Guanidine-II aptamer conformations and ligand binding modes through the lens of molecular simulation

Regulation of gene expression via riboswitches is a widespread mechanism in bacteria. Here, we investigate ligand binding of a member of the guanidine sensing riboswitch family, the guanidine-II riboswitch (Gd-II). It consists of two stem–loops forming a dimer upon ligand binding. Using extensive molecular dynamics simulations we have identified conformational states corresponding to ligand-bound and unbound states in a monomeric stem–loop of Gd-II and studied the selectivity of this binding. To characterize these states and ligand-dependent conformational changes we applied a combination of dimensionality reduction, clustering, and feature selection methods. In absence of a ligand, the shape of the binding pocket alternates between the conformation observed in presence of guanidinium and a collapsed conformation, which is associated with a deformation of the dimerization interface. Furthermore, the structural features responsible for the ability to discriminate against closely related analogs of guanidine are resolved. Based on these insights, we propose a mechanism that couples ligand binding to aptamer dimerization in the Gd-II system, demonstrating the value of computational methods in the field of nucleic acids research.     

Impact of low‐intensity pulsed ultrasound on the growth of Schizochytrium sp. for omega‐3 production

Schizochytrium sp. is a microalga that is known for its high content of oils or lipids. It has a high percentage of polyunsaturated fatty acids in the accumulated oil, especially docosahexaenoic acid (DHA). DHA is an important additive for the human diet. Large‐scale production of Schizochytrium sp. can serve as an alternative source of DHA for humans as well as for fish feed, decreasing the burden on aqua systems. Therefore, research on improving the productivity of Schizochytrium attracts a lot of attention. We studied the potential of using low‐intensity pulsed ultrasound (LIPUS) in the growth cycle of Schizochytrium sp. in shake flasks. Different intensities and treatment durations were tested. A positive effect of LIPUS on biomass accumulation was observed in the Schizochytrium sp. culture. Specifically, LIPUS stimulation at the ultrasound intensity of 400 mW/cm² with 20 min per treatment 10 times a day with equal intervals of 2.4 h between the treatments was found to enhance the growth of Schizochytrium biomass most effectively (by up to 20%). Due to the nature of cell division in Schizochytrium sp. which occurs via zoospore formation, LIPUS stimulation was inefficient if applied continuously during all 5 days of the growth cycle. Using microscopy, we studied the interval between zoospore formation in the culture and selected the optimal LIPUS application days (Days 0–1 and Days 4–5 of the 5‐day growth cycle). Microscopic images have also shown that LIPUS stimulation enhances zoospore formation in Schizochytrium sp., leading to more active cell division in the culture. This study shows that LIPUS can serve as an additional tool for cost‐efficiency improvement in the large‐scale production of Schizochytrium as a sustainable and environmentally friendly source of omega‐3 (DHA).     

Genetic variation of chloroplast and nuclear markers in natural populations of hazelnut (Corylus avellana L.) in Germany

Corylus avellana L. (hazel) is a long-lived, monoecious and wind-pollinated shrub species, widespread all over Europe. In Germany, hazel is intensively traded and planted, and thus is of central interest from a nature conservancy point of view. To assess the within- and between-population differentiation of hazel, 20 natural populations (18 from Germany, one from Italy and one from Hungary) were investigated genetically. Seven isozyme systems comprising 11 gene loci were analysed in up to 100 samples (average 92.6) per population, amplified fragment length polymorphisms (AFLP) were analysed in up to 50 samples (average 47.4) and nine cpDNA-SSR markers were assessed in 20 samples per population. Results for overall isozyme variability with Na 2.46 alleles per locus, allelic diversity (Ne) 1.39, expected heterozygosity He 21 % and 79 % polymorphic loci were in accordance with the findings of previous studies. The respective values for AFLPs were lower, but both marker systems revealed the same level of about 3.5 % differentiation between populations. For cpSSR only the Italian sample showed within-population variation and the two haplotypes were completely differentiated from all other populations expressing a unique genetic structure with one single haplotype. Among the three marker systems AFLPs showed the best ability to differentiate between populations. While only one isozyme locus revealed significant differentiation, 41 AFLP loci showed highly significant differentiation between all populations, but 26 loci when only German populations were considered. Consequently geographic differentiation analyses focused mainly on molecular markers. Mantel tests showed significant correlations between genetic and geographic distance, but in the unweighted pair-group method with arithmetic mean analyses, adjacent populations did not always form clusters. While chloroplast markers were able to clearly distinguish only the Hungarian population, the nuclear markers revealed clear spatial genetic structures. The correlations between geographic and genetic distance was high for AFLPs. The correlograms illustrate this effect for all populations as well as for the German populations.     

