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/.     

Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen,Legionella pneumophila

Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector–effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector–effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, to query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila‐translocated substrates. While capturing all known examples of effector–effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct—a hallmark of an emerging class of proteins called metaeffectors, or “effectors of effectors”. Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Metaeffectors, along with other, indirect, forms of effector–effector modulation, may be a common feature of many intracellular pathogens—with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell.     

Neuroprotective effects of Argon are mediated via an ERK‐1/2 dependent regulation of heme‐oxygenase‐1 in retinal ganglion cells

Retinal ischemia and reperfusion injuries (R‐IRI) damage neuronal tissue permanently. Recently, we demonstrated that Argon exerts anti‐apoptotic and protective properties. The molecular mechanism remains unclear. We hypothesized that Argon inhalation exert neuroprotective effects in rats retinal ganglion cells (RGC) via an ERK‐1/2 dependent regulation of heat‐shock proteins. Inhalation of Argon (75 Vol%) was performed after R‐IRI on the rats′ left eyes for 1 h immediately or with delay. Retinal tissue was harvested after 24 h to analyze mRNA and protein expression of heat‐shock proteins ?70, ?90 and heme‐oxygenase‐1, mitogen‐activated protein kinases (p38, JNK, ERK‐1/2) and histological changes. To analyze ERK dependent effects, the ERK inhibitor PD98059 was applicated prior to Argon inhalation. RGC count was analyzed 7 days after injury. Statistics were performed using anova . Argon significantly reduced the R‐IRI‐affected heat‐shock protein expression (p < 0.05). While Argon significantly induced ERK‐1/2 expression (p < 0.001), inhibition of ERK‐1/2 before Argon inhalation resulted in significantly lower vital RGCs (p < 0.01) and increase in heme‐oxygenase‐1 (p < 0.05). R‐IRI‐induced RGC loss was reduced by Argon inhalation (p < 0.001). Immunohistochemistry suggested ERK‐1/2 activation in Müller cells. We conclude, that Argon treatment protects R‐IRI‐induced apoptotic loss of RGC via an ERK‐1/2 dependent regulation of heme‐oxygenase‐1.

     

A Systems-Level Interrogation Identifies Regulators of Drosophila Blood Cell Number and Survival

In multicellular organisms, cell number is typically determined by a balance of intracellular signals that positively and negatively regulate cell survival and proliferation. Dissecting these signaling networks facilitates the understanding of normal development and tumorigenesis. Here, we study signaling by the Drosophila PDGF/VEGF Receptor (Pvr) in embryonic blood cells (hemocytes) and in the related cell line Kc as a model for the requirement of PDGF/VEGF receptors in vertebrate cell survival and proliferation. The system allows the investigation of downstream and parallel signaling networks, based on the ability of Pvr to activate Ras/Erk, Akt/TOR, and yet-uncharacterized signaling pathway/s, which redundantly mediate cell survival and contribute to proliferation. Using Kc cells, we performed a genome wide RNAi screen for regulators of cell number in a sensitized, Pvr deficient background. We identified the receptor tyrosine kinase (RTK) Insulin-like receptor (InR) as a major Pvr Enhancer, and the nuclear hormone receptors Ecdysone receptor (EcR) and ultraspiracle (usp), corresponding to mammalian Retinoid X Receptor (RXR), as Pvr Suppressors. In vivo analysis in the Drosophila embryo revealed a previously unrecognized role for EcR to promote apoptotic death of embryonic blood cells, which is balanced with pro-survival signaling by Pvr and InR. Phosphoproteomic analysis demonstrates distinct modes of cell number regulation by EcR and RTK signaling. We define common phosphorylation targets of Pvr and InR that include regulators of cell survival, and unique targets responsible for specialized receptor functions. Interestingly, our analysis reveals that the selection of phosphorylation targets by signaling receptors shows qualitative changes depending on the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems.     

