Exploiting the noise: improving biomarkers with ensembles of data analysis methodologies.

BackgroundThe advent of personalized medicine requires robust, reproducible biomarkers that indicate which treatment will maximize therapeutic benefit while minimizing side effects and costs. Numerous molecular signatures have been developed over the past decade to fill this need, but their validation and up-take into clinical settings has been poor. Here, we investigate the technical reasons underlying reported failures in biomarker validation for non-small cell lung cancer (NSCLC).MethodsWe evaluated two published prognostic multi-gene biomarkers for NSCLC in an independent 442-patient dataset. We then systematically assessed how technical factors influenced validation success.ResultsBoth biomarkers validated successfully (biomarker #1: hazard ratio (HR) 1.63, 95% confidence interval (CI) 1.21 to 2.19, P = 0.001; biomarker #2: HR 1.42, 95% CI 1.03 to 1.96, P = 0.030). Further, despite being underpowered for stage-specific analyses, both biomarkers successfully stratified stage II patients and biomarker #1 also stratified stage IB patients. We then systematically evaluated reasons for reported validation failures and find they can be directly attributed to technical challenges in data analysis. By examining 24 separate pre-processing techniques we show that minor alterations in pre-processing can change a successful prognostic biomarker (HR 1.85, 95% CI 1.37 to 2.50, P < 0.001) into one indistinguishable from random chance (HR 1.15, 95% CI 0.86 to 1.54, P = 0.348). Finally, we develop a new method, based on ensembles of analysis methodologies, to exploit this technical variability to improve biomarker robustness and to provide an independent confidence metric.ConclusionsBiomarkers comprise a fundamental component of personalized medicine. We first validated two NSCLC prognostic biomarkers in an independent patient cohort. Power analyses demonstrate that even this large, 442-patient cohort is under-powered for stage-specific analyses. We then use these results to discover an unexpected sensitivity of validation to subtle data analysis decisions. Finally, we develop a novel algorithmic approach to exploit this sensitivity to improve biomarker robustness

Unravelling the functions of biogenic volatiles in boreal and temperate forest ecosystems

Living trees are the main source of biogenic volatile organic compounds (BVOCs) in forest ecosystems, but substantial emissions originate from leaf and wood litter, the rhizosphere and from microorganisms. This review focuses on temperate and boreal forest ecosystems and the roles of BVOCs in ecosystem function, from the leaf to the forest canopy and from the forest soil to the atmosphere level. Moreover, emphasis is given to the question of how BVOCs will help forests adapt to environmental stress, particularly biotic stress related to climate change. Trees use their vascular system and emissions of BVOCs in internal communication, but emitted BVOCs have extended the communication to tree population and whole community levels and beyond. Future forestry practices should consider the importance of BVOCs in attraction and repulsion of attacking bark beetles, but also take an advantage of herbivore-induced BVOCs to improve the efficiency of natural enemies of herbivores. BVOCs are extensively involved in ecosystem services provided by forests including the positive effects on human health. BVOCs have a key role in ozone formation but also in ozone quenching. Oxidation products form secondary organic aerosols that disperse sunlight deeper into the forest canopy, and affect cloud formation and ultimately the climate. We also discuss the technical side of reliable BVOC sampling of forest trees for future interdisciplinary studies that should bridge the gaps between the forest sciences, health sciences, chemical ecology, conservation biology, tree physiology and atmospheric science

Differential utilization of NF-kappaB RELA and RELB in response to extracellular versus intracellular polyIC stimulation in HT1080 cells

Abstract Background Pattern recognition receptors (PRRs) for double-stranded RNA (dsRNA) are components of innate immunity that recognize the presence of viral infection and initiate efficient defense mechanisms. In addition to previously well-characterized signaling pathways that are mediated by PKR and TLR3, new intracellular dsRNA sensors, that are members of CARD and DExD/H box helicase family, have been identified. However, the molecular mechanisms involved in the signaling pathways mediated by these new dsRNA sensors have not been extensively characterized. Results Here, we studied an intracellular dsRNA pathway in the human fibrosarcoma cell line HT1080, which is distinct from the TLR3-mediated extracellular dsRNA pathway. Particularly, the NF-kB subunits RELA and RELB were differentially utilized by these two dsRNA signaling pathways. In TLR3-mediated dsRNA signaling, siRNA knock-down studies suggested a limited role for RELA on regulation of interferon beta and other cytokines whereas RELB appeared to have a negative regulatory role. By contrast, intracellular dsRNA signaling was dependent on RELA, but not RELB. Conclusions Our study suggests that extracellular and intracellular dsRNA signaling pathways may utilize different NF-kB members, and particularly the differential utilization of RELB may be a key mechanism for powerful inductions of NF-kB regulated genes in the intracellular dsRNA signaling pathway

Needs assessment to strengthen capacity in water and sanitation research in Africa:experiences of the African SNOWS consortium

Despite its contribution to global disease burden, diarrhoeal disease is still a relatively neglected area for research funding, especially in low-income country settings. The SNOWS consortium (Scientists Networked for Outcomes from Water and Sanitation) is funded by the Wellcome Trust under an initiative to build the necessary research skills in Africa. This paper focuses on the research training needs of the consortium as identified during the first three years of the project

Genomic DNA functions as a universal external standard in quantitative real-time PCR

Real-time quantitative PCR (qPCR) is a powerful tool for quantifying specific DNA target sequences. Although determination of relative quantity is widely accepted as a reliable means of measuring differences between samples, there are advantages to being able to determine the absolute copy numbers of a given target. One approach to absolute quantification relies on construction of an accurate standard curve using appropriate external standards of known concentration. We have validated the use of tissue genomic DNA as a universal external standard to facilitate quantification of any target sequence contained in the genome of a given species, addressing several key technical issues regarding its use. This approach was applied to validate mRNA expression of gene candidates identified from microarray data and to determine gene copies in transgenic mice. A simple method that can assist achieving absolute quantification of gene expression would broadly enhance the uses of real-time qPCR and in particular, augment the evaluation of global gene expression studies

