STAT3-dependent analysis reveals PDK4 as independent predictor of recurrence in prostate cancer.

Prostate cancer (PCa) has a broad spectrum of clinical behavior; hence, biomarkers are urgently needed for risk stratification. Here, we aim to find potential biomarkers for risk stratification, by utilizing a gene co-expression network of transcriptomics data in addition to laser-microdissected proteomics from human and murine prostate FFPE samples. We show up-regulation of oxidative phosphorylation (OXPHOS) in PCa on the transcriptomic level and up-regulation of the TCA cycle/OXPHOS on the proteomic level, which is inversely correlated to STAT3 expression. We hereby identify gene expression of pyruvate dehydrogenase kinase 4 (PDK4), a key regulator of the TCA cycle, as a promising independent prognostic marker in PCa. PDK4 predicts disease recurrence independent of diagnostic risk factors such as grading, staging, and PSA level. Therefore, low PDK4 is a promising marker for PCa with dismal prognosis.

Human Placenta Laminin-111 as a Multifunctional Protein for Tissue Engineering and Regenerative Medicine.

Laminins are major components of all basement membranes surrounding nerve or vascular tissues. In particular laminin-111, the prototype of the family, facilitates a large spectrum of fundamental cellular responses in all eukaryotic cells. Laminin-111 is a biomaterial frequently used in research, however it is primarily isolated from non-human origin or produced with time-intensive recombinant techniques at low yield.Here, we describe an effective method for isolating laminin-111 from human placenta, a clinical waste material, for various tissue engineering applications. By extraction with Tris-NaCl buffer combined with non-protein-denaturation ammonium sulfate precipitation and rapid tangential flow filtration steps, we could effectively isolate native laminin-111 within only 4 days. The resulting material was biochemically characterized using a combination of dot blot, SDS-PAGE, Western blot and HPLC-based amino acid analysis. Cytocompatibility studies demonstrated that the isolated laminin-111 promotes rapid and efficient adhesion of primary Schwann cells. In addition, the bioactivity of the isolated laminin-111 was demonstrated by (a) using the material as a substrate for outgrowth of NG 108-15 neuronal cell lines and (b) promoting the formation of interconnected vascular networks by GFP-expressing human umbilical vein endothelial cells.In summary, the isolation procedure of laminin-111 as described here from human placenta tissue, fulfills many demands for various tissue engineering and regenerative medicine approaches and therefore may represent a human alternative to various classically used xenogenic standard materials.

Model-Based Methods in the Biopharmaceutical Process Lifecycle.

Model-based methods are increasingly used in all areas of biopharmaceutical process technology. They can be applied in the field of experimental design, process characterization, process design, monitoring and control. Benefits of these methods are lower experimental effort, process transparency, clear rationality behind decisions and increased process robustness. The possibility of applying methods adopted from different scientific domains accelerates this trend further. In addition, model-based methods can help to implement regulatory requirements as suggested by recent Quality by Design and validation initiatives. The aim of this review is to give an overview of the state of the art of model-based methods, their applications, further challenges and possible solutions in the biopharmaceutical process life cycle. Today, despite these advantages, the potential of model-based methods is still not fully exhausted in bioprocess technology. This is due to a lack of (i) acceptance of the users, (ii) user-friendly tools provided by existing methods, (iii) implementation in existing process control systems and (iv) clear workflows to set up specific process models. We propose that model-based methods be applied throughout the lifecycle of a biopharmaceutical process, starting with the set-up of a process model, which is used for monitoring and control of process parameters, and ending with continuous and iterative process improvement via data mining techniques.

A control strategy to investigate the relationship between specific productivity and high-mannose glycoforms in CHO cells.

The integration of physiological knowledge into process control strategies is a cornerstone for the improvement of biopharmaceutical cell culture technologies. The present contribution investigates the applicability of specific productivity as a physiological control parameter in a cell culture process producing a monoclonal antibody (mAb) in CHO cells. In order to characterize cell physiology, the on-line oxygen uptake rate (OUR) was monitored and the time-resolved specific productivity was calculated as physiological parameters. This characterization enabled to identify the tight link between the deprivation of tyrosine and the decrease in cell respiration and in specific productivity. Subsequently, this link was used to control specific productivity by applying different feeding profiles. The maintenance of specific productivity at various levels enabled to identify a correlation between the rate of product formation and the relative abundance of high-mannose glycoforms. An increase in high mannose content was assumed to be the result of high specific productivity. Furthermore, the high mannose content as a function of cultivation pH and specific productivity was investigated in a design of experiment approach. This study demonstrated how physiological parameters could be used to understand interactions between process parameters, physiological parameters, and product quality attributes.

