M3 cholinoreceptors alter electrical activity of rat left atrium via suppression of L-type Ca<Superscript>2+</Superscript> current without affecting K<Superscript>+</Superscript> conductance

Electrophysiological effects produced by selective activation of M3 cholinoreceptors were studied in isolated left atrium preparations from rat using the standard sharp glass microelectrode technique. The stimulation of M3 receptors was obtained by application of muscarinic agonist pilocarpine (10^?5 M) in the presence of selective M2 antagonist methoctramine (10^?7 M). Stimulation of M3 receptors induced marked reduction of action potential duration by 14.4 ± 2.4% and 16.1 ± 2.5% of control duration measured at 50 and 90% of repolarization, respectively. This effect was completely abolished by selective M3 blocker 4-DAMP (10^?8 M). In isolated myocytes obtained from the rat left atrium, similar pharmacological stimulation of M3 receptors led to suppression of peak L-type calcium current by 13.9 ± 2.6% of control amplitude (measured at +10 mV), but failed to affect K⁺ currents I _to, I _Kur, and I _Kir. In the absence of M2 blocker methoctramine, pilocarpine (10^?5 M) produced stronger attenuation of I _CaL and induced an increase in I _Kir. This additive inward rectifier current could be abolished by highly selective blocker of K_ir3.1/3.4 channels tertiapin-Q (10^?6 M) and therefore was identified as I _KACh. Thus, in the rat atrial myocardium activation of M3 receptors leads to shortening of action potentials via suppression of I _CaL, but does not enhance the major potassium currents involved in repolarization. Joint stimulation of M2 and M3 receptors produces stronger action potential shortening due to M2-mediated activation of I _KACh.     

Climate change and alpine stream biology: progress,challenges, and opportunities for the future

In alpine regions worldwide, climate change is dramatically altering ecosystems and affecting biodiversity in many ways. For streams, receding alpine glaciers and snowfields, paired with altered precipitation regimes, are driving shifts in hydrology, species distributions, basal resources, and threatening the very existence of some habitats and biota. Alpine streams harbour substantial species and genetic diversity due to significant habitat insularity and environmental heterogeneity. Climate change is expected to affect alpine stream biodiversity across many levels of biological resolution from micro‐ to macroscopic organisms and genes to communities. Herein, we describe the current state of alpine stream biology from an organism‐focused perspective. We begin by reviewing seven standard and emerging approaches that combine to form the current state of the discipline. We follow with a call for increased synthesis across existing approaches to improve understanding of how these imperiled ecosystems are responding to rapid environmental change. We then take a forward‐looking viewpoint on how alpine stream biologists can make better use of existing data sets through temporal comparisons, integrate remote sensing and geographic information system (GIS) technologies, and apply genomic tools to refine knowledge of underlying evolutionary processes. We conclude with comments about the future of biodiversity conservation in alpine streams to confront the daunting challenge of mitigating the effects of rapid environmental change in these sentinel ecosystems.     

Effects of landscape features on gene flow of valley oaks (<Emphasis Type="Italic">Quercus lobata</Emphasis>)

Landscape features affect habitat connectivity and patterns of gene flow and hence influence genetic structure among populations. We studied valley oak (Quercus lobata), a threatened species of California (USA) savannas and oak woodlands, with a distribution forming a ring around the Central Valley grasslands. Our main goal was to determine the role of topography and land cover on patterns of gene flow and to test whether elevation or land cover forms stronger barriers to gene flow among valley oak populations. We sampled valley oaks in 12 populations across the range of this species, genotyped each tree at eight nuclear microsatellite loci, and created a series of resistance surfaces by assigning different resistance values to land cover type and elevation. We also estimated recent migration rates and evaluated them with regard to landscape features. There was a significant but weak relationship between Euclidian distance and genetic distance. There was no relationship between genetic distances and land cover, but a significant relationship between genetic distances and elevation resistance. We conclude that gene flow is restricted by high elevations in the northern part of the valley oak range and by high elevations and the Central Valley further south. Migration rate analysis indicated some gene flow occurring east–west but we suggest that the high connectivity in the northern Central Valley is facilitating the formation of these links. We predict that southern populations may become more differentiated in the future through genetic isolation and local adaptation taking place in the face of climate change.     

