EDISON-WMW: Exact Dynamic Programing Solution of the Wilcoxon–Mann–Whitney Test

In many research disciplines, hypothesis tests are applied to evaluate whether findings are statistically significant or could be explained by chance. The Wilcoxon–Mann–Whitney(WMW) test is among the most popular hypothesis tests in medicine and life science to analyze if two groups of samples are equally distributed. This nonparametric statistical homogeneity test is commonly applied in molecular diagnosis. Generally, the solution of the WMW test takes a high combinatorial effort for large sample cohorts containing a significant number of ties. Hence, P value is frequently approximated by a normal distribution. We developed EDISON-WMW, a new approach to calculate the exact permutation of the two-tailed unpaired WMW test without any corrections required and allowing for ties. The method relies on dynamic programing to solve the combinatorial problem of the WMW test efficiently. Beyond a straightforward implementation of the algorithm, we presented different optimization strategies and developed a parallel solution. Using our program,the exact P value for large cohorts containing more than 1000 samples with ties can be calculated within minutes. We demonstrate the performance of this novel approach on randomly-generated data, benchmark it against 13 other commonly-applied approaches and moreover evaluate molecular biomarkers for lung carcinoma and chronic obstructive pulmonary disease(COPD). We foundthat approximated P values were generally higher than the exact solution provided by EDISONWMW. Importantly, the algorithm can also be applied to high-throughput omics datasets, where hundreds or thousands of features are included. To provide easy access to the multi-threaded version of EDISON-WMW, a web-based solution of our algorithm is freely available at http://www.ccb.uni-saarland.de/software/wtest/.     

Post-translational regulation of CND41 protease activity in senescent tobacco leaves

The degradation of chloroplast proteins is an important occurrence in the mobilization of nutrients from senescing leaves to reproductive organs during senescence. Recently, we proved that tobacco CND41 protease is involved in Rubisco degradation and the translocation of nitrogen during senescence. In this study, we show the post-translational regulation of CND41 protease. Using very specific antibodies that were prepared against CND41-specific peptide (anti-Val 186 to Ser 206), immunoblot analysis clearly indicated a change in the accumulation and processing of CND41 during the maturation of leaves in whole plants. The developmental modification of CND41 was also observed in transgenic tobacco with constitutive expression of CND41 under cauliflower mosaic virus 35S promoter. Further studies of seedlings under senescence induced by combined treatment with nitrogen-starvation and high sucrose confirmed that the processing of CND41 was important for protease activity and senescence. A possible mechanism for the regulation of CND41 activity is discussed.     

Modeling Harvest Intensity of Sooty Shearwater Chicks by Rakiura Māori in New Zealand

Abstract: Cultural evidence suggests that sooty shearwater (Puffinus griseus) chicks have been harvested by Rakiura Māori on islands in southern New Zealand since prehistoric times. Concerns exist that modern harvests may be impacting sooty shearwater abundance. We modeled human-related and ecological determinants of harvest (total no. of individuals harvested) of sooty shearwater chicks on 11 islands and examined the relationship between shearwater abundance and harvesting rates (chicks/hr) and harvester behavior throughout the harvesting season. Models best explaining variation in harvest between harvesting areas (manu), for both the early and late parts of the harvesting season, included harvester-days (included in all models with change in deviance information criteria [ΔDIC], ΔDIC < 8.36 and ΔDIC < 11.5, for the early and late periods, respectively). Other harvest determinants included shearwater density, size of the manu, and number of people helping harvesters (all included in the top 5 models within ΔDIC = 2.25 for the late period). Areas harvested by several families under a common-property harvesting system had higher harvest intensity for their size (24% points higher, 95% credible interval 11–36%) than those managed as an exclusive resource for one family. The slowest harvesters spent more time harvesting but on average only harvested 36% (95% credible interval 15–65%) and 34% (95% credible interval 12–63%) of the harvest taken by the fastest harvesters during the early and late periods, respectively. Our results highlight the possibility of elevated harvest intensity as the population of harvesters increases. However, our models suggested that a corresponding reduction in harvesting rate at low prey densities during the most productive period could potentially regulate harvest intensity. Future research will integrate these results into prospective shearwater demographic models to assess the utility of a range of harvesting strategies in ensuring harvest sustainability.     