Prevocational exposure to public health in the Kimberley: a pathway to rural, remote and public health practice.

OBJECTIVE: To evaluate the Kimberley Population Health Unit (KPHU) prevocational public health placement in terms of its contribution to resident medical officers' (RMOs') knowledge, skills, career path and aspirations. DESIGN: All RMOs who had completed a public health placement at the KPHU (n=27) during 2001-2012 were invited to complete an online survey in September 2012. SETTING: The KPHU, based in Broome, provides population health services to the Kimberley region, far north Western Australia. MAIN OUTCOME MEASURES: The extent to which RMOs perceived the development of public health skills and knowledge during the placement, and the degree to which RMOs believe this placement influenced future career pathways and their current practice. RESULTS: Twenty-three RMOs (85%) completed the survey. Sixty per cent are currently working in general practice or public health medicine; of these, 43% have returned to the Kimberley. Over 70% reported that the placement developed their knowledge of public health and Aboriginal health to a 'great' or 'very great' extent. Sixty-one per cent felt that their placement influenced their future desire to work in public health 'a lot' or 'a great extent'. CONCLUSION: This placement provides a unique opportunity for RMOs to undertake public health and Aboriginal health work in a remote setting. Given the increasing demand for prevocational placements, the value of imparting sound public health knowledge to the next generation of doctors and the urgent need to recruit and retain rural doctors, this placement provides a potential model that could be expanded to other locations.

Continuity of care: why it matters and what we can do.

Continuity of care matters; however, expansion and specialisation of the health system tends to fragment care. Continuity of care is accompanied by a range of patient benefits, including reduced all-cause mortality; lower rates of hospital presentation and preventable admission; and improved patient satisfaction. Potential concerns have been raised about some aspects of continuity of care, but these are outweighed by the perceived benefits. There are many barriers to achieving continuity, especially in rural and remote settings. Some practical solutions have been proposed that include adapting clinic procedures, utilising a small team approach, improving staff retention and ongoing advocacy.

Lecithin is the key material attribute in soy bean oil affecting filamentous bioprocesses.

Complex raw materials are widely used as supplements in biopharmaceutical production processes due to their positive effect on biomass growth and productivity at low cost. However, their use negatively impacts process reproducibility due to high lot-to-lot variability which contradicts current regulatory guidelines. In this study we investigated crude soy bean oil (SBO) which is a common complex raw material for filamentous fungi. We demonstrated that lecithin, which we define as phosphatidylcholines, is in fact the key material attribute in crude SBO positively affecting fungal growth and consequently productivity. The methodological toolbox we present here allows the straightforward isolation of lecithin from crude SBO, its semi-quantification by HPLC and the consequent supplementation thereof in defined amounts. Thus, over-dosage and potential resulting negative impacts on fungal growth and productivity can be omitted.

Amino Acid Signature in Human Melanoma Cell Lines from Different Disease Stages.

Cancer cells rewire metabolism to sustain high proliferation rates. Beside glycolysis and glutaminolysis, amino acids substitute as energy source, feed fatty acid biosynthesis and represent part of the secretome of transformed cells, including melanoma. We have therefore investigated acetate, pyruvate and the amino acid composition of the secretome of human melanoma cells representing the early slow (WM35, WM278, WM793b and VM21) and metastatic fast (A375, 518a2, 6F and WM8) growth phase in order to identify possible signalling components within these profiles. Proliferation assays and a principle component analysis revealed a stringent difference between the fast and slow growing melanoma cells. Moreover, upon inhibition of the mevalonate pathway, glutamic acid and alanine were identified as the central difference in the conditional media. A supplementation of the media with glutamic acid and the combination with alanine significantly accelerated the proliferation, migration and invasion of early stage melanoma cells, but not metastatic cells. Finally, the inhibition of the mevalonate pathway abolished the growth advantage of the melanoma cells in a time dependent manner. Taken together, these data corroborate a stage specific response in growth and aggressiveness to extracellular glutamic acid and alanine, indicative for microenvironmental signalling of individual amino acids.

Prediction of filamentous process performance attributes by CSL quality assessment using mid-infrared spectroscopy and chemometrics.