Osmotic stress decreases complexity underlying the electrophysiological dynamic in soybean

• Studies on plant electrophysiology are mostly focused on specific traits of single cells. Inspired by the complexity of the signalling network in plants, and by analogy with neurons in human brains, we sought evidence of high complexity in the electrical dynamics of plant signalling and a likely relationship with environmental cues.
• An EEG‐like standard protocol was adopted for high‐resolution measurements of the electrical signal in Glycine max seedlings. The signals were continuously recorded in the same plants before and after osmotic stimuli with a ?2 MPa mannitol solution. Non‐linear time series analyses methods were used as follows: auto‐correlation and cross‐correlation function, power spectra density function, and complexity of the time series estimated as Approximate Entropy (ApEn).
• Using Approximate Entropy analysis we found that the level of temporal complexity of the electrical signals was affected by the environmental conditions, decreasing when the plant was subjected to a low osmotic potential. Electrical spikes observed only after stimuli followed a power law distribution, which is indicative of scale invariance.
• Our results suggest that changes in complexity of the electrical signals could be associated with water stress conditions in plants. We hypothesised that the power law distribution of the spikes could be explained by a self‐organised critical state (SOC) after osmotic stress.

     

The Development of a Novel Mycobacterium-Escherichia coli Shuttle Vector System Using pMyong2, a Linear Plasmid from Mycobacterium yongonense DSM 45126T

The Mycobacterium-Escherichia coli shuttle vector system, equipped with the pAL5000 replicon, is widely used for heterologous gene expression and gene delivery in mycobacteria. Despite its extensive use, this system has certain limitations, which has led to the development of alternative mycobacterial vector systems. The present study describes the molecular structure and expression profiles of a novel 18-kb linear plasmid, pMyong2, from Mycobacterium yongonense. Sixteen open reading frames and a putative origin of replication were identified, and the compatibility of the pMyong2 and pAL5000 vector systems was demonstrated. In recombinant Mycobacterium smegmatis (rSmeg), the pMyong2 vector system showed a copy number that was approximately 37 times greater than that of pAL5000. Furthermore, pMyong2 increased the mRNA and protein expression of the human macrophage migration inhibitory factor (hMIF) over pAL5000 levels by approximately 10-fold and 50-fold, respectively, demonstrating the potential utility of the pMyong2 vector system in heterologous gene expression in mycobacteria. Successful delivery of the EGFP gene into mammalian cells via rSmeg carrying the pMyong2 vector system was also observed, demonstrating the feasibility of this system for DNA delivery. In conclusion, the pMyong2 vector system could be effectively used not only for the in vivo delivery of recombinant protein and DNA but also for mycobacterial genetic studies as an alternative or a complement to the pAL5000 vector system.     

Biosorption of Cr(VI) by Ceratocystis paradoxa MSR2 Using Isotherm Modelling,Kinetic Study and Optimization of Batch Parameters Using Response Surface Methodology

This study is focused on the possible use of Ceratocystis paradoxa MSR2 native biomass for Cr(VI) biosorption. The influence of experimental parameters such as initial pH, temperature, biomass dosage, initial Cr(VI) concentration and contact time were optimized using batch systems as well as response surface methodology (RSM). Maximum Cr(VI) removal of 68.72% was achieved, at an optimal condition of biomass dosage 2g L⁻¹, initial Cr(VI) concentration of 62.5 mg L⁻¹ and contact time of 60 min. The closeness of the experimental and the predicted values exhibit the success of RSM. The biosorption mechanism of MSR2 biosorbent was well described by Langmuir isotherm and a pseudo second order kinetic model, with a high regression coefficient. The thermodynamic study also revealed the spontaneity and exothermic nature of the process. The surface characterization using FT-IR analysis revealed the involvement of amine, carbonyl and carboxyl groups in the biosorption process. Additionally, desorption efficiency of 92% was found with 0.1 M HNO₃. The Cr(VI) removal efficiency, increased with increase in metal ion concentration, biomass concentration, temperature but with a decrease in pH. The size of the MSR2 biosorbent material was found to be 80 μm using particle size analyzer. Atomic force microscopy (AFM) visualizes the distribution of Cr(VI) on the biosorbent binding sites with alterations in the MSR2 surface structure. The SEM-EDAX analysis was also used to evaluate the binding characteristics of MSR2 strain with Cr(VI) metals. The mechanism of Cr(VI) removal of MSR2 biomass has also been proposed.     

