Understanding osmotic stress tolerance in leaves and nodules of two <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> cultivars with contrasting drought tolerance

Drought and salinity are environmental constraints that affect crop yields worldwide. In nature, both stresses are multifaceted problems that are usually associated with other adverse circumstances which limit plant performance such as water shortage and nutrient deficits. In order to assess common features of both stresses, the effects of mannitol-induced osmotic stress were monitored using two Phaseolus vulgaris cultivars, Cv. ‘Flamingo’ (tolerant) and Cv. ‘Coco Blanc’ (sensitive) which differed in their drought and salinity tolerance. Growth, water relations, organic and inorganic compound accumulation and soluble protein contents were measured in leaves and nodules of these N₂-fixing plants. The aim of the present study was to check whether osmotic stress tolerance is associated with accumulation of some of these compounds either in leaves, nodules or both organs. At the whole-plant level, Cv. ‘Flamingo’ showed a better maintenance of plant biomass and shoot water status. At the cell level, this was related to a better osmotic adjustment ability both in leaves and nodules and also to a better adjustment of the cell wall elasticity. At the metabolic level, the contrasting accumulation of the different amino acids in nodules of each cultivar suggested that amino acids pathways can be regulated to different degrees under stress conditions. At the metabolic level, it seems that symbiosis in the sink organ (the nodule) plays a crucial role in conferring drought and salinity tolerance in the common bean.     

Evolutionary transitions to new DNA methyltransferases through target site expansion and shrinkage

DNA-binding and modifying proteins show high specificity but also exhibit a certain level of promiscuity. Such latent promiscuous activities comprise the starting points for new protein functions, but this hypothesis presents a paradox: a new activity can only evolve if it already exists. How then, do novel activities evolve? DNA methyltransferases, for example, are highly divergent in their target sites, but how transitions toward novel sites occur remains unknown. We performed laboratory evolution of the DNA methyltransferase M.HaeIII. We found that new target sites emerged primarily through expansion of the original site, GGCC, and the subsequent shrinkage of evolved expanded sites. Variants evolved for sites that are promiscuously methylated by M.HaeIII [GG(^A/_T)CC and GGCGCC] carried mutations in ‘gate-keeper’ residues. They could thereby methylate novel target sites such as GCGC and GGATCC that were neither selected for nor present in M.HaeIII. These ‘generalist’ intermediates were further evolved to obtain variants with novel target specificities. Our results demonstrate the ease by which new DNA-binding and modifying specificities evolve and the mechanism by which they occur at both the protein and DNA levels.     

Mast cell deficiency attenuates progression of atherosclerosis and hepatic steatosis in apolipoprotein E-null mice

Mast cells are important cells of the immune system and are recognized as participants in the pathogenesis of atherosclerosis. In this study, we evaluated the role of mast cells on the progression of atherosclerosis and hepatic steatosis using the apolipoprotein E-deficient (ApoE(-/-)) and ApoE(-/-)/mast cell-deficient (Kit(W-sh/W-sh)) mouse models maintained on a high-fat diet. The en face analyses of aortas showed a marked reduction in plaque coverage in ApoE(-/-)/Kit(W-sh/W-sh) compared with ApoE(-/-) after a 6-mo regimen with no significant change noted after 3 mo. Quantification of intima/media thickness on hematoxylin and eosin-stained histological cross sections of the aortic arch revealed no significant difference between ApoE(-/-) and ApoE(-/-)/Kit(W-sh/W-sh) mice. The high-fat regimen did not induce atherosclerosis in either Kit(W-sh/W-sh) or wild-type mice. Mast cells with indications of degranulation were seen only in the aortic walls and heart of ApoE(-/-) mice. Compared with ApoE(-/-) mice, the serum levels of total cholesterol, low-density lipoprotein and high-density lipoprotein were decreased by 50% in ApoE(-/-)/Kit(W-sh/W-sh) mice, whereas no appreciable differences were noted in serum levels of triglycerides or very low density lipoprotein. ApoE(-/-)/Kit(W-sh/W-sh) mice developed significantly less hepatic steatosis than ApoE(-/-) mice after the 3-mo regimen. The analysis of Th1/Th2/Th17 cytokine profile in the sera revealed significant reduction of interleukin (IL)-6 and IL-10 in ApoE(-/-)/Kit(W-sh/W-sh) mice compared with ApoE(-/-) mice. The assessment of systemic generation of thromboxane A(2) (TXA(2)) and prostaglandin I(2) (PGI(2)) revealed significant decrease in the production of PGI(2) in ApoE(-/-)/Kit(W-sh/W-sh) mice with no change in TXA(2). The decrease in PGI(2) production was found to be associated with reduced levels of cyclooxygenase-2 mRNA in the aortic tissues. A significant reduction in T-lymphocytes and macrophages was noted in the atheromas of the ApoE(-/-)/Kit(W-sh/W-sh) mice. These results demonstrate the direct involvement of mast cells in the progression of atherosclerosis and hepatic steatosis.     

Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis

The ability to redesign enzymes to catalyze noncognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of metalloenzyme active site functional groups to catalyze new reactions. Using this method, we engineered a zinc-containing mouse adenosine deaminase to catalyze the hydrolysis of a model organophosphate with a catalytic efficiency (k(cat)/K(m)) of ~10(4) M(-1) s(-1) after directed evolution. In the high-resolution crystal structure of the enzyme, all but one of the designed residues adopt the designed conformation. The designed enzyme efficiently catalyzes the hydrolysis of the R(P) isomer of a coumarinyl analog of the nerve agent cyclosarin, and it shows marked substrate selectivity for coumarinyl leaving groups. Computational redesign of native enzyme active sites complements directed evolution methods and offers a general approach for exploring their untapped catalytic potential for new reactivities.     

Low levels of quantitative and molecular genetic differentiation among natural populations of Medicago ciliaris Kroch. (Fabaceae) of different Tunisian eco-geographical origin

In this paper, we analyze the genetic variability in four Tunisian natural populations of Medicago ciliaris using 19 quantitative traits and six polymorphic microsatellite loci. We investigated the amplification transferability of 30 microsatellites developed in the model legume M. truncatula to M. ciliaris. Results revealed that about 56.66% of analyzed markers are valuable genetic markers for M. ciliaris. The most genetic diversity at quantitative traits and microsatellite loci was found to occur within populations (>80%). Low differentiations among populations at quantitative traits Q _ST  = 0.146 and molecular markers F _ST  = 0.18 were found. The majority of measured traits exhibited no significant difference in the level of Q _ST and F _ST . Furthermore, significant correlations established between these traits and eco-geographical factors suggested that natural selection should be invoked to explain the level of phenotypic divergence among populations rather than drift. There was no significant correlation between population differentiation at quantitative traits and molecular markers. Significant spatial genetic structure consistent with models of isolation by distance was detected within all studied populations. The site-of-origin environmental factors explain about 9.07% of total phenotypic genetic variation among populations. The eco-geographical factors that influence more the variation of measured traits among populations are the soil texture and altitude. Nevertheless, there were no consistent pattern of associations between gene diversity (He) and environmental factors.     

An improved intrafemoral injection with minimized leakage as an orthotopic mouse model of osteosarcoma

Osteosarcoma, the most common type of primary bone cancer, is the second highest cause of cancer-related death in pediatric patients. To understand the mechanisms behind osteosarcoma progression and to discover novel therapeutic strategies for this disease, a reliable and appropriate mouse model is essential. For this purpose, osteosarcoma cells need to be injected into the bone marrow. Previously, the intratibial and intrafemoral injection methods were reported; however, the major drawback of these methods is the potential leakage of tumor cells from the injection site during or after these procedures. To overcome this, we have established an improved method to minimize leakage in an orthotopic mouse model of osteosarcoma. By taking advantage of the anatomical benefits of the femur with less bowing and larger medullary cavity than those of the tibia, osteosarcoma cells are injected directly into the femoral cavity following reaming of its intramedullary space. To prevent potential leakage of tumor cells during and after the surgery, the injection site is sealed with bone wax. This method requires a minor surgery of approximately 15 min under anesthesia. Our established orthotopic osteosarcoma model could serve as a valuable and reliable tool for examining progression of various types of bone tumors.     

