Influence of extracellular pH on the cytotoxicity, cellular accumulation, and DNA interaction of novel pH-sensitive 2-aminoalcoholatoplatinum(II) complexes

Extracellular acidity is a frequent pathophysiological condition of solid tumors offering possibilities for improving the tumor selectivity of molecular therapy. This might be accomplished by prodrugs with low systemic toxicity, attaining their full antitumor potency only under acidic conditions, such as bis(2-aminoalcoholato-κ²N,O)platinum(II) complexes that are activated by protonation of alcoholato oxygen, resulting in cleavage of platinum–oxygen bonds. In this work, we examined whether the pH dependency of such compounds is reflected in differential biological activity in vitro. In particular, the pH dependence of cytotoxicity, cellular accumulation, DNA platination, GMP binding, effects on DNA secondary structure, cell cycle alterations, and induction of apoptosis was investigated. Enhanced cytotoxicity of five of these complexes in non-small-cell lung cancer (A549) and colon carcinoma (HT-29) cells at pH 6.0 in comparison with pH 7.4 was confirmed: 50 % growth inhibition concentrations ranged from 42 to 214 μM in A549 cells and from 35 to 87 μM in HT-29 cells at pH 7.4 and decreased at pH 6.0 to 11–50 and 7.3–25 μM, respectively. The effects induced by all five pH-sensitive compounds involve increased 5′-GMP binding, cellular accumulation, and DNA platination as well as stronger effects on DNA secondary structure at pH 6.0 than at pH 7.4. As exemplified by treatment of A549 cells with a 2-amino-4-methyl-1-pentanolato complex, induction of apoptosis is enhanced at pH 6.5. These results confirm the increased reactivity and in vitro activity of these compounds under slightly acidic conditions, encouraging further evaluation of ring-closed aminoalcoholatoplatinum(II) derivatives in solid tumors in vivo.     

Matrix effects during monitoring of antibody and host cell proteins using attenuated total reflection spectroscopy

Production of recombinant proteins, e.g. antibodies, requires constant real‐time monitoring to optimize yield and quality attributes and to respond to changing production conditions, such as host cell protein (HCP) titers. To date, this monitoring of mammalian cell culture‐based processes is done using laborious and time consuming enzyme‐linked immunosorbent assays (ELISA), two‐dimensional sodium dodecylsulphate polyacrylamide gel electrophoresis, and chromatography‐based systems. Measurements are usually performed off‐line, requiring regular sample withdrawal associated with increased contamination risk. As information is obtained retrospectively, the reaction time to adapt to process changes is too long, leading to lower yield and higher costs. To address the resulting demand for continuous online‐monitoring systems, we present a feasibility study using attenuated total reflection spectroscopy (ATR) to monitor mAb and HCP levels of NS0 cell culture in situ, taking matrix effects into account. Fifty‐six NS0 cell culture samples were treated with polyelectrolytes for semi‐selective protein precipitation. Additionally, part of the samples was subjected to filtration prior to analysis, to change the background matrix and evaluate effects on chemometric quantification models. General models to quantify HCP and mAb in both filtered and unfiltered matrix showed lower prediction accuracy compared to models designed for a specific matrix. HCP quantification in the range of 2,000–55,000 ng mL^?1 using specific models was accurate for most samples, with results within the accepted limit of an ELISA assay. In contrast, mAb prediction was less accurate, predicting mAb in the range of 0.2–1.7 g L^?1. As some samples deviated substantially from reference values, further investigations elucidating the suitability of ATR for monitoring are required. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Perturbation of the mutated EGFR interactome identifies vulnerabilities and resistance mechanisms

We hypothesized that elucidating the interactome of epidermal growth factor receptor (EGFR) forms that are mutated in lung cancer, via global analysis of protein–protein interactions, phosphorylation, and systematically perturbing the ensuing network nodes, should offer a new, more systems‐level perspective of the molecular etiology. Here, we describe an EGFR interactome of 263 proteins and offer a 14‐protein core network critical to the viability of multiple EGFR‐mutated lung cancer cells. Cells with acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) had differential dependence of the core network proteins based on the underlying molecular mechanisms of resistance. Of the 14 proteins, 9 are shown to be specifically associated with survival of EGFR‐mutated lung cancer cell lines. This included EGFR, GRB2, MK12, SHC1, ARAF, CD11B, ARHG5, GLU2B, and CD11A. With the use of a drug network associated with the core network proteins, we identified two compounds, midostaurin and lestaurtinib, that could overcome drug resistance through direct EGFR inhibition when combined with erlotinib. Our results, enabled by interactome mapping, suggest new targets and combination therapies that could circumvent EGFR TKI resistance.     

