating with ingenuity pathway analysis (IPA) was applied to characterize the tissue specific metabolic perturbation of rats that was induced by indomethacin. The selective pattern recognition analyses were applied to analyze global metabolic profiling of urine of rats treated by indomethacin at an acute dosage of reference that has been proven to induce tissue disorders in rats, evaluated throughout the time-course of -24-72 h. The results preliminarily revealed that modifications of amino acid metabolism, fatty acid metabolism and energetically associated metabolic pathways accounted for metabolic perturbation of the rats that was induced by indomethacin. Furthermore, IPA was applied to deeply analyze the biomarkers and their relations with the metabolic perturbations evidenced by pattern recognition analyses. Specific biochemical functions affected by indomethacin suggested that there is an important correlation of its effects in kidney and liver metabolism, based on the determined metabolites and their pathway-based analysis. The IPA correlation of the three major biomarkers, identified as creatinine, prostaglandin E2 and guanosine, suggested that the administration of indomethacin induced certain levels of toxicity in the kidneys and liver. The changes in the levels of biomarker metabolites allowed the phenotypical determination of the metabolic perturbations induced by indomethacin in a time-dependent manner.

Thyroxine and reserpine-induced changes in metabolic profiles of rat urine and the therapeutic effect of Liu Wei Di Huang Wan detected by UPLC-HDMS

The promise of metabonomics, a new "omics" technique, to validate Chinese medicines and the compatibility of Chinese formulas has been appreciated. The present study was undertaken to explore the excretion pattern of low molecular mass metabolites in the male Wistar-derived rat model of kidney yin deficiency induced with thyroxine and reserpine as well as the therapeutic effect of Liu Wei Di Huang Wan (LW) and its separated prescriptions, a classic traditional Chinese medicine formula for treating kidney yin deficiency in China. The study utilized ultra-performance liquid chromatography/electrospray ionization synapt high definition mass spectrometry (UPLC/ESI-SYNAPT-HDMS) in both negative and positive electrospray ionization (ESI). At the same time, blood biochemistry was examined to identify specific changes in the kidney yin deficiency. Distinct changes in the pattern of metabolites, as a result of daily administration of thyroxine and reserpine, were observed by UPLC-HDMS combined with a principal component analysis (PCA). The changes in metabolic profiling were restored to their baseline values after treatment with LW according to the PCA score plots. Altogether, the current metabonomic approach based on UPLC-HDMS and orthogonal projection to latent structures discriminate analysis (OPLS-DA) indicated 20 ions (14 in the negative mode, 8 in the positive mode, and 2 in both) as "differentiating metabolites".

Effect of induced airway inflammation in asthma : the expression of trefoil factors, secretoglobins and genes involved in histone acetylation

