Insights into xanthine riboswitch structure and metal ion-mediated ligand recognition

Riboswitches are conserved functional domains in mRNA that mostly exist in bacteria. They regulate gene expression in response to varying concentrations of metabolites or metal ions. Recently, the NMT1 RNA motif has been identified to selectively bind xanthine and uric acid, respectively, both are involved in the metabolic pathway of purine degradation. Here, we report a crystal structure of this RNA bound to xanthine. Overall, the riboswitch exhibits a rod-like, continuously stacked fold composed of three stems and two internal junctions. The binding-pocket is determined by the highly conserved junctional sequence J1 between stem P1 and P2a, and engages a long-distance Watson–Crick base pair to junction J2. Xanthine inserts between a G–U pair from the major groove side and is sandwiched between base triples. Strikingly, a Mg²⁺ ion is inner-sphere coordinated to O6 of xanthine and a non-bridging oxygen of a backbone phosphate. Two further hydrated Mg²⁺ ions participate in extensive interactions between xanthine and the pocket. Our structure model is verified by ligand binding analysis to selected riboswitch mutants using isothermal titration calorimetry, and by fluorescence spectroscopic analysis of RNA folding using 2-aminopurine-modified variants. Together, our study highlights the principles of metal ion-mediated ligand recognition by the xanthine riboswitch.     

Adaptive divergence between lake and stream populations of an East African cichlid fish

Divergent natural selection acting in different habitats may build up barriers to gene flow and initiate speciation. This speciation continuum can range from weak or no divergence to strong genetic differentiation between populations. Here, we focus on the early phases of adaptive divergence in the East African cichlid fish Astatotilapia burtoni, which occurs in both Lake Tanganyika (LT) and inflowing rivers. We first assessed the population structure and morphological differences in A. burtoni from southern LT. We then focused on four lake–stream systems and quantified body shape, ecologically relevant traits (gill raker and lower pharyngeal jaw) as well as stomach contents. Our study revealed the presence of several divergent lake–stream populations that rest at different stages of the speciation continuum, but show the same morphological and ecological trajectories along the lake–stream gradient. Lake fish have higher bodies, a more superior mouth position, longer gill rakers and more slender pharyngeal jaws, and they show a plant/algae and zooplankton‐biased diet, whereas stream fish feed more on snails, insects and plant seeds. A test for reproductive isolation between closely related lake and stream populations did not detect population‐assortative mating. Analyses of F1 offspring reared under common garden conditions indicate that the detected differences in body shape and gill raker length do not constitute pure plastic responses to different environmental conditions, but also have a genetic basis. Taken together, the A. burtoni lake–stream system constitutes a new model to study the factors that enhance and constrain progress towards speciation in cichlid fishes.     

Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples

We have developed a robust RNA sequencing method for generating complete de novo assemblies with intra-host variant calls of Lassa and Ebola virus genomes in clinical and biological samples. Our method uses targeted RNase H-based digestion to remove contaminating poly(rA) carrier and ribosomal RNA. This depletion step improves both the quality of data and quantity of informative reads in unbiased total RNA sequencing libraries. We have also developed a hybrid-selection protocol to further enrich the viral content of sequencing libraries. These protocols have enabled rapid deep sequencing of both Lassa and Ebola virus and are broadly applicable to other viral genomics studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0519-7) contains supplementary material, which is available to authorized users.     

Impact on survival of intensive follow up after curative resection for colorectal cancer: systematic review and meta-analysis of randomised trials

ObjectiveTo review the evidence from clinical trials of follow up of patients after curative resection for colorectal cancer.DesignSystematic review and meta-analysis of randomised controlled trials of intensive compared with control follow up.ResultsFive trials, which included 1342 patients, met the inclusion criteria. Intensive follow up was associated with a reduction in all cause mortality (combined risk ratio 0.81, 95% confidence interval 0.70 to 0.94, P=0.007). The effect was most pronounced in the four extramural detection trials that used computed tomography and frequent measurements of serum carcinoembryonic antigen (risk ratio 0.73, 0.60 to 0.89, P=0.002). Intensive follow up was associated with significantly earlier detection of all recurrences (difference in means 8.5 months, 7.6 to 9.4 months, P<0.001) and an increased detection rate for isolated local recurrences (risk ratio 1.61, 1.12 to 2.32, P=0.011).ConclusionsIntensive follow up after curative resection for colorectal cancer improves survival. Large trials are required to identify which components of intensive follow up are most beneficial.