Bioreactors in tissue engineering—principles,applications and commercial constraints

Bioreactor technology is vital for tissue engineering. Usually, bioreactors are used to provide a tissue-specific physiological in vitro environment during tissue maturation. In addition to this most obvious application, bioreactors have the potential to improve the efficiency of the overall tissue-engineering concept. To date, a variety of bioreactor systems for tissue-specific applications have been developed. Of these, some systems are already commercially available. With bioreactor technology, various functional tissues of different types were generated and cultured in vitro. Nevertheless, these efforts and achievements alone have not yet led to many clinically successful tissue-engineered implants. We review possible applications for bioreactor systems within a tissue-engineering process and present basic principles and requirements for bioreactor development. Moreover, the use of bioreactor systems for the expansion of clinically relevant cell types is addressed. In contrast to cell expansion, for the generation of functional three-dimensional tissue equivalents, additional physical cues must be provided. Therefore, bioreactors for musculoskeletal tissue engineering are discussed. Finally, bioreactor technology is reviewed in the context of commercial constraints.     

The Absence of Mrp4 Has No Effect on the Recruitment of Neutrophils and Eosinophils into the Lung after LPS,Cigarette Smoke or Allergen Challenge

The multidrug resistance protein 4 (Mrp4) is an ATP-binding cassette transporter that is capable of exporting the second messenger cAMP from cells, a process that might regulate cAMP-mediated anti-inflammatory processes. However, using LPS- or cigarette smoke (CS)-inflammation models, we found that neutrophil numbers in the bronchoalveolar lavage fluid (BALF) were similar in Mrp4^−/− and Mrp4^+/+ mice treated with LPS or CS. Similarly, neutrophil numbers were not reduced in the BALF of LPS-challenged wt mice after treatment with 10 or 30 mg/kg of the Mrp1/4 inhibitor MK571. The absence of Mrp4 also had no impact on the influx of eosinophils or IL-4 and IL-5 levels in the BALF after OVA airway challenge in mice sensitized with OVA/alum. LPS-induced cytokine release in whole blood ex vivo was also not affected by the absence of Mrp4. These data clearly suggest that Mrp4 deficiency alone is not sufficient to reduce inflammatory processes in vivo. We hypothesized that in combination with PDE4 inhibitors, used at suboptimal concentrations, the anti-inflammatory effect would be more pronounced. However, LPS-induced neutrophil recruitment into the lung was no different between Mrp4^−/− and Mrp4^+/+ mice treated with 3 mg/kg Roflumilast. Finally, the single and combined administration of 10 and 30 mg/kg MK571 and the specific breast cancer resistance protein (BCRP) inhibitor KO143 showed no reduction of LPS-induced TNFα release into the BALF compared to vehicle treated control animals. Similarly, LPS-induced TNFα release in murine whole blood of Mrp4^+/+ or Mrp4^−/− mice was not reduced by KO143 (1, 10 µM). Thus, BCRP seems not to be able to compensate for the absence or inhibition of Mrp4 in the used models. Taken together, our data suggest that Mrp4 is not essential for the recruitment of neutrophils into the lung after LPS or CS exposure or of eosinophils after allergen exposure.     

Long-Term Results after Proximal Thoracic Aortic Redo Surgery

Objective

To evaluate early and mid-term results in patients undergoing proximal thoracic aortic redo surgery.

Methods

We analyzed 60 patients (median age 60 years, median logistic EuroSCORE 40) who underwent proximal thoracic aortic redo surgery between January 2005 and April 2012. Outcome and risk factors were analyzed.

Results

In hospital mortality was 13%, perioperative neurologic injury was 7%. Fifty percent of patients underwent redo surgery in an urgent or emergency setting. In 65%, partial or total arch replacement with or without conventional or frozen elephant trunk extension was performed. The preoperative logistic EuroSCORE I confirmed to be a reliable predictor of adverse outcome- (ROC 0.786, 95%CI 0.64–0.93) as did the new EuroSCORE II model: ROC 0.882 95%CI 0.78–0.98. Extensive individual logistic EuroSCORE I levels more than 67 showed an OR of 7.01, 95%CI 1.43–34.27. A EuroSCORE II larger than 28 showed an OR of 4.44 (95%CI 1.4–14.06). Multivariate logistic regression analysis identified a critical preoperative state (OR 7.96, 95%CI 1.51–38.79) but not advanced age (OR 2.46, 95%CI 0.48–12.66) as the strongest independent predictor of in-hospital mortality. Median follow-up was 23 months (1–52 months). One year and five year actuarial survival rates were 83% and 69% respectively. Freedom from reoperation during follow-up was 100%.

