Advantages and limitations in the use of hairy root cultures for the production of tropane alkaloids: Use of anti-auxins in the maintenance of normal root morphology

Summary Brugmansia candida hairy roots, obtained by infection withAgrobacterium rhizogenes LBA 9402, exhibit, after subculturing in liquid media, a tendency towards dedifferentiation. It has been found that the following strategies can be applied to inhibit this dedifferentiation and preserve normal root morphology: (a) lowering both the mineral and sucrose concentration in the media employed so as to diminish osmotic stress (a condition to which these roots appear to be particularly susceptible); (b) employing antiauxins in appropriate concentrations; and (c) maintaining the hairy roots on solid media prior to use in production processes in liquid media. The first strategy suggested does not favor alkaloid productivity, but in this case a two-step method could be attempted: biomass with normal root morphology could be obtained in a first stage using low sucrose concentrations, and in a second stage, sucrose could be increased in order to achieve higher productivity. In all the clones ofB. candida obtained, alkaloid production was biased towards scopolamine.     

Interpretability of bi-level variable selection methods

Variable selection is usually performed to increase interpretability, as sparser models are easier to understand than full models. However, a focus on sparsity is not always suitable, for example, when features are related due to contextual similarities or high correlations. Here, it may be more appropriate to identify groups and their predictive members, a task that can be accomplished with bi-level selection procedures. To investigate whether such techniques lead to increased interpretability, group exponential LASSO (GEL), sparse group LASSO (SGL), composite minimax concave penalty (cMCP), and least absolute shrinkage, and selection operator (LASSO) as reference methods were used to select predictors in time-to-event, regression, and classification tasks in bootstrap samples from a cohort of 1001 patients. Different groupings based on prior knowledge, correlation structure, and random assignment were compared in terms of selection relevance, group consistency, and collinearity tolerance. The results show that bi-level selection methods are superior to LASSO in all criteria. The cMCP demonstrated superiority in selection relevance, while SGL was convincing in group consistency. An all-round capacity was achieved by GEL: the approach jointly selected correlated and content-related predictors while maintaining high selection relevance. This method seems recommendable when variables are grouped, and interpretation is of primary interest.     

Identification of the sequences required for chromosomal replicator function in Kluyveromyces lactis

The analysis of replication intermediates of a Kluyveromyces lactis chromosomal autonomous replicating sequence (ARS), KARS101, has shown that it is active as a chromosomal replicator. KARS101 contains a 50 bp sequence conserved in two other K. lactis ARS elements. The deletion of the conserved sequence in KARS101 completely abolished replicator activity, in both the plasmids and the chromosome. Gel shift assays indicated that this sequence binds proteins present in K. lactis nuclear extracts, and a 40 bp sequence, previously defined as the core essential for K. lactis ARS function, is required for efficient binding. Reminiscent of the origin replication complex (ORC), the binding appears to be ATP dependent. A similar pattern of protection of the core was seen with in vitro footprinting. KARS101 also functions as an ARS sequence in Saccharomyces cerevisiae. A comparative study using S. cerevisiae nuclear extracts revealed that the sequence required for binding is a dodecanucleotide related to the S. cerevisiae ARS consensus sequence and essential for S. cerevisiae ARS activity.     

Evaluating mortality rates and causalities in a critically endangered felid across its whole distribution range

The conservation of endangered species requires accurate data, and knowledge of cause-specific mortality rates is one of the most important issues. In recent years, conservation programs for the critically endangered Iberian lynx Lynx pardinus have been developed on the basis of mortality data derived 30 years ago from the small Doñana population. Thus, there is an urgent need for an update of mortality rates and causes in both populations (Sierra Morena and Doñana). Here we use radio-tracking information from the whole range of the Iberian lynx to quantify mortality rates and identify their causes. Between 2006 and 2011, we radio-tagged 78 Iberian lynxes from its two remaining populations (39 from Sierra Morena and 39 from Doñana). Mortality events were evaluated to identify causes, and cause-specific annual mortality rates (AMR) were obtained using the nonparametric cumulative incidence function estimator. Overall, AMR was estimated at 0.16?±?0.05 (0.19?±?0.09 in Sierra Morena and 0.12?±?0.07 in Doñana). Disease was the main cause of mortality both for the whole population and the Doñana population. Poaching was the main cause of mortality in Sierra Morena. Our results suggest that the best strategy for conserving this species is to focus action on decreasing the fatal effect of disease and poaching. Given the possible existence of an underlying inbreeding-mediated immunosuppression, genetic management aimed at increasing the genetic diversity of this population is also recommended.     

