The structural basis of cooperative regulation at an alternate genetic switch

Bacteriophage lambda is a paradigm for understanding the role of cooperativity in gene regulation. Comparison of the regulatory regions of lambda and the unrelated temperate bacteriophage 186 provides insight into alternate ways to assemble functional genetic switches. The structure of the C-terminal domain of the 186 repressor, determined at 2.7 A resolution, reveals an unusual heptamer of dimers, consistent with presented genetic studies. In addition, the structure of a cooperativity mutant of the full-length 186 repressor, identified by genetic screens, was solved to 1.95 A resolution. These structures provide a molecular basis for understanding lysogenic regulation in 186. Whereas the overall fold of the 186 and lambda repressor monomers is remarkably similar, the way the two repressors cooperatively assemble is quite different and explains in part the differences in their regulatory activity.     

Development and evaluation of different normalization strategies for gene expression studies in Candida albicans biofilms by real-time PCR

Background  

Candida albicans biofilms are commonly found on indwelling medical devices. However, the molecular basis of biofilm formation and development is not completely understood. Expression analysis of genes potentially involved in these processes, such as the ALS (Agglutinine Like Sequence) gene family can be performed using quantitative PCR (qPCR). In the present study, we investigated the expression stability of eight housekeeping genes potentially useful as reference genes to study gene expression in Candida albicans (C. albicans) biofilms, using the geNorm Visual Basic Application (VBA) for Microsoft Excel. To validate our normalization strategies we determined differences in ALS1 and ALS3 expression levels between C. albicans biofilm cells and their planktonic counterparts.     

A flexible framework for multi-particle refinement in cryo-electron tomography

Cryo-electron tomography (cryo-ET) and subtomogram averaging (STA) are increasingly used for macromolecular structure determination in situ. Here, we introduce a set of computational tools and resources designed to enable flexible approaches to STA through increased automation and simplified metadata handling. We create a bidirectional interface between the Dynamo software package and the Warp-Relion-M pipeline, providing a framework for ab initio and geometrical approaches to multiparticle refinement in M. We illustrate the power of working within this framework by applying it to EMPIAR-10164, a publicly available dataset containing immature HIV-1 virus-like particles (VLPs), and a challenging in situ dataset containing chemosensory arrays in bacterial minicells. Additionally, we provide a comprehensive, step-by-step guide to obtaining a 3.4-Å reconstruction from EMPIAR-10164. The guide is hosted on https://teamtomo.org/, a collaborative online platform we establish for sharing knowledge about cryo-ET.

Employing optimal computational methodology in cryo-electron tomography is not always easy; this article provides a set of tools and a complete guide to obtaining high-resolution structures from cryo-ET data.     

Composition and chemical polymorphism of the essential oils from Piper lanceaefolium

Essential oils obtained by hydrodistillation from leaves and spikes of Piper lanceaefolium H.B.K. of Costa Rica were analysed by GC-FID, GC-MS and 13C-NMR methods. Main constituents found in the oil from leaves were sesquiterpene hydrocarbons - especially beta-caryophyllene and germacrene D - and phenylpropanoids, of which elemicin and parsley apiol were the major ones. The volatile oil from spikes showed monoterpene hydrocarbons, namely alpha- and beta-pinene, and the same phenylpropanoids as in the oil from leaves as the major constituents. Results obtained in the analysis by GC-FID and GC-MS of the essential oils from individual plants of different geographic origin were submitted to chemometric cluster analysis and principal component analysis, showing the presence of three different types of oils (i) parsley apiol/elemicin, (ii) elemicin/parsley apiol/dill apiol, and (iii) parsley apiol/dill apiol.     

Use of the gyrB gene for the identification of Pandoraea species

The recently described genus Pandoraea consists of five named species and four unnamed genomospecies, several of which have been identified in clinical specimens including respiratory secretions from persons with cystic fibrosis. We investigated whether it is possible to distinguish species of the genus Pandoraea by means of restriction fragment length polymorphism (RFLP) analysis and direct sequencing of the gyrB gene. Sixty-seven Pandoraea isolates were included. Species-specific RFLP patterns were obtained following digestion of the PCR-amplified gyrB gene with MspI. Specificity of RFLP groupings was confirmed by direct sequencing of several representative isolates. Our results indicate that RFLP analysis and sequencing of the gyrB gene are useful for the identification of Pandoraea species. We also found that further taxonomic studies within the beta-Proteobacteria using the gyrB gene would benefit from the development of additional primers allowing more efficient amplification of the gyrB gene. Our data also indicate that the taxonomic status of Pandoraea genomospecies 2 should be reinvestigated.     