Every biopharmaceutical production process aims for control strategies to achieve process robustness in order to ensure consistent product quality. Process variability can origin from process parameters, the biological nature as well as from high lot-to-lot variability of raw materials. In filamentous processes raw materials with very complex matrices, such as corn steep liquor (CSL), are used, which are especially challenging to characterize. In this study, CSL was characterized in detail for its ingredients presenting an overall composition of its matrix of 50 analyzed components (19 amino acids, 5 organic acids, 8 reducing sugars, 7 water-soluble vitamins and 11 trace elements/minerals) in order to facilitate analytical reduction to fingerprinting methods FT-MIR was evaluated as fast and non-destructive spectroscopic fingerprinting method for adequate assessment of CSL quality. Feasibility of this method was shown by the correlation of certain bands in the spectra to substance groups present in CSL, such as the Amide I and II band and amino acids, respectively. Additionally, applicability of FT-MIR could be shown for classification of different CSL lots differing in provider and corn quality as well as for predictability of process performance attributes. The latter was demonstrated on a fed-batch filamentous fungi process for the production of antibiotics. By multivariate data analysis, it could be shown that CSL quality assessment via FT-MIR can be used for the prediction of maximal biomass generated in the process, with a correlation coefficient R2 of 0.964, as well as for the prediction of an unwanted impurity. The combination of a fast and easy method for CSL quality assessment and correlations of this quality with process performance attributes may facilitate the establishment of a risk-based acceptance criteria for raw material quality release of CSL. As CSL is a frequent used raw material, we believe that this method will also be useful for other processes and that CSL quality assessment is of high relevance in academia and industry.

An Effective Method of Atelocollagen Type 1/3 Isolation from Human Placenta and Its In Vitro Characterization in Two-Dimesional and Three-Dimensional Cell Culture Applications.

Pepsin-solubilized atelocollagen can be used to form highly complex three-dimensional matrices for a broad spectrum of tissue engineering applications. Moreover, it has a long history as a favorable biomaterial in pharmaceutical and medical industries. So far, the main sources for these approaches are collagens from xenogenic sources. Yet, these nonhuman collagens carry a risk of provoking immune reactions in patients. Here we describe an effective method of isolating atelocollagen type 1/3 (COL1/3) from human placenta. By combining a single pepsin digestion step with tangential flow filtration and further precipitation steps, we could purify COL1/3 within only 4 days of processing. The resulting COL1/3 was biochemically characterized by determining residual DNA content, proving the absence of impurities by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis combined with total amino acid quantification, identifying the isolated collagen types by Western blot analysis, and analyzing the spontaneous formation of fibrous structures on freeze-drying via scanning electron microscopy. Finally, the cytocompatibility of the isolated collagen was demonstrated in two dimensional using primary rat hepatocytes and in three dimensional by a sprouting assay of human umbilical vein endothelial cell. The isolation method described not only fulfills demands for cost-efficient bioengineering using a human waste material but also potentially increases overall safety for patients by use of homologous products.

Contribution of aldehyde dehydrogenase to mitochondrial bioactivation of nitroglycerin: evidence for the activation of purified soluble guanylate cyclase through direct formation of nitric oxide.

Vascular relaxation to GTN (nitroglycerin) and other antianginal nitrovasodilators requires bioactivation of the drugs to NO or a related activator of sGC (soluble guanylate cyclase). Conversion of GTN into 1,2-GDN (1,2-glycerol dinitrate) and nitrite by mitochondrial ALDH2 (aldehyde dehydrogenase 2) may be an essential pathway of GTN bioactivation in blood vessels. In the present study, we characterized the profile of GTN biotransformation by purified human liver ALDH2 and rat liver mitochondria, and we used purified sGC as a sensitive detector of GTN bioactivity to examine whether ALDH2-catalysed nitrite formation is linked to sGC activation. In the presence of mitochondria, GTN activated sGC with an EC50 (half-maximally effective concentration) of 3.77+/-0.83 microM. The selective ALDH2 inhibitor, daidzin (0.1 mM), increased the EC50 of GTN to 7.47+/-0.93 microM. Lack of effect of the mitochondrial poisons, rotenone and myxothiazol, suggested that nitrite reduction by components of the respiratory chain is not essential to sGC activation. However, since co-incubation of sGC with purified ALDH2 led to significant stimulation of cGMP formation by GTN that was completely inhibited by 0.1 mM daidzin and NO scavengers, ALDH2 may convert GTN directly into NO or a related species. Studies with rat aortic rings suggested that ALDH2 contributes to GTN bioactivation and showed that maximal relaxation to GTN occurred at cGMP levels that were only 3.4% of the maximal levels obtained with NO. Comparison of sGC activation in the presence of mitochondria with cGMP accumulation in rat aorta revealed a slightly higher potency of GTN to activate sGC in vitro compared with blood vessels. Our results suggest that ALDH2 catalyses the mitochondrial bioactivation of GTN by the formation of a reactive NO-related intermediate that activates sGC. In addition, the previous conflicting notion of the existence of a high-affinity GTN-metabolizing pathway operating in intact blood vessels but not in tissue homogenates is explained.

