Statistical analysis of amplified fragment length polymorphism data: a toolbox for molecular ecologists and evolutionists

Recently, the amplified fragment length polymorphism (AFLP) technique has gained a lot of popularity, and is now frequently applied to a wide variety of organisms. Technical specificities of the AFLP procedure have been well documented over the years, but there is on the contrary little or scattered information about the statistical analysis of AFLPs. In this review, we describe the various methods available to handle AFLP data, focusing on four research topics at the population or individual level of analysis: (i) assessment of genetic diversity; (ii) identification of population structure; (iii) identification of hybrid individuals; and (iv) detection of markers associated with phenotypes. Two kinds of analysis methods can be distinguished, depending on whether they are based on the direct study of band presences or absences in AFLP profiles ('band-based' methods), or on allelic frequencies estimated at each locus from these profiles ('allele frequency-based' methods). We investigate the characteristics and limitations of these statistical tools; finally, we appeal for a wider adoption of methodologies borrowed from other research fields, like for example those especially designed to deal with binary data.     

Genetic constraints on protein evolution

Evolution requires the generation and optimization of new traits ("adaptation") and involves the selection of mutations that improve cellular function. These mutations were assumed to arise by selection of neutral mutations present at all times in the population. Here we review recent evidence that indicates that deleterious mutations are more frequent in the population than previously recognized and that these mutations play a significant role in protein evolution through continuous positive selection. Positively selected mutations include adaptive mutations, i.e. mutations that directly affect enzymatic function, and compensatory mutations, which suppress the pleiotropic effects of adaptive mutations. Compensatory mutations are by far the most frequent of the two and would allow potentially adaptive but deleterious mutations to persist long enough in the population to be positively selected during episodes of adaptation. Compensatory mutations are, by definition, context-dependent and thus constrain the paths available for evolution. This provides a mechanistic basis for the examples of highly constrained evolutionary landscapes and parallel evolution reported in natural and experimental populations. The present review article describes these recent advances in the field of protein evolution and discusses their implications for understanding the genetic basis of disease and for protein engineering in vitro.     

Environmental decision support systems: A new approach to support the operation and maintenance of horizontal subsurface flow constructed wetlands

This paper describes the development and operation of an Environmental Decision Support System (EDSS) to improve the operation and maintenance of horizontal subsurface-flow constructed wetlands (EDSS-maintenance). Constructed wetlands (CWs) allow wastewater treatment in a sustainable manner since they involve low energy consumption, low construction and functioning costs and low environmental impact. However, operation and maintenance activities are essential to guarantee reliability in CWs performance. The definition of operation and maintenance protocols depends on several quantitative and qualitative aspects such as wastewater treatment plant configuration, CW design, influent characteristics, sensitivity of the receiving media, etc. Bearing this in mind and considering the limited technical knowledge about CWs, the need for a new tool to support CW performance is clear. In this sense, EDSSs offer a new approach because they can tackle problems of complex and uncertain systems. The EDSS-maintenance provides operation and maintenance manuals specifically defined for every CW. To achieve it, the required knowledge was implemented within a rule-based system, which forms the backbone of the EDSS. Several features presented in this paper demonstrate how the EDSS-maintenance provides a proper platform to support the necessary collaborative work in the ecological engineering problem of horizontal subsurface flow CWs operation and maintenance.     

Notch signaling is essential for ventricular chamber development

Ventricular chamber morphogenesis, first manifested by trabeculae formation, is crucial for cardiac function and embryonic viability and depends on cellular interactions between the endocardium and myocardium. We show that ventricular Notch1 activity is highest at presumptive trabecular endocardium. RBPJk and Notch1 mutants show impaired trabeculation and marker expression, attenuated EphrinB2, NRG1, and BMP10 expression and signaling, and decreased myocardial proliferation. Functional and molecular analyses show that Notch inhibition prevents EphrinB2 expression, and that EphrinB2 is a direct Notch target acting upstream of NRG1 in the ventricles. However, BMP10 levels are found to be independent of both EphrinB2 and NRG1 during trabeculation. Accordingly, exogenous BMP10 rescues the myocardial proliferative defect of in vitro-cultured RBPJk mutants, while exogenous NRG1 rescues differentiation in parallel. We suggest that during trabeculation Notch independently regulates cardiomyocyte proliferation and differentiation, two exquisitely balanced processes whose perturbation may result in congenital heart disease.     