Single atom substitution in mouse protein kinase G eliminates oxidant sensing to cause hypertension

Blood pressure regulation is crucial for the maintenance of health, and hypertension is a risk factor for myocardial infarction, heart failure, stroke and renal disease. Nitric oxide (NO) and prostacyclin trigger well-defined vasodilator pathways; however, substantial vasorelaxation in response to agents such as acetylcholine persists when the synthesis of these molecules is prevented. This remaining vasorelaxation activity, termed endothelium-derived hyperpolarizing factor (EDHF), is more prevalent in resistance than in conduit blood vessels and is considered a major mechanism for blood pressure control. Hydrogen peroxide (H2O2) has been shown to be a major component of EDHF in several vascular beds in multiple species, including in humans. H2O2 causes the formation of a disulfide bond between the two α subunits of protein kinase G I-α (PKGI-α), which activates the kinase independently of the NO-cyclic guanosine monophosphate (cGMP) pathway and is coupled to vasodilation. To test the importance of PKGI-α oxidation in the EDHF mechanism and blood pressure control in vivo, we generated a knock-in mouse expressing only a C42S 'redox-dead' version of PKGI-α. This amino acid substitution, a single-atom change (an oxygen atom replacing a sulfur atom), blocked the vasodilatory action of H2O2 on resistance vessels and resulted in hypertension in vivo.     

Structural elements that govern Sec14-like PITP sensitivities to potent small molecule inhibitors

Sec14-like phosphatidylinositol transfer proteins (PITPs) play important biological functions in integrating multiple aspects of intracellular lipid metabolism with phosphatidylinositol-4-phosphate signaling. As such, these proteins offer new opportunities for highly selective chemical interference with specific phosphoinositide pathways in cells. The first and best characterized small molecule inhibitors of the yeast PITP, Sec14, are nitrophenyl(4-(2-methoxyphenyl)piperazin-1-yl)methanones (NPPMs), and a hallmark feature of NPPMs is their exquisite targeting specificities for Sec14 relative to other closely related Sec14-like PITPs. Our present understanding of Sec14::NPPM binding interactions is based on computational docking and rational loss-of-function approaches. While those approaches have been informative, we still lack an adequate understanding of the basis for the high selectivity of NPPMs among closely related Sec14-like PITPs. Herein, we describe a Sec14 motif, which we term the VV signature, that contributes significantly to the NPPM sensitivity/resistance of Sec14-like phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer proteins. The data not only reveal previously unappreciated determinants that govern Sec14-like PITP sensitivities to NPPMs, but enable predictions of which Sec14-like PtdIns/PtdCho transfer proteins are likely to be NPPM resistant or sensitive based on primary sequence considerations. Finally, the data provide independent evidence in support of previous studies highlighting the importance of Sec14 residue Ser173 in the mechanism by which NPPMs engage and inhibit Sec14-like PITPs.     

Molecular cloning of CoA Synthase. The missing link in CoA biosynthesis

Coenzyme A functions as a carrier of acetyl and acyl groups in living cells and is essential for numerous biosynthetic, energy-yielding, and degradative metabolic pathways. There are five enzymatic steps in CoA biosynthesis. To date, molecular cloning of enzymes involved in the CoA biosynthetic pathway in mammals has been only reported for pantothenate kinase. In this study, we present cDNA cloning and functional characterization of CoA synthase. It has an open reading frame of 563 aa and encodes a protein of approximately 60 kDa. Sequence alignments suggested that the protein possesses both phosphopantetheine adenylyltransferase and dephospho-CoA kinase domains. Biochemical assays using wild type recombinant protein confirmed the gene product indeed contained both these enzymatic activities. The presence of intrinsic phosphopantetheine adenylyltransferase activity was further confirmed by site-directed mutagenesis. Therefore, this study describes the first cloning and characterization of a mammalian CoA synthase and confirms this is a bifunctional enzyme containing the last two components of CoA biosynthesis.     

Tree rings provide no evidence of a CO2 fertilization effect in old‐growth subalpine forests of western Canada

Atmospheric CO₂ concentrations are now 1.7 times higher than the preindustrial values. Although photosynthetic rates are hypothesized to increase in response to rising atmospheric CO₂ concentrations, results from in situ experiments are inconsistent in supporting a CO₂ fertilization effect of tree growth. Tree‐ring data provide a historical record of tree‐level productivity that can be used to evaluate long‐term responses of tree growth. We use tree‐ring data from old‐growth, subalpine forests of western Canada that have not had a stand‐replacing disturbance for hundreds of years to determine if growth has increased over 19th and 20th centuries. Our sample consisted of 5,858 trees belonging to five species distributed over two sites in the coastal zone and two in the continental climate of the interior. We calculated annual increments in tree basal area, adjusted these increments for tree size and age, and tested whether there was a detectable temporal trend in tree growth over the 19th and 20th centuries. We found a similar pattern in 20th century growth trends among all species at all sites. Growth during the 19th century was mostly stable or increasing, with the exception of one of the coastal sites, where tree growth was slightly decreasing; whereas growth during the 20th century consistently decreased. The unexpected decrease in growth during the 20th century indicates that there was no CO₂ fertilization effect on photosynthesis. We compared the growth trends from our four sites to the trends simulated by seven Earth System Models, and saw that most of the models did not predict these growth declines. Overall, our results indicate that these old‐growth forests are unlikely to increase their carbon storage capacity in response to rising atmospheric CO₂, and thus are unlikely to contribute substantially to offsetting future carbon emissions.