Anti-apoptotic genes Aven and E1B-19K enhance performance of BHK cells engineered to express recombinant factor VIII in batch and low perfusion cell culture

The engineering of production cell lines to express anti-apoptotic genes has been pursued in recent years due to potential process benefits, including enhanced cell survival, increased protein expression, and improved product quality. In this study, a baby hamster kidney cell line secreting recombinant factor VIII (BHK-FVIII) was engineered to express the anti-apoptotic genes Aven and E1B-19K. In high cell density shake flask culture evaluation, 11 clonal cell lines expressing either E1B-19K or a combination of Aven and E1B-19K showed improved survival compared to both parental and blank vector cell line controls. These cell lines exhibited lower caspase-3 activation and reduced Annexin-V binding compared to the controls. Parental and blank vector cell lines were less than 50% viable after 48 h of exposure to thapsigargin while cell lines expressing E1B-19K with or without Aven maintained viabilities approaching 90%. Subsequently, the best Aven-E1B-19K candidate cell line was compared to the parental cell line in 12-L perfusion bioreactor studies. Choosing the appropriate perfusion rates in bioreactors is a bioprocess optimization issue, so the bioreactors were operated at sequentially lower specific perfusion rates, while maintaining a cell density of 2 x 10(7) viable cells/mL. The viability of the parental cell line declined from nearly 100% at a perfusion rate of 0.5 nL/cell/day to below 80% viability, with caspase-3 activity exceeding 15%, at its lower perfusion limit of 0.15 nL/cell/day. In contrast, the Aven-E1B-19K cell line maintained an average viability of 94% and a maximum caspase-3 activity of 2.5% even when subjected to a lower perfusion minimum of 0.1 nL/cell/day. Factor VIII productivity, specific growth rate, and cell size decreased for both cell lines at lower perfusion rates, but the drop in all cases was larger for the parental cell line. Specific consumption of glucose and glutamine and production of lactate were consistently lower for the Aven-E1B-19K culture. Furthermore, the yield of ammonia from glutamine increased for the Aven-E1B-19K cell line relative to the parent to suggest altered metabolic pathways following anti-apoptosis engineering. These results demonstrate that expression of anti-apoptotic genes Aven and E1B-19K can increase the stability and robustness of an industrially relevant BHK-FVIII mammalian cell line over a wide range of perfusion rates.     

How to keep proliferative neural stem/progenitor cells

It has long been argued that cell cycle regulators such as cyclins, cyclin-dependent kinases and their inhibitors affect the fate of neuronal progenitor cells. Recently, we identified that cyclin D2, which localizes at the basal tip of the radial glial cell (i.e., the neural progenitor in the developing neocortex), functions to give differential cell fates to its daughter cells just after cell division. This basally biased localization is due to transportation of cyclin D2 mRNA via its unique cis-regulatory sequence and local translation into cyclin D2 protein at the basal endfoot. During division of the neural progenitor cells, cyclin D2 protein is inherited by the daughter cell that retain the basal process, resulting in asymmetric distribution of cyclin D2 protein between the two daughter cells. Cyclin D2 is similarly localized in the human fetal cortical primordium, suggesting a common mechanism for the maintenance of neural progenitors and a possible scenario in evolution of primate brains. Here we introduce our recent findings and discuss how cyclin D2 functions in mammalian brain development and evolution.     

The yeast autophagy protease Atg4 is regulated by thioredoxin

Autophagy is a membrane-trafficking process whereby double-membrane vesicles called autophagosomes engulf and deliver intracellular material to the vacuole for degradation. Atg4 is a cysteine protease with an essential function in autophagosome formation. Mounting evidence suggests that reactive oxygen species may play a role in the control of autophagy and could regulate Atg4 activity but the precise mechanisms remain unclear. In this study, we showed that reactive oxygen species activate autophagy in the model yeast Saccharomyces cerevisiae and unraveled the molecular mechanism by which redox balance controls Atg4 activity. A combination of biochemical assays, redox titrations, and site-directed mutagenesis revealed that Atg4 is regulated by oxidoreduction of a single disulfide bond between Cys338 and Cys394. This disulfide has a low redox potential and is very efficiently reduced by thioredoxin, suggesting that this oxidoreductase plays an important role in Atg4 regulation. Accordingly, we found that autophagy activation by rapamycin was more pronounced in a thioredoxin mutant compared with wild-type cells. Moreover, in vivo studies indicated that Cys338 and Cys394 are required for the proper regulation of autophagosome biogenesis, since mutation of these cysteines resulted in increased recruitment of Atg8 to the phagophore assembly site. Thus, we propose that the fine-tuning of Atg4 activity depending on the intracellular redox state may regulate autophagosome formation.