Vascular endothelial growth factor receptor-3 directly interacts with phosphatidylinositol 3-kinase to regulate lymphangiogenesis

Background Dysfunctional lymphatic vessel formation has been implicated in a number of pathological conditions including cancer metastasis, lymphedema, and impaired wound healing. The vascular endothelial growth factor (VEGF) family is a major regulator of lymphatic endothelial cell (LEC) function and lymphangiogenesis. Indeed, dissemination of malignant cells into the regional lymph nodes, a common occurrence in many cancers, is stimulated by VEGF family members. This effect is generally considered to be mediated via VEGFR-2 and VEGFR-3. However, the role of specific receptors and their downstream signaling pathways is not well understood. Methods and Results Here we delineate the VEGF-C/VEGF receptor (VEGFR)-3 signaling pathway in LECs and show that VEGF-C induces activation of PI3K/Akt and MEK/Erk. Furthermore, activation of PI3K/Akt by VEGF-C/VEGFR-3 resulted in phosphorylation of P70S6K, eNOS, PLCc1, and Erk1/2. Importantly, a direct interaction between PI3K and VEGFR-3 in LECs was demonstrated both in vitro and in clinical cancer specimens. This interaction was strongly associated with the presence of lymph node metastases in primary small cell carcinoma of the lung in clinical specimens. Blocking PI3K activity abolished VEGF-C-stimulated LEC tube formation and migration. Conclusions Our findings demonstrate that specific VEGFR-3 signaling pathways are activated in LECs by VEGF-C. The importance of PI3K in VEGF-C/VEGFR-3-mediated lymphangiogenesis provides a potential therapeutic target for the inhibition of lymphatic metastasis.

NT-ProBNP levels in saliva and its clinical relevance to heart failure

Background: Current blood based diagnostic assays to detect heart failure (HF) have large intra-individual and inter-individual variations which have made it difficult to determine whether the changes in the analyte levels reflect an actual change in disease activity. Human saliva mirrors the body's health and well being and similar to 20% of proteins that are present in blood are also found in saliva. Saliva has numerous advantages over blood as a diagnostic fluid which allows for a non-invasive, simple, and safe sample collection. The aim of our study was to develop an immunoassay to detect NT-proBNP in saliva and to determine if there is a correlation with blood levels. Methods: Saliva samples were collected from healthy volunteers (n = 40) who had no underlying heart conditions and HF patients (n = 45) at rest. Samples were stored at -80 degrees C until analysis. A customised homogeneous sandwich AlphaLISA((R)) immunoassay was used to quantify NT-proBNP levels in saliva. Results: Our NT-proBNP immunoassay was validated against a commercial Roche assay on plasma samples collected from HF patients (n = 37) and the correlation was r(2) = 0.78 (p<0.01, y = 1.705 x +1910.8). The median salivary NT-proBNP levels in the healthy and HF participants were <16 pg/mL and 76.8 pg/mL, respectively. The salivary NT-proBNP immunoassay showed a clinical sensitivity of 82.2% and specificity of 100%, positive predictive value of 100% and negative predictive value of 83.3%, with an overall diagnostic accuracy of 90.6%. Conclusion: We have firstly demonstrated that NT-proBNP can be detected in saliva and that the levels were higher in heart failure patients compared with healthy control subjects. Further studies will be needed to demonstrate the clinical relevance of salivary NT-proBNP in unselected, previously undiagnosed populations.

The MyD88+ phenotype is an adverse prognostic factor in epithelial ovarian cancer