Extending networks of protected areas to optimize connectivity and population growth rate

Protected areas (PAs) are recognized as the flagship tool to offset biodiversity loss on Earth. Spatial conservation planning seeks optimal designs of PAs that meet multiple targets such as biodiversity representation and population persistence. Since connectivity between PAs is a fundamental requirement for population persistence, several methods have been developed to include connectivity into PA design algorithms. Among these, the eigenvalue decomposition of the connectivity matrix allows for identifying clusters of strongly connected sites and selecting the sites contributing the most to population persistence. So far, this method was only suited to optimize an entire network of PAs without considering existing PAs in the new design. However, a more cost‐effective and realistic approach is to optimize the design of an extended network to improve its connectivity and thus population persistence. Here, we develop a flexible algorithm based on eigenvalue decomposition of connectivity matrices to extend existing networks of PAs while optimizing connectivity and population growth rate. We also include a splitting algorithm to improve cluster identification. The new algorithm accounts for the change in connectivity due to the increased biological productivity often observed in existing PAs. We illustrate the potential of our algorithm by proposing an extension of the network of ～100 Mediterranean marine PAs to reach the targeted 10% surface area protection from the current 1.8%. We identify differences between the clean slate scenario, where all sites are available for protection, irrespective of their current protection status, and the scenario where existing PAs are forced to be included into the optimized solution. By integrating this algorithm to existing multi‐objective and multi‐specific algorithms of PA selection, the demographic effects of connectivity can be explicitly included into conservation planning.     

Crocin Prevents Patulin‐Induced Acute Toxicity in Cardiac Tissues via the Regulation of Oxidative Damage and Apoptosis

Patulin (PAT) is a mycotoxin produced by several species of the genera of Penicillium, Aspergillus, and Byssochlamys principally by Penicillium expansum. This mycotoxin is suspected to affect several organs including kidney and liver. However, its toxic effect on heart remains unknown. The present study investigated for the first time the cardiotoxic effect of PAT in mice. We demonstrated that PAT increased creatinin phosphokinase (CPK) level, induced lipoperoxydation and protein oxidation, and triggered the antioxidant enzymes such as superoxide dismutase and catalase activities. We also demonstrated that acute administration of PAT triggers apoptosis via P53 overexpression and caspase 3 activation. We further investigated the antioxidant efficiency of crocin (CRO), a carotenoid pigment, against PAT‐induced cardiotoxicity. We found that pretreatment with CRO prevents cardiac impairment by reducing CPK levels, restoring the redox statute and suppressing apoptosis. Collectively, our data provide new preventive effect of CRO toward PAT‐induced cardiotoxicity in mice.     

Enzymatic hydrolysis of olive wastewater for hydroxytyrosol enrichment

Aspergillus niger broth culture on wheat bran was assessed for olive wastewater (OW) hydrolysis in order to release hydroxytyrosol (HT). The enzyme profiles of this culture broth gave essentially (IU/L): 3000 β-glucosidase and 100 esterase. Hydrolysis activity of A. niger enzyme preparation was evaluated by using three substrates: raw OW, phenolic fraction extracted from OW by ethyl acetate and its corresponding exhausted fraction. Large amounts of free simple phenolics were released from exhausted fraction and raw OW after enzymatic treatment. HPLC analyses show that HT was the main phenolic compound. One step of ethyl acetate extraction of hydrolysed OW allowed the recovery of 0.8 g of HT per litre of OW. The antioxidant activity of extracts from OW and exhausted fraction, measured by DPPH method, was drastically enhanced after hydrolysis treatment. This study demonstrates that hydrolysed OW is a potential source of bioactive phenolic compounds with promising applications in food and pharmaceutical industries.     

Detection of Olive‐infecting Viruses in Tunisia

During a virus survey in autumn 2007 and spring 2008 of two Tunisian olive mother blocks, 175 olive samples were collected from 19 different cultivars and tested by RT‐PCR for the presence of Arabis mosaic virus (ArMV), Cherry leaf roll virus (CLRV), Cucumber mosaic virus (CMV), Olive latent ringspot virus (OLRSV), Olive latent virus 1 (OLV‐1), Olive latent virus 2 (OLV‐2), Olive leaf yellowing‐associated virus (OLYaV) and Strawberry latent ringspot virus (SLRSV), using specific sets of primers. The PCR‐negative samples were also subjected to dsRNA and mechanical transmission tests. PCR results indicated that c. 86% of the trees were infected with at least one virus, whereas visible bands were shown by 3 of 24 PCR‐negative samples in dsRNA analysis. OLYaV was the most prevalent virus (49.1%), followed by OLV‐1 (34.3%), CMV (25.7%), OLRSV (16.6%), CLRV (13.1%), SLRSV (7.4%) and OLV‐2 (6.9%), whereas ArMV was not detected. Very high infection rates were found in the two main oil cvs. Chemlali (84.6%) and Chétoui (86.9%).