A German e-learning-training in the context of early preventive intervention and child protection: preliminary findings of a pre-post evaluation.

BACKGROUND: In recent years, a number of government-sponsored initiatives have been implemented in Germany that are focused on early preventive intervention in child protection. In response to the need for interdisciplinary training in this area, the internet-based e-learning program "Early Preventive Intervention and Child Protection" was developed for professionals in the child welfare and health care systems working with families with infants and toddlers. The program is currently undergoing evaluation for effectiveness and user satisfaction. METHODS: In a pre-post design, users are requested to complete questionnaires that assess three measures of expertise: theoretical knowledge of relevant fields, the ability to correctly identify subtle signals of infant communication, and the ability to assess maternal sensitivity. This article presents the contents of the program and the pre-training results (N = 1.294 participants). Descriptive analyses as well as Pearson correlations and Bonferroni corrections of error were conducted using the statistical program SPSS v. 21.0. RESULTS: The findings show that a wide range of professionals are making use of the program, and that their existing theoretical knowledge about early preventive intervention, as well as their ability to identify subtle signals of infant communication, is relatively good. However, their ability to assess maternal sensitivity, which is considered a crucial indicator for the risk of child abuse, was low. CONCLUSIONS: The outcome of the pre-training results indicates that professionals working in the area of child protection need to develop more capability in recognizing maternal sensitivity, in order to ensure early detection of families who are at risk and thus in need of support. Finally, the number of years of professional experience did not correlate with the scores on any of the three measures, which emphasizes the importance of providing interdisciplinary training in this area for all those working in child and family services, regardless of background.

Mortality due to rheumatic heart disease in the Kimberley 2001­2010.

OBJECTIVE: To determine the mortality burden of rheumatic heart disease (RHD) in the Kimberley. METHODS: A retrospective medical record audit was conducted for patients identified by the Western Australian (WA) RHD Program as deceased between 2001 and 2010. Patients with documented evidence strongly suggesting or confirming RHD were included in the study. Crude and age-standardised death rates were calculated. RESULTS: A total of 34 patients were identified as having RHD, 15 of whom died of RHD-attributable causes and 93% of whom were Aboriginal. The most common causes of death were arrhythmias and heart failure. The mean age at death of Aboriginal people was 41 years. The age-standardised death rate in Aboriginal people attributable to RHD in the Kimberley was 12.5 per 100,000 people per year, which is 1.6 times the rate of Indigenous Australians nationally. RHD contributed to 342 potential life years lost over the 10-year period. CONCLUSION: RHD contributes to significant premature mortality and higher rates of death in Aboriginal people in the Kimberley, which is consistent with other areas of northern Australia. While the recent establishment of the WA RHD Program will endeavour to improve mortality and morbidity due to RHD in the Kimberley, further research and investment is needed to address this disease of socioeconomic disadvantage.

Invasive Staphylococcus aureus Infections in Children in Tropical Northern Australia.

BACKGROUND: Despite a high burden of staphylococcal skin disease in children and high incidence of Staphylococcus aureus bacteremia in adult Indigenous populations in northern Australia, there are few studies describing incidence or clinical information of invasive S aureus (ISA) infections in children. METHODS: We conducted a retrospective review for all cases of S aureus bacteremia and sterile site infections, for children under 15 years, in northern Australia over a 4-year period (2007-2010). Cases were categorized as neonatal (<28 days) and pediatric (≥28 days). RESULTS: Forty-four cases (9 neonatal, 35 pediatric) were identified. The annual incidence of ISA was 27.9 cases per 100 000 population. Among pediatric cases, the annual incidence was significantly higher in the Indigenous (46.6) compared with the non-Indigenous (4.4) population (IRR: 10.6 [95% confidence interval, 3.8-41.4]). Pediatric infections were predominantly community-associated (86%). Clinical infection sites included osteoarticular (66%), pleuropulmonary (29%), and endocarditis (9%), and multifocal disease was common (20%). Eighty-three percent of pediatric cases presented with sepsis; 34% resulted in intensive care admission. Neonatal cases were all born prematurely; 89% were late-onset infections. Overall, 27% of infections were due to methicillin-resistant S aureus (MRSA). Compared with methicillin-sensitive S aureus (MSSA), there was no difference in severity or presentation in pediatric MRSA cases, but a higher proportion of MRSA cases were readmitted. CONCLUSIONS: The annual incidence of ISA infection in this study is among the highest described, largely due to a disproportionate burden in Indigenous children. Infections are frequently severe and infection with MRSA is common. Children presenting with suspected ISA in this region should be treated empirically for MRSA.