The prognosis of epithelial ovarian cancer is poor in part due to the high frequency of chemoresistance. Recent evidence points to the Toll-like receptor-4 (TLR4), and particularly its adaptor protein MyD88, as one potential mediator of this resistance. This study aims to provide further evidence that MyD88 positive cancer cells are clinically significant, stem-like and reproducibly detectable for the purposes of prognostic stratification. Expression of TLR4 and MyD88 was assessed immunohistochemically in 198 paraffin-embedded ovarian tissues and in an embryonal carcinoma model of cancer stemness. In parallel, expression of TLR4 and MyD88 mRNA and regulatory microRNAs (miR-21 and miR-146a) was assessed, as well as in a series of chemosensitive and resistant cancer cells lines. Functional analysis of the pathway was assessed in chemoresistant SKOV-3 ovarian cancer cells. TLR4 and MyD88 expression can be reproducibly assessed via immunohistochemistry using a semi-quantitative scoring system. TLR4 expression was present in all ovarian epithelium (normal and neoplastic), whereas MyD88 was restricted to neoplastic cells, independent of tumour grade and associated with reduced progression-free and overall survival, in an immunohistological specific subset of serous carcinomas, p<0.05. MiR-21 and miR-146a expression was significantly increased in MyD88 negative cancers (p<0.05), indicating their participation in regulation. Significant alterations in MyD88 mRNA expression were observed between chemosensitive and chemoresistant cells and tissue. Knockdown of TLR4 in SKOV-3 ovarian cells recovered chemosensitivity. Knockdown of MyD88 alone did not. MyD88 expression was down-regulated in differentiated embryonal carcinoma (NTera2) cells, supporting the MyD88+ cancer stem cell hypothesis. Our findings demonstrate that expression of MyD88 is associated with significantly reduced patient survival and altered microRNA levels and suggest an intact/functioning TLR4/MyD88 pathway is required for acquisition of the chemoresistant phenotype. Ex vivo manipulation of ovarian cancer stem cell (CSC) differentiation can decrease MyD88 expression, providing a potentially valuable CSC model for ovarian cancer.

Differential effects of tissue culture coating substrates on prostate cancer cell adherence, morphology and behavior

Weak cell-surface adhesion of cell lines to tissue culture surfaces is a common problem and presents technical limitations to the design of experiments. To overcome this problem, various surface coating protocols have been developed. However, a comparative and precise real-time measurement of their impact on cell behavior has not been conducted. The prostate cancer cell line LNCaP, derived from a patient lymph node metastasis, is a commonly used model system in prostate cancer research. However, the cells’ characteristically weak attachment to the surface of tissue culture vessels and cover slips has impeded their manipulation and analysis and use in high throughput screening. To improve the adherence of LNCaP cells to the culture surface, we compared different coating reagents (poly-L-lysine, poly-L-ornithine, collagen type IV, fibronectin, and laminin) and culturing conditions and analyzed their impact on cell proliferation, adhesion, morphology, mobility and gene expression using real-time technologies. The results showed that fibronectin, poly-L-lysine and poly-L-ornithine improved LNCaP cells adherence and provoked cell morphology alterations, such as increase of nuclear and cellular area. These coating reagents also induced a higher expression of F-actin and reduced cell mobility. In contrast, laminin and collagen type IV did not improve adherence but promoted cell aggregation and affected cell morphology. Cells cultured in the presence of laminin displayed higher mobility than control cells. All the coating conditions significantly affected cell viability; however, they did not affect the expression of androgen receptor-regulated genes. Our comparative findings provide important insight for the selection of the ideal coating reagent and culture conditions for the cancer cell lines with respect to their effect on proliferation rate, attachment, morphology, migration, transcriptional response and cellular cytoskeleton arrangement.

The global epidemiology and contribution of cannabis use and dependence to the global burden of disease: results from the GBD 2010 study

Aim Estimate the prevalence of cannabis dependence and its contribution to the global burden of disease. Methods Systematic reviews of epidemiological data on cannabis dependence (1990-2008) were conducted in line with PRISMA and meta-analysis of Observational Studies in Epidemiology (MOOSE) guidelines. Culling and data extraction followed protocols, with cross-checking and consistency checks. DisMod-MR, the latest version of generic disease modelling system, redesigned as a Bayesian meta-regression tool, imputed prevalence by age, year and sex for 187 countries and 21 regions. The disability weight associated with cannabis dependence was estimated through population surveys and multiplied by prevalence data to calculate the years of life lived with disability (YLDs) and disability-adjusted life years (DALYs). YLDs and DALYs attributed to regular cannabis use as a risk factor for schizophrenia were also estimated. Results There were an estimated 13.1 million cannabis dependent people globally in 2010 (point prevalence0.19% (95% uncertainty: 0.17-0.21%)). Prevalence peaked between 20-24 yrs, was higher in males (0.23% (0.2-0.27%)) than females (0.14% (0.12-0.16%)) and in high income regions. Cannabis dependence accounted for 2 million DALYs globally (0.08%; 0.05-0.12%) in 2010; a 22% increase in crude DALYs since 1990 largely due to population growth. Countries with statistically higher age-standardised DALY rates included the United States, Canada, Australia, New Zealand and Western European countries such as the United Kingdom; those with lower DALY rates were from Sub-Saharan Africa-West and Latin America. Regular cannabis use as a risk factor for schizophrenia accounted for an estimated 7,000 DALYs globally. Conclusion Cannabis dependence is a disorder primarily experienced by young adults, especially in higher income countries. It has not been shown to increase mortality as opioid and other forms of illicit drug dependence do. Our estimates suggest that cannabis use as a risk factor for schizophrenia is not a major contributor to population-level disease burden.