NSC666715 and Its Analogs Inhibit Strand-Displacement Activity of DNA Polymerase β and Potentiate Temozolomide-Induced DNA Damage,Senescence and Apoptosis in Colorectal Cancer Cells

Recently approved chemotherapeutic agents to treat colorectal cancer (CRC) have made some impact; however, there is an urgent need for newer targeted agents and strategies to circumvent CRC growth and metastasis. CRC frequently exhibits natural resistance to chemotherapy and those who do respond initially later acquire drug resistance. A mechanism to potentially sensitize CRC cells is by blocking the DNA polymerase β (Pol-β) activity. Temozolomide (TMZ), an alkylating agent, and other DNA-interacting agents exert DNA damage primarily repaired by a Pol-β-directed base excision repair (BER) pathway. In previous studies, we used structure-based molecular docking of Pol-β and identified a potent small molecule inhibitor (NSC666715). In the present study, we have determined the mechanism by which NSC666715 and its analogs block Fen1-induced strand-displacement activity of Pol-β-directed LP-BER, cause apurinic/apyrimidinic (AP) site accumulation and induce S-phase cell cycle arrest. Induction of S-phase cell cycle arrest leads to senescence and apoptosis of CRC cells through the p53/p21 pathway. Our initial findings also show a 10-fold reduction of the IC₅₀ of TMZ when combined with NSC666715. These results provide a guide for the development of a target-defined strategy for CRC chemotherapy that will be based on the mechanisms of action of NSC666715 and TMZ. This combination strategy can be used as a framework to further reduce the TMZ dosages and resistance in CRC patients.     

Subgroups of Paediatric Acute Lymphoblastic Leukaemia Might Differ Significantly in Genetic Predisposition to Asparaginase Hypersensitivity

L-asparaginase (ASP) is a key element in the treatment of paediatric acute lymphoblastic leukaemia (ALL). However, hypersensitivity reactions (HSRs) to ASP are major challenges in paediatric patients. Our aim was to investigate genetic variants that may influence the risk to Escherichia coli-derived ASP hypersensitivity. Sample and clinical data collection was carried out from 576 paediatric ALL patients who were treated according to protocols from the Berlin—Frankfurt—Münster Study Group. A total of 20 single nucleotide polymorphisms (SNPs) in GRIA1 and GALNT10 genes were genotyped. Patients with GRIA1 rs4958351 AA/AG genotype showed significantly reduced risk to ASP hypersensitivity compared to patients with GG genotype in the T-cell ALL subgroup (OR = 0.05 (0.01–0.26); p = 4.70E-04), while no such association was found in pre-B-cell ALL. In the medium risk group two SNPs of GRIA1 (rs2055083 and rs707176) were associated significantly with the occurrence of ASP hypersensitivity (OR = 0.21 (0.09–0.53); p = 8.48E-04 and OR = 3.02 (1.36–6.73); p = 6.76E-03, respectively). Evaluating the genders separately, however, the association of rs707176 with ASP HSRs was confined only to females. Our results suggest that genetic variants of GRIA1 might influence the risk to ASP hypersensitivity, but subgroups of patients can differ significantly in this respect.     

Towards a Supertree of Arthropoda: A Species-Level Supertree of the Spiny,Slipper and Coral Lobsters (Decapoda: Achelata)

While supertrees have been built for many vertebrate groups (notably birds, mammals and dinosaurs), invertebrates have attracted relatively little attention. The paucity of supertrees of arthropods is particularly surprising given their economic and ecological importance, as well as their overwhelming contribution to biodiversity. The absence of comprehensive archives of machine-readable source trees, coupled with the need for software implementing repeatable protocols for managing them, has undoubtedly impeded progress. Here we present a supertree of Achelata (spiny, slipper and coral lobsters) as a proof of concept, constructed using new supertree specific software (the Supertree Toolkit; STK) and following a published protocol. We also introduce a new resource for archiving and managing published source trees. Our supertree of Achelata is synthesised from morphological and molecular source trees, and represents the most complete species-level tree of the group to date. Our findings are consistent with recent taxonomic treatments, confirming the validity of just two families: Palinuridae and Scyllaridae; Synaxidae were resolved within Palinuridae. Monophyletic Silentes and Stridentes lineages are recovered within Palinuridae, and all sub-families within Scyllaridae are found to be monophyletic with the exception of Ibacinae. We demonstrate the feasibility of building larger supertrees of arthropods, with the ultimate objective of building a complete species-level phylogeny for the entire phylum using a divide and conquer strategy.