The role of very small fragments in conserving genetic diversity of a common tree in a hyper fragmented Brazilian Atlantic forest landscape

In hyper fragmented biomes, conservation of extant biota relies on preservation and proper management of remnants. The maintenance of genetic diversity and functional connectivity in a landscape context is probably key to long-term conservation of remnant populations. We measured the genetic diversity in seedlings and adults of tree Copaifera langsdorffii and evaluated whether edge and density-dependent effects drive natural regeneration in a set of very small and degraded Brazilian Atlantic forest fragments. We evaluated the role of small remnants in the conservation of genetic diversity in a hyper fragmented landscape and discuss the challenge of long-term population sustainability of such altered habitats. High genetic diversity in adults indicated these fragments are valuable targets for C. langsdorffii in situ conservation, but both genetic diversity and divergence among patches decreased in seedlings. In our landscape, regeneration increased as it neared edges and adults; suggesting this population is resilient to fragmentation. However, at a broader scale, current levels of gene flow have not been sufficient to prevent the loss of genetic diversity across generations. Restoration plans, even at a small scale, are necessary to promote fragment connectivity and spatially expand opportunities for the fairly restricted gene flow observed in this severely fragmented Brazilian Atlantic forest region.     

Combined metabolomics and proteomics reveals hypoxia as a cause of lower productivity on scale‐up to a 5000‐liter CHO bioprocess

Large‐scale bioprocessing is key to the successful manufacturing of a biopharmaceutical. However, cell viability and productivity are often lower in the scale‐up from laboratory to production. In this study, we analyzed CHO cells, which showed lower percent viabilities and productivity in a 5‐KL production scale bioreactor compared to a 20‐L bench‐top scale under seemingly identical process parameters. An increase in copper concentration in the media from 0.02 µM to 0.4 µM led to a doubling of percent viability in the production scale albeit still at a lower level than the bench‐top scale. Combined metabolomics and proteomics revealed the increased copper reduced the presence of reactive oxygen species (ROS) in the 5‐KL scale process. The reduction in oxidative stress was supported by the increased level of glutathione peroxidase in the lower copper level condition. The excess ROS was shown to be due to hypoxia (intermittent), as evidenced by the reduction in fibronectin with increased copper. The 20‐L scale showed much less hypoxia and thus less excess ROS generation, resulting in little to no impact to productivity with the increased copper in the media. The study illustrates the power of 'Omics in aiding in the understanding of biological processes in biopharmaceutical production.     

Active and adaptive Legionella CRISPR‐Cas reveals a recurrent challenge to the pathogen

Clustered regularly interspaced short palindromic repeats with CRISPR‐associated gene (CRISPR‐Cas) systems are widely recognized as critical genome defense systems that protect microbes from external threats such as bacteriophage infection. Several isolates of the intracellular pathogen Legionella pneumophila possess multiple CRISPR‐Cas systems (type I‐C, type I‐F and type II‐B), yet the targets of these systems remain unknown. With the recent observation that at least one of these systems (II‐B) plays a non‐canonical role in supporting intracellular replication, the possibility remained that these systems are vestigial genome defense systems co‐opted for other purposes. Our data indicate that this is not the case. Using an established plasmid transformation assay, we demonstrate that type I‐C, I‐F and II‐B CRISPR‐Cas provide protection against spacer targets. We observe efficient laboratory acquisition of new spacers under ‘priming’ conditions, in which initially incomplete target elimination leads to the generation of new spacers and ultimate loss of the invasive DNA. Critically, we identify the first known target of L. pneumophila CRISPR‐Cas: a 30 kb episome of unknown function whose interbacterial transfer is guarded against by CRISPR‐Cas. We provide evidence that the element can subvert CRISPR‐Cas by mutating its targeted sequences – but that primed spacer acquisition may limit this mechanism of escape. Rather than generally impinging on bacterial fitness, this element drives a host specialization event – with improved fitness in Acanthamoeba but a reduced ability to replicate in other hosts and conditions. These observations add to a growing body of evidence that host range restriction can serve as an existential threat to L. pneumophila in the wild.