Effects of anaerobiosis on carbohydrate oxidation by roots of Pisum sativum

The aim of this work was to discover the effects of anaerobiosis on the breakdown of sugars by the apical 6 mm of the roots of 5-day-old seedlings of Pisum sativum. Estimates of the maximum catalytic activities of alcohol dehydrogenase, lactate dehydrogenase, phosphoenolpyruvate carboxylase and NADP-specific malic enzyme showed them to be comparable to that of phosphofructokinase. Metabolism of sucrose-[U-¹⁴C] by excised apices was restricted by anoxia mainly to conversion to ethanol, CO₂ alanine and glycolytic intermediates. Measurements of metabolites over a period of 240 min after transfer of excised apices to nitrogen showed a marked and continual accumulation of ethanol, a smaller continual accumulation of alanine, a small initial rise in lactate and no detectable accumulation of malate or pyruvate. The rates of CO₂ production, of accumulation of ethanol and alanine, and of the labelling of these compounds by sucrose-[¹⁴C] declined markedly during the first 240 min of anaerobiosis. The conclusion is that under anaerobic conditions carbohydrate metabolism in the pea root apex is largely restricted to alcoholic fermentation, and, to a lesser degree, to alanine production.     

Polar bear (Ursus maritimus) cub mortality at a den site in northern Alaska

In March 2009, we documented the death of one member of a triplet polar bear (Ursus maritimus) litter at its den site in the southern Beaufort Sea (SBS) of Alaska. We used a self-contained video camera unit to document activity between den emergence and departure. All three cubs showed low activity levels relative to other cubs observed, and one died within one week of den emergence. Necropsy confirmed that the dead cub had a low body weight and was malnourished. Capture later confirmed that the two surviving cubs were also undersized. Polar bear cub survival is influenced by many factors including litter size and sea ice conditions. Triplet litters are often smaller and suffer higher mortality rates than singletons and twins. This cub was not only a triplet but also born following 2 years of record minimum sea ice extent, both of which may have played a role in this cub’s demise.     

Using dot blot with immunochemical detection to evaluate global changes in SUMO-2/3 conjugation

Here we describe a non-invasive method for rapid and highly reproducible genotyping of transgenic mammals with ubiquitous expression of fluorophore reporters. Hair samples from transgenic mice and pigs with systemic expression of the fluorophore reporter Venus were analyzed with a fluorescence microscope in few minutes. The hair samples can be preserved for long-term storage at ambient temperature conditions. This non-invasive method is useful for genotyping of transgenic large animals and contributes to animal welfare by reducing stress and discomfort of the animals during sample collection.     

Does history repeat itself? Wavelets and the phylodynamics of influenza A

Unprecedented global surveillance of viruses will result in massive sequence data sets that require new statistical methods. These data sets press the limits of Bayesian phylogenetics as the high-dimensional parameters that comprise a phylogenetic tree increase the already sizable computational burden of these techniques. This burden often results in partitioning the data set, for example, by gene, and inferring the evolutionary dynamics of each partition independently, a compromise that results in stratified analyses that depend only on data within a given partition. However, parameter estimates inferred from these stratified models are likely strongly correlated, considering they rely on data from a single data set. To overcome this shortfall, we exploit the existing Monte Carlo realizations from stratified Bayesian analyses to efficiently estimate a nonparametric hierarchical wavelet-based model and learn about the time-varying parameters of effective population size that reflect levels of genetic diversity across all partitions simultaneously. Our methods are applied to complete genome influenza A sequences that span 13 years. We find that broad peaks and trends, as opposed to seasonal spikes, in the effective population size history distinguish individual segments from the complete genome. We also address hypotheses regarding intersegment dynamics within a formal statistical framework that accounts for correlation between segment-specific parameters.