The epidemiological modelling of major depressive disorder : application for the global burden of disease study 2010

Background Although the detrimental impact of major depressive disorder (MDD) at the individual level has been described, its global epidemiology remains unclear given limitations in the data. Here we present the modelled epidemiological profile of MDD dealing with heterogeneity in the data, enforcing internal consistency between epidemiological parameters and making estimates for world regions with no empirical data. These estimates were used to quantify the burden of MDD for the Global Burden of Disease Study 2010 (GBD 2010). Method Analyses drew on data from our existing literature review of the epidemiology of MDD. DisMod-MR, the latest version of the generic disease modelling system redesigned as a Bayesian meta-regression tool, derived prevalence by age, year and sex for 21 regions. Prior epidemiological knowledge, study- and country-level covariates adjusted sub-optimal raw data. Results There were over 298 million cases of MDD globally at any point in time in 2010, with the highest proportion of cases occurring between 25 and 34 years. Global point prevalence was very similar across time (4.4% (95% uncertainty: 4.2–4.7%) in 1990, 4.4% (4.1–4.7%) in 2005 and 2010), but higher in females (5.5% (5.0–6.0%) compared to males (3.2% (3.0–3.6%) in 2010. Regions in conflict had higher prevalence than those with no conflict. The annual incidence of an episode of MDD followed a similar age and regional pattern to prevalence but was about one and a half times higher, consistent with an average duration of 37.7 weeks. Conclusion We were able to integrate available data, including those from high quality surveys and sub-optimal studies, into a model adjusting for known methodological sources of heterogeneity. We were also able to estimate the epidemiology of MDD in regions with no available data. This informed GBD 2010 and the public health field, with a clearer understanding of the global distribution of MDD.

Burden of depressive disorders by country, sex, age, and year : findings from the Global Burden of Disease Study 2010

Background Depressive disorders were a leading cause of burden in the Global Burden of Disease (GBD) 1990 and 2000 studies. Here, we analyze the burden of depressive disorders in GBD 2010 and present severity proportions, burden by country, region, age, sex, and year, as well as burden of depressive disorders as a risk factor for suicide and ischemic heart disease. Methods and Findings Burden was calculated for major depressive disorder (MDD) and dysthymia. A systematic review of epidemiological data was conducted. The data were pooled using a Bayesian meta-regression. Disability weights from population survey data quantified the severity of health loss from depressive disorders. These weights were used to calculate years lived with disability (YLDs) and disability adjusted life years (DALYs). Separate DALYs were estimated for suicide and ischemic heart disease attributable to depressive disorders.Depressive disorders were the second leading cause of YLDs in 2010. MDD accounted for 8.2% (5.9%-10.8%) of global YLDs and dysthymia for 1.4% (0.9%-2.0%). Depressive disorders were a leading cause of DALYs even though no mortality was attributed to them as the underlying cause. MDD accounted for 2.5% (1.9%-3.2%) of global DALYs and dysthymia for 0.5% (0.3%-0.6%). There was more regional variation in burden for MDD than for dysthymia; with higher estimates in females, and adults of working age. Whilst burden increased by 37.5% between 1990 and 2010, this was due to population growth and ageing. MDD explained 16 million suicide DALYs and almost 4 million ischemic heart disease DALYs. This attributable burden would increase the overall burden of depressive disorders from 3.0% (2.2%-3.8%) to 3.8% (3.0%-4.7%) of global DALYs. Conclusions GBD 2010 identified depressive disorders as a leading cause of burden. MDD was also a contributor of burden allocated to suicide and ischemic heart disease. These findings emphasize the importance of including depressive disorders as a public-health priority and implementing cost-effective interventions to reduce its burden.Please see later in the article for the Editors' Summary.