Asthma is an incapacitating disease of the respiratory system, which causes extensive morbidity and mortality worldwide. Asthma affects more than 300 million people globally(Masoli et al. 2004). In Australia, it affects 10.2% of the population (Masoli et al. 2004) and causes 60,000 people to be hospitalised annually. Health care expenditure due to asthma in Australia was $606 million in 2004–2005. There are four primary biological factors that function in the initiation and exacerbation of asthma. Airway inflammation is important as it is often the first response to an airway insult, initiating the three other components: bronchoconstriction, mucus hyper-secretion and hyper-reactivity. The mediators involved in asthma are still not well understood, and current anti-inflammatory corticosteroid treatments are not effective with all asthmatics. As there is currently no cure for asthma, and airway inflammation is the primary component of the disease, it is important that we understand and investigate the mediators of airway inflammation to look for a potential cure and to produce better therapeutics to treat the inflammation. Trefoil factors (TFFs) and secretoglobins (SCGBs) are small secreted proteins involved in the mediation of inflammation and epithelial restitution. TFFs are pro-inflammatory and SCGBs anti-inflammatory by nature. The hypothesis of this study is that in response to induced acute airway inflammation, the expression of TFF1 and TFF3 will increase and expression of SCGB1A1 and SCGB3A2 will decrease in non-asthmatics (N-A), asthmatics medicating with bronchodilators (A-BD) and asthmatics medicating with corticosteroids (A-ST). When comparing the three groups, we expect to see higher expression of the TFFs in the A-BD group compared to the N-A and A-ST groups, indicating that inflammation is mediated by TFFs in asthma and that corticosteroid medication controls their expression as part of the control of inflammation. We expect to see the opposite with SCGBs, with a greater decrease in the A-BD group compared to the other two groups, suggesting that the A-BD group has the least anti-inflammatory activity in response to inflammatory insult. Epigenetic modification plays a role in the regulation of genes that initiate disease states such as inflammatory conditions and cancers. Histone acetylation is one such modification, which involves the acetylation of histones in chromatin by histone acetyltransferases (HATs). This increases the transcription of genes involved with inflammation or enrols histone deacetylases (HDACs) to down-regulate the transcription of inflammatory genes. These HATs and HDACs work in a homeostatic fashion; however, in the event of inflammation, increased HAT activity can stimulate further inflammation, which is believed to be the mechanism involved in some inflammatory diseases. This study hypothesises that in response to inflammation, the expression of HDACs (HDAC1-5) will decrease and the expression of HATs (NCOA1-3, HAT-1 and CREBBP) will increase in all groups. When comparing the expression between the groups, it was expected that a greater decrease in HDACs and a greater increase in HATs will be seen in the A-BD group compared to the other two groups. This would identify histone acetylation as a mechanism involved in the inflammatory condition of asthma and indicate that corticosteroids may treat the inflammation in asthma at least in part by controlling histone acetylation. The aim of the project was to compare the expression of inflammatory genes TFF1, TFF3, SCGB1A1 and SCGB3A2, as well as to compare the gene expression of HDAC1-5, NCOA1-3, HAT-1 and CREBBP within and between N-A (n=15), A-BD (n=15) and A-ST (n=15) groups in response to inflammation. This was performed by collecting airway cells and proteins by sputum induction in three sessions. The sessions were coordinated into an initial baseline collection (SI-1), followed by a second session at least one week later (SI-2) and a third session, six hours after SI-2 to collect a sample containing the resultant acute inflammation caused in SI-2 (SI-3). Analysis of the SI-1 and SI-2 samples in all three groups had high amounts of variability between samples. The samples were taken at least one weak apart and the environmental stimuli on each participant outside of the testing sessions could not be controlled. For this reason, the SI-1 samples were not used for analysis; instead SI-2 and SI-3 samples were compared as they were same-day collections, reducing the probability of differences being due to anything other than the sputum induction. The gene expressions of the TFFs, SCGBs, HDACs and HATs were analysed using real-time PCR. Western blot analysis was performed to analyse the protein concentrations of the TFFs and SCGBs in secreted fractions of the sputum collection. Both the secreted and intracellular protein fractions collected from the sputum inductions for pre- and post-inflammation (SI-2, SI-3) samples of the N-A and A-BD groups were analysed using a proteomic method called iTRAQ. This allowed the comparison of the change in protein expression as a result of airway inflammation in each group. This technique was used as a discovery method to identify novel proteins that are modulated by induced acute airway inflammation. Any proteins of interest would then be further validated and used for future research. Inflammation was achieved in the SI-3 samples of the N-A group with a 21% unit increase in % neutrophils compared to SI-2 (p=0.01). The N-A group had a marked 5.5-fold decrease in HDAC1 gene expression in SI-3 compared to SI-2 (p=0.03). No differences were seen in any of the TFFs, SCGBs or any of the rest of the HDACs and HATs. Western blot data did not display any significant changes in the protein levels of the TFFs and SCGBs analysed. However, non-significant analysis of the data displayed increases in TFF1 and TFF3, and decreases in SCGB1A1 and SCGB3A2 for the majority of SI-3 samples compared to SI-2. The A-BD group also presented a marked increase in neutrophils in the SI-3 samples compared to SI-2 (27% unit increase, p=0.04). The A-BD group had a significant increase in TFF3 and SCGB1A1 gene expression concomitant with induced acute airway inflammation. A 7.3-fold increase