Conclusions

Despite a substantial early attrition rate in patients presenting with a critical preoperative state, proximal thoracic aortic redo surgery provides excellent early and mid-term results. Higher EuroSCORE I and II levels and a critical preoperative state but not advanced age are independent predictors of in-hospital mortality. As a consequence, age alone should no longer be regarded as a contraindication for surgical treatment in this particular group of patients.     

SNPflow: A Lightweight Application for the Processing,Storing and Automatic Quality Checking of Genotyping Assays

Single nucleotide polymorphisms (SNPs) play a prominent role in modern genetics. Current genotyping technologies such as Sequenom iPLEX, ABI TaqMan and KBioscience KASPar made the genotyping of huge SNP sets in large populations straightforward and allow the generation of hundreds of thousands of genotypes even in medium sized labs. While data generation is straightforward, the subsequent data conversion, storage and quality control steps are time-consuming, error-prone and require extensive bioinformatic support. In order to ease this tedious process, we developed SNPflow. SNPflow is a lightweight, intuitive and easily deployable application, which processes genotype data from Sequenom MassARRAY (iPLEX) and ABI 7900HT (TaqMan, KASPar) systems and is extendible to other genotyping methods as well. SNPflow automatically converts the raw output files to ready-to-use genotype lists, calculates all standard quality control values such as call rate, expected and real amount of replicates, minor allele frequency, absolute number of discordant replicates, discordance rate and the p-value of the HWE test, checks the plausibility of the observed genotype frequencies by comparing them to HapMap/1000-Genomes, provides a module for the processing of SNPs, which allow sex determination for DNA quality control purposes and, finally, stores all data in a relational database. SNPflow runs on all common operating systems and comes as both stand-alone version and multi-user version for laboratory-wide use. The software, a user manual, screenshots and a screencast illustrating the main features are available at http://genepi-snpflow.i-med.ac.at.     

Trade‐offs between land and water requirements for large‐scale bioenergy production

Bioenergy is expected to play an important role in the future energy mix as it can substitute fossil fuels and contribute to climate change mitigation. However, large‐scale bioenergy cultivation may put substantial pressure on land and water resources. While irrigated bioenergy production can reduce the pressure on land due to higher yields, associated irrigation water requirements may lead to degradation of freshwater ecosystems and to conflicts with other potential users. In this article, we investigate the trade‐offs between land and water requirements of large‐scale bioenergy production. To this end, we adopt an exogenous demand trajectory for bioenergy from dedicated energy crops, targeted at limiting greenhouse gas emissions in the energy sector to 1100 Gt carbon dioxide equivalent until 2095. We then use the spatially explicit global land‐ and water‐use allocation model MAgPIE to project the implications of this bioenergy target for global land and water resources. We find that producing 300 EJ yr^?1 of bioenergy in 2095 from dedicated bioenergy crops is likely to double agricultural water withdrawals if no explicit water protection policies are implemented. Since current human water withdrawals are dominated by agriculture and already lead to ecosystem degradation and biodiversity loss, such a doubling will pose a severe threat to freshwater ecosystems. If irrigated bioenergy production is prohibited to prevent negative impacts of bioenergy cultivation on water resources, bioenergy land requirements for meeting a 300 EJ yr^?1 bioenergy target increase substantially (+ 41%) – mainly at the expense of pasture areas and tropical forests. Thus, avoiding negative environmental impacts of large‐scale bioenergy production will require policies that balance associated water and land requirements.     

Turning Up the Heat on a Hotspot: DNA Barcodes Reveal 80% More Species of Geometrid Moths along an Andean Elevational Gradient

We sampled 14,603 geometrid moths along a forested elevational gradient from 1020–3021 m in the southern Ecuadorian Andes, and then employed DNA barcoding to refine decisions on species boundaries initially made by morphology. We compared the results with those from an earlier study on the same but slightly shorter gradient that relied solely on morphological criteria to discriminate species. The present analysis revealed 1857 putative species, an 80% increase in species richness from the earlier study that detected only 1010 species. Measures of species richness and diversity that are less dependent on sample size were more than twice as high as in the earlier study, even when analysis was restricted to an identical elevational range. The estimated total number of geometrid species (new dataset) in the sampled area is 2350. Species richness at single sites was 32–43% higher, and the beta diversity component rose by 43–51%. These impacts of DNA barcoding on measures of richness reflect its capacity to reveal cryptic species that were overlooked in the first study. The overall results confirmed unique diversity patterns reported in the first investigation. Species diversity was uniformly high along the gradient, declining only slightly above 2800 m. Species turnover also showed little variation along the gradient, reinforcing the lack of evidence for discrete faunal zones. By confirming these major biodiversity patterns, the present study establishes that incomplete species delineation does not necessarily conceal trends of biodiversity along ecological gradients, but it impedes determination of the true magnitude of diversity and species turnover.