The burden attributable to mental and substance use disorders as risk factors for suicide : findings from the Global Burden of Disease Study 2010

Background The Global Burden of Disease Study 2010 (GBD 2010) identified mental and substance use disorders as the 5th leading contributor of burden in 2010, measured by disability adjusted life years (DALYs). This estimate was incomplete as it excluded burden resulting from the increased risk of suicide captured elsewhere in GBD 2010's mutually exclusive list of diseases and injuries. Here, we estimate suicide DALYs attributable to mental and substance use disorders. Methods Relative-risk estimates of suicide due to mental and substance use disorders and the global prevalence of each disorder were used to estimate population attributable fractions. These were adjusted for global differences in the proportion of suicide due to mental and substance use disorders compared to other causes then multiplied by suicide DALYs reported in GBD 2010 to estimate attributable DALYs (with 95% uncertainty). Results Mental and substance use disorders were responsible for 22.5 million (14.8-29.8 million) of the 36.2 million (26.5-44.3 million) DALYs allocated to suicide in 2010. Depression was responsible for the largest proportion of suicide DALYs (46.1% (28.0%-60.8%)) and anorexia nervosa the lowest (0.2% (0.02%-0.5%)). DALYs occurred throughout the lifespan, with the largest proportion found in Eastern Europe and Asia, and males aged 20-30 years. The inclusion of attributable suicide DALYs would have increased the overall burden of mental and substance use disorders (assigned to them in GBD 2010 as a direct cause) from 7.4% (6.2%-8.6%) to 8.3% (7.1%-9.6%) of global DALYs, and would have changed the global ranking from 5th to 3rd leading cause of burden. Conclusions Capturing the suicide burden attributable to mental and substance use disorders allows for more accurate estimates of burden. More consideration needs to be given to interventions targeted to populations with, or at risk for, mental and substance use disorders as an effective strategy for suicide prevention.

Towards a quantitative theory of epidermal calcium profile formation in unwounded skin

We propose and mathematically examine a theory of calcium profile formation in unwounded mammalian epidermis based on: changes in keratinocyte proliferation, fluid and calcium exchange with the extracellular fluid during these cells' passage through the epidermal sublayers, and the barrier functions of both the stratum corneum and tight junctions localised in the stratum granulosum. Using this theory, we develop a mathematical model that predicts epidermal sublayer transit times, partitioning of the epidermal calcium gradient between intracellular and extracellular domains, and the permeability of the tight junction barrier to calcium ions. Comparison of our model's predictions of epidermal transit times with experimental data indicates that keratinocytes lose at least 87% of their volume during their disintegration to become corneocytes. Intracellular calcium is suggested as the main contributor to the epidermal calcium gradient, with its distribution actively regulated by a phenotypic switch in calcium exchange between keratinocytes and extracellular fluid present at the boundary between the stratum spinosum and the stratum granulosum. Formation of the extracellular calcium distribution, which rises in concentration through the stratum granulosum towards the skin surface, is attributed to a tight junction barrier in this sublayer possessing permeability to calcium ions that is less than 15 nm/s in human epidermis and less than 37 nm/s in murine epidermis. Future experimental work may refine the presented theory and reduce the mathematical uncertainty present in the model predictions.

Standardising and assessing digital images for use in clinical trials : a practical, reproducible method that blinds the assessor to treatment allocation

With the increasing availability of high quality digital cameras that are easily operated by the non-professional photographer, the utility of using digital images to assess endpoints in clinical research of skin lesions has growing acceptance. However, rigorous protocols and description of experiences for digital image collection and assessment are not readily available, particularly for research conducted in remote settings. We describe the development and evaluation of a protocol for digital image collection by the non-professional photographer in a remote setting research trial, together with a novel methodology for assessment of clinical outcomes by an expert panel blinded to treatment allocation.