What is already known on this topic

There is a lack of direct evidence that intensive follow up after initial curative treatment for colorectal cancer leads to increased survivalGuidelines are inconclusive and clinical practice varies widely

What this study adds

The cumulative analysis of available data supports the view that intensive follow up after curative resection for colorectal cancer improves survivalIf computed tomography and frequent measurements of serum carcinoembryonic antigen are used during follow up mortality related to cancer is reduced by 9-13%This survival benefit is partly attributable to the earlier detection of all recurrences, particularly the increased detection of isolated recurrent disease     

Epidemiology of Rhodococcus equi Strains on Thoroughbred Horse Farms

Pulsed-field gel electrophoresis of restriction endonuclease-digested genomic DNA from a large collection of clinical isolates of Rhodococcus equi, an important pathogen of foals, was used to compare strain distribution between farms and over time. Forty-four strains were found among 209 isolates, with 5 of these accounting for over half the isolates and the 22 strains isolated more than once accounting for 90% of the isolates. The average genotypic diversity on each farm and in each year was found to be less than the genotypic diversity of the isolates taken as a whole, with 5.2% of the total diversity being due to differences between farms and 5.5% to differences between years. A small number of strains on each farm were found to have caused at least half the clinical cases of disease, and these varied between farms and, to a lesser extent, years. Most strains were found on more than one farm, and some very similar restriction patterns were found among isolates from different continents, indicating that strains can be very widespread. Multiple strains were isolated in five of the six cases in which more than one isolate from a single foal was examined, indicating that disease may commonly be caused by simultaneous infection with multiple strains. It was concluded that there are a number of different strains of R. equi which carry the vapA gene, and these strains tend to be widespread, but individual farms tend to have particular strains associated with them.     

Annotating DNA Variants Is the Next Major Goal for Human Genetics

Clinical genetic testing has undergone a dramatic transformation in the past two decades. Diagnostic laboratories that previously tested for well-established disease-causing DNA variants in a handful of genes have evolved into sequencing factories identifying thousands of variants of known and unknown medical consequence. Sorting out what does and does not cause disease in our genomes is the next great challenge in making genetics a central feature of healthcare. I propose that closing the gap in our ability to interpret variation responsible for Mendelian disorders provides a grand and unprecedented opportunity for geneticists. Human geneticists are well placed to coordinate a systematic evaluation of variants in collaboration with basic scientists and clinicians. Sharing of knowledge, data, methods, and tools will aid both researchers and healthcare workers in achieving their common goal of defining the pathogenic potential of variants. Generation of variant annotations will inform genetic testing and will deepen our understanding of gene and protein function, thereby aiding the search for molecular targeted therapies.     

Peptide entry inhibitors of enveloped viruses: The importance of interfacial hydrophobicity

There are many peptides known that inhibit the entry of enveloped viruses into cells, including one peptide that is successfully being used in the clinic as a drug. In this review, we discuss the discovery, antiviral activity and mechanism of action of such peptides. While peptide entry inhibitors have been discovered by a wide variety of approaches (structure-based, accidental, intentional, rational and brute force) we show here that they share a common physical chemical property: they are at least somewhat hydrophobic and/or amphipathic and have a propensity to interact with membrane interfaces. We propose that this propensity drives a shared mechanism of action for many peptide entry inhibitors, involving direct interactions with viral and cellular membranes, as well as interactions with the complex hydrophobic protein/lipid interfaces that are exposed, at least transiently, during virus–cell fusion. By interacting simultaneously with the membrane interfaces and other critical hydrophobic surfaces, we hypothesize that peptide entry inhibitors can act by changing the physical chemistry of the membranes, and the fusion protein interfaces bridging them, and by doing so interfere with the fusion of cellular and viral membranes. Based on this idea, we propose that an approach that focuses on the interfacial hydrophobicity of putative entry inhibitors could lead to the efficient discovery of novel, broad-spectrum viral entry inhibitors. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.