Customized media based on miniaturized screening improve growth rate and cell yield of methane-oxidizing bacteria of the genus Methylomonas

The growth of twelve methanotrophic strains within the genus Methylomonas, including the type strains of Methylomonas methanica and Methylomonas koyamae, was evaluated with 40 different variations of standard diluted nitrate mineral salts medium in 96-well microtiter plates. Unique profiles of growth preference were observed for each strain, showing a strong strain dependency for optimal growth conditions, especially with regards to the preferred concentration and nature of the nitrogen source. Based on the miniaturized screening results, a customized medium was designed for each strain, allowing the improvement of the growth of several strains in a batch setup, either by a reduction of the lag phase or by faster biomass accumulation. As such, the maintenance of fastidious strains could be facilitated while the growth of fast-growing Methylomonas strains could be further improved. Methylomonas sp. R-45378 displayed a 50 % increase in cell dry weight when grown in its customized medium and showed the lowest observed nitrogen and oxygen requirement of all tested strains. We demonstrate that the presented miniaturized approach for medium optimization is a simple tool allowing the quick generation of strain-specific growth preference data that can be applied downstream of an isolation campaign. This approach can also be applied as a first step in the search for strains with biotechnological potential, to facilitate cultivation of fastidious strains or to steer future isolation campaigns.     

Distinctive roles of receptor‐interacting protein kinases 1 and 3 in caspase‐independent cell death of L929

Upon tumour necrosis factor alpha (TNFα) stimulation, cells respond actively by way of cell survival, apoptosis or programmed necrosis. The receptor‐interacting proteins 1 (RIP1) and 3 (RIP3) are responsible for TNFα‐mediated programmed necrosis. To delineate the differential contributions of RIP3 and RIP1 to programmed necrosis, L929 cells were stimulated with TNFα, carbobenzoxy‐valyl‐alanyl‐aspartyl‐[O‐methyl]‐fluoromethylketone (zVAD) or zVAD along with TNFα following RNA interference against RIP1 and RIP3, respectively. RIP1 silencing did not protect cells from TNFα‐mediated cell death, while RIP3 down‐regulation made them refractory to TNFα. The heat shock protein 90 inhibitor geldanamycin (GA) down‐regulated both RIP1 and RIP3 expression, which rendered cells resistant to zVAD/TNFα‐mediated cell death but not to TNFα‐mediated cell death alone. Therefore, the protective effect of GA on zVAD/TNFα‐stimulated necrosis might be attributed to RIP3, not RIP1, down‐regulation. Pretreatment of L929 cells with rapamycin mitigated zVAD‐mediated cell death, while the autophagy inhibitor chloroquine did not affect necrotic cell death. Meanwhile, necrotic cell death by zVAD and TNFα was caused by reactive oxygen species generation and effectively diminished by lipid‐soluble butylated hydroxyanisole. Taken together, the results indicate that RIP1 and RIP3 can independently mediate death signals being transduced by two different death stimuli, zVAD and TNFα. Copyright © 2013 John Wiley & Sons, Ltd.     

Reliably Detecting Clinically Important Variants Requires Both Combined Variant Calls and Optimized Filtering Strategies

A diversity of tools is available for identification of variants from genome sequence data. Given the current complexity of incorporating external software into a genome analysis infrastructure, a tendency exists to rely on the results from a single tool alone. The quality of the output variant calls is highly variable however, depending on factors such as sequence library quality as well as the choice of short-read aligner, variant caller, and variant caller filtering strategy. Here we present a two-part study first using the high quality ‘genome in a bottle’ reference set to demonstrate the significant impact the choice of aligner, variant caller, and variant caller filtering strategy has on overall variant call quality and further how certain variant callers outperform others with increased sample contamination, an important consideration when analyzing sequenced cancer samples. This analysis confirms previous work showing that combining variant calls of multiple tools results in the best quality resultant variant set, for either specificity or sensitivity, depending on whether the intersection or union, of all variant calls is used respectively. Second, we analyze a melanoma cell line derived from a control lymphocyte sample to determine whether software choices affect the detection of clinically important melanoma risk-factor variants finding that only one of the three such variants is unanimously detected under all conditions. Finally, we describe a cogent strategy for implementing a clinical variant detection pipeline; a strategy that requires careful software selection, variant caller filtering optimizing, and combined variant calls in order to effectively minimize false negative variants. While implementing such features represents an increase in complexity and computation the results offer indisputable improvements in data quality.     

Relationship between Initial Telomere Length,Initial Telomerase Activity,Age, and Replicative Capacity of Nucleus Pulposus Chondrocytes in Human Intervertebral Discs: What Is a Predictor of Replicative Potential?

There is evidence that telomere length (TL), telomerase activity (TA), and age are related to the replicative potential of human nucleus pulposus chondrocytes (NPCs). However, it has not yet been established if any of these factors can serve as predictors of the replicative potential of NPCs. To establish predictors of the replicative potential of NPCs, we evaluated potential relationships between replicative capacity of NPCs, initial TL (telomere length at the first passage), initial TA (telomerase activity at the first passage), and age. Nucleus pulposus specimens were obtained from 14 patients of various ages undergoing discectomy. NPCs were serially cultivated until the end of their replicative lifespans. Relationships among cumulative population doubling level (PDL), initial TL, initial TA, and age were analyzed. Initial TA was negatively correlated with age (r = -0.674, P = 0.008). However, no correlation between initial TL and age was observed. Cumulative PDL was also negatively correlated with age (r = -0.585, P = 0.028). Although the cumulative PDL appeared to increase with initial TL or initial TA, this trend was not statistically significant. In conclusion, age is the sole predictor of the replicative potential of human NPCs, and replicative potential decreases with age. Initial TL and initial TA are not predictors of replicative potential, and can serve only as reference values.     

Growth medium sterilization using decomposition of peracetic acid for more cost-efficient production of omega-3 fatty acids by Aurantiochytrium

Bioprocess and Biosystems Engineering - Aurantiochytrium can produce significant amounts of omega-3 fatty acids, specifically docosahexaenoic acid and docosapentaenoic acid. Use of a glucose-based...