PAX6 haplotypes are associated with high myopia in Han chinese

Background

The paired box 6 (PAX6) gene is considered as a master gene for eye development. Linkage of myopia to the PAX6 region on chromosome 11p13 was shown in several studies, but the results for association between myopia and PAX6 were inconsistent so far.

Methodology/Principal Findings

We genotyped 16 single nucleotide polymorphisms (SNPs) in the PAX6 gene and its regulatory regions in an initial study for 300 high myopia cases and 300 controls (Group 1), and successfully replicated the positive results with another independent group of 299 high myopia cases and 299 controls (Group 2). Five SNPs were genotyped in the replication study. The spherical equivalent of subjects with high myopia was ≤−8.0 dioptres. The PLINK package was used for genetic data analysis. No association was found between each of the SNPs and high myopia. However, exhaustive sliding-window haplotype analysis highlighted an important role for rs12421026 because haplotypes containing this SNP were found to be associated with high myopia. The most significant results were given by the 4-SNP haplotype window consisting of rs2071754, rs3026393, rs1506 and rs12421026 (P = 3.54×10⁻¹⁰, 4.06×10⁻¹¹ and 1.56×10⁻¹⁸ for Group 1, Group 2 and Combined Group, respectively) and the 3-SNP haplotype window composed of rs3026393, rs1506 and rs12421026 (P = 5.48×10⁻¹⁰, 7.93×10⁻¹² and 6.28×10⁻²³ for the three respective groups). The results remained significant after correction for multiple comparisons by permutations. The associated haplotyes found in a previous study were also successfully replicated in this study.

Conclusions/Significance

PAX6 haplotypes are associated with susceptibility to the development of high myopia in Chinese. The PAX6 locus plays a role in high myopia.     

In vitro and in vivo growth and casein gene expression of mouse mammary tumor epithelial cells in response to hormones

Cells from autochthonous mouse mammary carcinomas which display estrogen-independent growth _vivo were studied for their hormonal responses in primary culture. A culture system employing insulin-supplemented, serum-free medium and basement membrane Matrigel as a substratum was used to cultivate tumor cells. The cells did not exhibit in vitro estrogenor prolactin-dependent growth. Primary tumors still displayed a constitutional expression of α-, β-, and γ-casein mRNAs. These messages were dramatically reduced during the culture period. However, seven to eightfold increases in α- and β-casein mRNAs were inducible in the 5-day cultures by treatment with prolactin and hydrocortisone. If the hormones were present through a 2-week culture period, the levels of α-, β-, and γ-casein mRNAs in the cells were maintained and displayed in a time-dependent increase with a peak at 10–14 days. The accumulation of β-casein mRNA in vitro did not require DNA synthesis. Administration of prolactin directly into the growing tumors in vivo could also enhance β-casein mRNA levels in the tumor cells. Morphological studies of the cells cultured in the presence of prolactin and hydrocortisone did not reveal visible changes compared with those without hormonal treatment. Transplantation of tumor cells cultured in the presence or absence of hormones resulted in the development of tumors in mice at approximately the same time. The current studies suggest that the autochthonous mammary tumor cells, independent of estrogen for cell growth, were still inducible for casein gene expression in vitro and in vivo by appropriate hormones. The induction and maintenance of casein messages by a single hormonal treatment did not appear to correlate with morphology and DNA synthesis of cells in vitro or with tumor-producing capacities in vivo.