A comparison between conductive and infrared devices for measuring mean skin temperature at rest, during exercise in the heat, and recovery

Purpose: Skin temperature assessment has historically been undertaken with conductive devices affixed to the skin. With the development of technology, infrared devices are increasingly utilised in the measurement of skin temperature. Therefore, our purpose was to evaluate the agreement between four skin temperature devices at rest, during exercise in the heat, and recovery. Methods: Mean skin temperature (T̅sk) was assessed in thirty healthy males during 30 min rest (24.0± 1.2°C, 56 ± 8%), 30 min cycle in the heat (38.0 ± 0.5°C, 41 ± 2%), and 45 min recovery(24.0 ± 1.3°C, 56 ± 9%). T̅sk was assessed at four sites using two conductive devices(thermistors, iButtons) and two infrared devices (infrared thermometer, infrared camera). Results: Bland–Altman plots demonstrated mean bias ± limits of agreement between the thermistors and iButtons as follows (rest, exercise, recovery): -0.01 ± 0.04, 0.26 ± 0.85, -0.37 ± 0.98°C; thermistors and infrared thermometer: 0.34 ± 0.44, -0.44 ± 1.23, -1.04 ± 1.75°C; thermistors and infrared camera (rest, recovery): 0.83 ± 0.77, 1.88 ± 1.87°C. Pairwise comparisons of T̅sk found significant differences (p < 0.05) between thermistors and both infrared devices during resting conditions, and significant differences between the thermistors and all other devices tested during exercise in the heat and recovery. Conclusions: These results indicate poor agreement between conductive and infrared devices at rest, during exercise in the heat, and subsequent recovery. Infrared devices may not be suitable for monitoring T̅sk in the presence of, or following, metabolic and environmental induced heat stress.

Voluntary exercise decreases atherosclerosis in nephrectomised ApoE knockout mice

Cardiovascular disease is the main cause of morbidity and mortality in patients with kidney disease. The effectiveness of exercise for cardiovascular disease that is accelerated by the presence of chronic kidney disease remains unknown. The present study utilized apolipoprotein E knockout mice with 5/6 nephrectomy as a model of combined kidney disease and cardiovascular disease to investigate the effect of exercise on aortic plaque formation, vascular function and systemic inflammation. Animals were randomly assigned to nephrectomy or control and then to either voluntary wheel running exercise or sedentary. Following 12-weeks, aortic plaque area was significantly (p<0.05, d=1.2) lower in exercising nephrectomised mice compared to sedentary nephrectomised mice. There was a strong, negative correlation between average distance run each week and plaque area in nephrectomised and control mice (r=–0.76, p=0.048 and r=–0.73, p=0.062; respectively). In vitro aortic contraction and endothelial-independent and endothelial-dependent relaxation were not influenced by exercise (p>0.05). Nephrectomy increased IL-6 and TNF-α concentrations compared with control mice (p<0.001 and p<0.05, respectively), while levels of IL-10, MCP-1 and MIP-1α were not significantly influenced by nephrectomy or voluntary exercise (p>0.05). Exercise was an effective non-pharmacologic approach to slow cardiovascular disease in the presence of kidney disease in the apolipoprotein E knockout mouse.

Variation in the complex carbohydrate biosynthesis loci of Acinetobacter baumannii genomes

Extracellular polysaccharides are major immunogenic components of the bacterial cell envelope. However, little is known about their biosynthesis in the genus Acinetobacter, which includes A. baumannii, an important nosocomial pathogen. Whether Acinetobacter sp. produce a capsule or a lipopolysaccharide carrying an O antigen or both is not resolved. To explore these issues, genes involved in the synthesis of complex polysaccharides were located in 10 complete A. baumannii genome sequences, and the function of each of their products was predicted via comparison to enzymes with a known function. The absence of a gene encoding a WaaL ligase, required to link the carbohydrate polymer to the lipid A-core oligosaccharide (lipooligosaccharide) forming lipopolysaccharide, suggests that only a capsule is produced. Nine distinct arrangements of a large capsule biosynthesis locus, designated KL1 to KL9, were found in the genomes. Three forms of a second, smaller variable locus, likely to be required for synthesis of the outer core of the lipid A-core moiety, were designated OCL1 to OCL3 and also annotated. Each K locus includes genes for capsule export as well as genes for synthesis of activated sugar precursors, and for glycosyltransfer, glycan modification and oligosaccharide repeat-unit processing. The K loci all include the export genes at one end and genes for synthesis of common sugar precursors at the other, with a highly variable region that includes the remaining genes in between. Five different capsule loci, KL2, KL6, KL7, KL8 and KL9 were detected in multiply antibiotic resistant isolates belonging to global clone 2, and two other loci, KL1 and KL4, in global clone 1. This indicates that this region is being substituted repeatedly in multiply antibiotic resistant isolates from these clones.