Financial Time Series Prediction Using Spiking Neural Networks

In this paper a novel application of a particular type of spiking neural network, a Polychronous Spiking Network, was used for financial time series prediction. It is argued that the inherent temporal capabilities of this type of network are suited to non-stationary data such as this. The performance of the spiking neural network was benchmarked against three systems: two “traditional”, rate-encoded, neural networks; a Multi-Layer Perceptron neural network and a Dynamic Ridge Polynomial neural network, and a standard Linear Predictor Coefficients model. For this comparison three non-stationary and noisy time series were used: IBM stock data; US/Euro exchange rate data, and the price of Brent crude oil. The experiments demonstrated favourable prediction results for the Spiking Neural Network in terms of Annualised Return and prediction error for 5-Step ahead predictions. These results were also supported by other relevant metrics such as Maximum Drawdown and Signal-To-Noise ratio. This work demonstrated the applicability of the Polychronous Spiking Network to financial data forecasting and this in turn indicates the potential of using such networks over traditional systems in difficult to manage non-stationary environments.     

Burkholderia pseudomallei γ-carbonic anhydrase is strongly activated by amino acids and amines

Activation of the γ-class carbonic anhydrase (CAs, EC 4.2.1.1) from the pathogenic bacterium Burkholderia pseudomallei (BpsγCA) with a series of natural and non-natural amino acids and aromatic/heterocyclic amines has been investigated. The best BpsγCA activators were d-His, l-DOPA, d-Trp, 4-amino-l-Phe, dopamine, 2-(2-aminoethyl)pyridine, 2-aminoethyl-piparazine/morpholine and l-adrenaline, which showed activation constants ranging between 9 and 86 nM. The least effective activators were l-His, l-Phe and 2-pyridyl-methylamine, with K_As in the range of 1.73–24.7 μM. As little is known about the role of γ-CAs in the lifecycle and virulence of this saprophytic bacterium, this study may shed some light on such phenomena. This is the first CA activation study of a γ-CA from a pathogenic bacterium, the only other such study being on the enzyme discovered in the archaeon Methanosarcina thermophila, Cam.     

A validated antibody panel for the characterization of tau post-translational modifications

Background

Tau is a microtubule-binding protein, which is subject to various post-translational modifications (PTMs) including phosphorylation, methylation, acetylation, glycosylation, nitration, sumoylation and truncation. Aberrant PTMs such as hyperphosphorylation result in tau aggregation and the formation of neurofibrillary tangles, which are a hallmark of Alzheimer’s disease (AD). In order to study the importance of PTMs on tau function, antibodies raised against specific modification sites are widely used. However, quality control of these antibodies is lacking and their specificity for particular modifications is often unclear.

Methods

In this study, we first designed an online tool called ‘TauPTM’, which enables the visualization of PTMs and their interactions on human tau. Using TauPTM, we next searched for commercially available antibodies against tau PTMs and characterized their specificity by peptide array, immunoblotting, electrochemiluminescence ELISA and immunofluorescence technologies.

Results

We demonstrate that commercially available antibodies can show a significant lack of specificity, and PTM-specific antibodies in particular often recognize non-modified versions of the protein. In addition, detection may be hindered by other PTMs in close vicinity, complicating the interpretation of results. Finally, we compiled a panel of specific antibodies and show that they are useful to detect PTM-modified endogenous tau in hiPSC-derived neurons and mouse brains.

Conclusion

This study has created a platform to reliably and robustly detect changes in localization and abundance of post-translationally modified tau in health and disease. A web-based version of TauPTM is fully available at http://www.tauptm.org.