The diversity and evolution of pollination systems in large plant clades: Apocynaceae as a case study

Background and Aims Large clades of angiosperms are often characterized by diverse interactions with pollinators, but how these pollination systems are structured phylogenetically and biogeographically is still uncertain for most families. Apocynaceae is a clade of >5300 species with a worldwide distribution. A database representing >10 % of species in the family was used to explore the diversity of pollinators and evolutionary shifts in pollination systems across major clades and regions. Methods The database was compiled from published and unpublished reports. Plants were categorized into broad pollination systems and then subdivided to include bimodal systems. These were mapped against the five major divisions of the family, and against the smaller clades. Finally, pollination systems were mapped onto a phylogenetic reconstruction that included those species for which sequence data are available, and transition rates between pollination systems were calculated. Key Results Most Apocynaceae are insect pollinated with few records of bird pollination. Almost three-quarters of species are pollinated by a single higher taxon (e.g. flies or moths); 7 % have bimodal pollination systems, whilst the remaining approx. 20 % are insect generalists. The less phenotypically specialized flowers of the Rauvolfioids are pollinated by a more restricted set of pollinators than are more complex flowers within the Apocynoids + Periplocoideae + Secamonoideae + Asclepiadoideae (APSA) clade. Certain combinations of bimodal pollination systems are more common than others. Some pollination systems are missing from particular regions, whilst others are over-represented. Conclusions Within Apocynaceae, interactions with pollinators are highly structured both phylogenetically and biogeographically. Variation in transition rates between pollination systems suggest constraints on their evolution, whereas regional differences point to environmental effects such as filtering of certain pollinators from habitats. This is the most extensive analysis of its type so far attempted and gives important insights into the diversity and evolution of pollination systems in large clades

A Concomitant Muscle Injury Does Not Worsen Traumatic Brain Injury Outcomes in Mice

Traumatic brain injury (TBI) often involves multitrauma in which concurrent extracranial injury occurs. We previously demonstrated that a long bone fracture exacerbates neuroinflammation and functional outcomes in mice given a TBI. Whether other forms of concomitant peripheral trauma that are common in the TBI setting, such as skeletal muscle injury, have similar effects is unknown. As such, here we developed a novel mouse multitrauma model by combining a closed-skull TBI with a cardiotoxin (CTX)-induced muscle injury to investigate whether muscle injury affects TBI outcomes. Adult male mice were assigned to four groups: sham-TBI + sham-muscle injury (SHAM); sham-TBI + CTX-muscle injury (CTX); TBI + sham-muscle injury (TBI); TBI + CTX-muscle injury (MULTI). Some mice were euthanized at 24 h post-injury to assess neuroinflammation and cerebral edema. The remaining mice underwent behavioral testing after a 30-day recovery period, and were euthanized at 35 days post-injury for post-mortem analysis. At 24 h post-injury, both TBI and MULTI mice had elevated edema, increased expression of GFAP (i.e., a marker for reactive astrocytes), and increased mRNA levels of inflammatory chemokines. There was also an effect of injury on cytokine levels at 35 days post-injury. However, the TBI and MULTI mice did not significantly differ on any of the measures assessed. These initial findings suggest that a concomitant muscle injury does not significantly affect preclinical TBI outcomes. Future studies should investigate the combination of different injury models, additional outcomes, and other post-injury time points

CpG Island microarray probe sequences derived from a physical library are representative of CpG Islands annotated on the human genome

An effective tool for the global analysis of both DNA methylation status and protein–chromatin interactions is a microarray constructed with sequences containing regulatory elements. One type of array suited for this purpose takes advantage of the strong association between CpG Islands (CGIs) and gene regulatory regions. We have obtained 20 736 clones from a CGI Library and used these to construct CGI arrays. The utility of this library requires proper annotation and assessment of the clones, including CpG content, genomic origin and proximity to neighboring genes. Alignment of clone sequences to the human genome (UCSC hg17) identified 9595 distinct genomic loci; 64% were defined by a single clone while the remaining 36% were represented by multiple, redundant clones. Approximately 68% of the loci were located near a transcription start site. The distribution of these loci covered all 23 chromosomes, with 63% overlapping a bioinformatically identified CGI. The high representation of genomic CGI in this rich collection of clones supports the utilization of microarrays produced with this library for the study of global epigenetic mechanisms and protein–chromatin interactions. A browsable database is available on-line to facilitate exploration of the CGIs in this library and their association with annotated genes or promoter elements

Studies of the ecology of the Benguela Current Upwelling System: The TRAFFIC approach

Under the umbrella of SPACES (Science Partnerships for the Adaptation to Complex Earth System Processes in Southern Africa), several marine projects have been conducted to study the coastal upwelling area off southwestern Africa, the Benguela Upwelling System (BUS). The BUS is economically important for the bordering countries due to its large fish stocks. We present results from the projects GENUS and TRAFFIC, which focused on the biogeochemistry and biology of this marine area. The physical drivers, the nutrient distributions, and the different ecosystem components were studied on numerous expeditions using different methods. The important aspects of the ecosystem, such as key species and food web complexity were studied for a later evaluation of trophic transfer efficiency and to forecast possible changes in this highly productive marine area. This chapter provides a literature review and analyses of own data of the main biological trophic components in the Benguela Upwelling System gathered during two cruises in February/March 2019 and October 2021