Modified MLVA for genotyping Queensland invasive Streptococcus pneumoniae

Background Globally, over 800 000 children under five die each year from infectious diseases caused by Streptococcus pneumoniae. To understand genetic relatedness between isolates, study transmission routes, assess the impact of human interventions e.g. vaccines, and determine infection sources, genotyping methods are required. The ‘gold standard’ genotyping method, Multi-Locus Sequence Typing (MLST), is useful for long-term and global studies. Another genotyping method, Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA), has emerged as a more discriminatory, inexpensive and faster technique; however there is no universally accepted method and it is currently suitable for short-term and localised epidemiology studies. Currently Australia has no national MLST database, nor has it adopted any MLVA method for short-term or localised studies. This study aims to improve S. pneumoniae genotyping methods by modifying the existing MLVA techniques to be more discriminatory, faster, cheaper and technically less demanding than previously published MLVA methods and MLST. Methods Four different MLVA protocols, including a modified method, were applied to 317 isolates of serotyped invasive S. pneumoniae isolated from sterile body sites of Queensland children under 15 years from 2007–2012. MLST was applied to 202 isolates for comparison. Results The modified MLVA4 is significantly more discriminatory than the ‘gold standard’ MLST method. MLVA4 has similar discrimination compared to other MLVA techniques in this study). The failure to amplify particular loci in previous MLVA methods were minimised in MLVA4. Failure to amplify BOX-13 and Spneu19 were found to be serotype specific. Conclusion We have modified a highly discriminatory MLVA technique for genotyping Queensland invasive S. pneumoniae. MLVA4 has the ability to enhance our understanding of the pneumococcal epidemiology and the changing genetics of the pneumococcus in localised and short-term studies.

Effects of resistance training on measures of muscular strength in people with Parkinson’s disease: A systematic review and meta-analysis

Objective The aim of this systematic review and meta-analysis was to determine the overall effect of resistance training (RT) on measures of muscular strength in people with Parkinson’s disease (PD). Methods Controlled trials with parallel-group-design were identified from computerized literature searching and citation tracking performed until August 2014. Two reviewers independently screened for eligibility and assessed the quality of the studies using the Cochrane risk-of-bias-tool. For each study, mean differences (MD) or standardized mean differences (SMD) and 95% confidence intervals (CI) were calculated for continuous outcomes based on between-group comparisons using post-intervention data. Subgroup analysis was conducted based on differences in study design. Results Nine studies met the inclusion criteria; all had a moderate to high risk of bias. Pooled data showed that knee extension, knee flexion and leg press strength were significantly greater in PD patients who undertook RT compared to control groups with or without interventions. Subgroups were: RT vs. control-without-intervention, RT vs. control-with-intervention, RT-with-other-form-of-exercise vs. control-without-intervention, RT-with-other-form-of-exercise vs. control-with-intervention. Pooled subgroup analysis showed that RT combined with aerobic/balance/stretching exercise resulted in significantly greater knee extension, knee flexion and leg press strength compared with no-intervention. Compared to treadmill or balance exercise it resulted in greater knee flexion, but not knee extension or leg press strength. RT alone resulted in greater knee extension and flexion strength compared to stretching, but not in greater leg press strength compared to no-intervention. Discussion Overall, the current evidence suggests that exercise interventions that contain RT may be effective in improving muscular strength in people with PD compared with no exercise. However, depending on muscle group and/or training dose, RT may not be superior to other exercise types. Interventions which combine RT with other exercise may be most effective. Findings should be interpreted with caution due to the relatively high risk of bias of most studies.