in TFF3 (p=0.05) in SI-3 indicated that TFF3 is linked to inflammation in asthmatics. A 2.8-fold increase in SCGB1A1 (p=0.03) indicated that this gene is also up-regulated, suggesting that this SCGB is expressed to try to combat induced acute airway inflammation. No significant changes were seen in any of the other genes analysed. Western blot data did not display any significant changes in the protein levels of the TFFs and SCGBs analysed. However, non-significant analysis of the data displayed an increase in TFF1 and TFF3, and a decrease in SCGB1A1 and SCGB3A2 in SI-3, similar to that seen in the N-A group. The A-ST group was different from the A-BD group, characterised by the use of inhaled corticosteroid medication to treat asthma symptoms. Inhaled corticosteroids are known to treat asthma symptoms through the control of inflammation. Therefore, it was expected that corticosteroid medication would also control the expression of TFFs, SCGBs, HATs and HDACs. Gene expression results only identified a 7.6-fold decrease in HDAC2 expression in SI-3 (p=0.001), which is proposed to be due to the up-regulation of HDAC2 protein that is known to be a function of corticosteroid use. Western blot data did not display any significant changes in the protein levels of the TFFs and SCGBs analysed. The gene expression in SI-2 and SI-3 in each group was compared. When comparing the A-BD group to the N-A group, a 9-fold increase in TFF3 (p=0.008) and a 34-fold increase in SCGB1A1 (p=0.03) were seen in the SI-3 samples. Comparisons of the A-ST group to the N-A group had an increased expression in SI-2 samples for HDAC5 (3.6-fold, p=0.04), NCOA2 (8.5-fold, p=0.04), NCOA3 (17-fold, p=0.01), HAT-1 (36-fold, p=0.003) and CREBBP (13-fold, p=0.001). The SI-3 samples in the A-ST group compared to the N-A group had increased expression for HDAC1 (6.4-fold, p=0.04), HDAC5 (5.2-fold, p=0.008), NCOA2 (9.6-fold, p=0.03), NCOA3 (16-fold, p=0.06), HAT-1 (41-fold, p<0.001) and CREBBP (31-fold, p=0.001). Comparisons of the A-ST group to the A-BD group had SI-2 increases in HDAC1 (3.8-fold, p=0.03), NCOA3 (4.5-fold, p=0.03), HAT-1 (5.3-fold, p=0.01) and CREBBP (23-fold, p=0.001), while SI-3 comparisons saw a decrease in HDAC2 (41-fold, p=0.008) and increases in HAT-1 (4.3-fold, p=0.003) and CREBBP (40-fold, p=0.001). Results showed that TFF3 and SCGB1A1 expression is higher in asthmatics than non-asthmatics and that histone acetylation is more active in the A-ST group than either the N-A or A-BD group, which suggests that histone acetylation activity may be positively correlated with asthma severity. The iTRAQ proteomic analysis of the secreted protein samples identified the SCGB1A1 protein and found it to be decreased in both the N-A and A-BD groups post-inflammation, but significantly so only in the A-BD group. Although no significant results were obtained from the western blot data, both groups displayed a decrease in SCGB1A1 concentration in SI-3 samples, suggesting a correlation with the proteomic data. Only 31 peptides were identified from the secreted samples. The intracellular iTRAQ analysis successfully identified 664 peptides, eight of which had differential expression in association with induced acute airway inflammation. Significant increases were seen in the A-BD group in SI-3 compared to SI-2 than in the N-A group in chloride intracellular channel protein 1, keratin-19, eosinophil cationic protein, calnexin, peroxiredoxin-5, and ATP-synthase delta subunit, while decreases were seen in cystatin-A and mucin-5AC. The iTRAQ analysis was only a discovery measure and further validation must be performed. In summary, the expression of TFFs and SCGBs differed between non-asthmatics and asthmatics. It is clear that TFF3 is active in the airway inflammation associated with asthma as indicated by an increase associated with inflammation in the A-BD group compared to the N-A group. Results for HDAC and HAT genes showed high HAT expression in the A-ST group compared to the N-A and A-BD groups, suggesting that histone acetyltransferases may be responsible for the characteristic unregulated inflammatory symptoms of asthmatics taking corticosteroids. Interestingly, corticosteroid medication did not seem to silence the expression of the analysed HAT genes, which indicates that corticosteroids may not control inflammation by direct regulation of HATs, but instead by competition, most probably with HDAC2 protein. As a discovery tool, iTRAQ is a potent method to both identify and compare the concentration of proteins between samples. The method is a powerful first step into the identification of novel proteins that are regulated in response to different treatments.

Current status of pharmacogenomics testing for anti-tumor drug therapies : approaches to non-melanoma skin cancer

Skin cancer is one of the most commonly occurring cancer types, with substantial social, physical, and financial burdens on both individuals and societies. Although the role of UV light in initiating skin cancer development has been well characterized, genetic studies continue to show that predisposing factors can influence an individual's susceptibility to skin cancer and response to treatment. In the future, it is hoped that genetic profiles, comprising a number of genetic markers collectively involved in skin cancer susceptibility and response to treatment or prognosis, will aid in more accurately informing practitioners' choices of treatment. Individualized treatment based on these profiles has the potential to increase the efficacy of treatments, saving both time and money for the patient by avoiding the need for extensive or repeated treatment. Increased treatment responses may in turn prevent recurrence of skin cancers, reducing the burden of this disease on society. Currently existing pharmacogenomic tests, such as those that assess variation in the metabolism of the anticancer drug fluorouracil, have the potential to reduce the toxic effects of anti-tumor drugs used in the treatment of non-melanoma skin cancer (NMSC) by determining individualized appropriate dosage. If the savings generated by reducing adverse events negate the costs of developing these tests, pharmacogenomic testing may increasingly inform personalized NMSC treatment.