Improved Activity of a Thermophilic Cellulase,Cel5A,from Thermotoga maritima on Ionic Liquid Pretreated Switchgrass

Ionic liquid pretreatment of biomass has been shown to greatly reduce the recalcitrance of lignocellulosic biomass, resulting in improved sugar yields after enzymatic saccharification. However, even under these improved saccharification conditions the cost of enzymes still represents a significant proportion of the total cost of producing sugars and ultimately fuels from lignocellulosic biomass. Much of the high cost of enzymes is due to the low catalytic efficiency and stability of lignocellulolytic enzymes, especially cellulases, under conditions that include high temperatures and the presence of residual pretreatment chemicals, such as acids, organic solvents, bases, or ionic liquids. Improving the efficiency of the saccharification process on ionic liquid pretreated biomass will facilitate reduced enzyme loading and cost. Thermophilic cellulases have been shown to be stable and active in ionic liquids but their activity is typically at lower levels. Cel5A_Tma, a thermophilic endoglucanase from Thermotoga maritima, is highly active on cellulosic substrates and is stable in ionic liquid environments. Here, our motivation was to engineer mutants of Cel5A_Tma with higher activity on 1-ethyl-3-methylimidazolium acetate ([C₂mim][OAc]) pretreated biomass. We developed a robotic platform to screen a random mutagenesis library of Cel5A_Tma. Twelve mutants with 25–42% improvement in specific activity on carboxymethyl cellulose and up to 30% improvement on ionic-liquid pretreated switchgrass were successfully isolated and characterized from a library of twenty thousand variants. Interestingly, most of the mutations in the improved variants are located distally to the active site on the protein surface and are not directly involved with substrate binding.     

Factors affecting incidence and severity of leaf spot disease on lettuce caused by Septoria birgitae

In the 1990s during wet seasons a new disease causing brown leaf spots on lettuce (Lactuca sativa) was found for the first time in many lettuce‐growing areas of Austria and Germany. The causal agent, a new pathogenic species called Septoria birgitae, may be responsible for total crop loss. To study how temperature, inoculum density and leaf wetness period influence disease incidence and severity of leaf spot on lettuce caused by S. birgitae, we carried out in vivo experiments in growth chambers and in the field. Additionally, we evaluated the relevance of infected plant debris acting as a primary inoculum source in soil for subsequent crops. S. birgitae produces spores over a wide temperature range between 5°C and 30°C, and can infect plants at temperatures between 10°C and 30°C, with an optimum between 20°C and 30°C. Spores of S. birgitae at a density of at least 10³ conidia mL^–1 are essential for disease outbreak on lettuce. Because leaf wetness is crucial for releasing conidia from pycnidia, we studied the impact of leaf wetness duration on disease development under various temperature conditions. For relevant leaf spot disease development on lettuce in vivo, a leaf wetness duration of at least 24 h and temperatures higher than 10°C were necessary. Leaf spot disease development in the field required several leaf wetness periods longer than 20 h at approximately 15°C at the beginning of crop cultivation. Incorporating S. birgitae infected plant debris in soil as a primary inoculum was not relevant for leaf spot disease outbreak in the next year. However, in cases of continuous cropping of lettuce on the same field and in the same season, Septoria‐infected lettuce debris may become more relevant.     

Maltodextrins as Chiral Selectors in CE: Molecular Structure Effect of Basic Chiral Compounds on the Enantioseparation

Prediction of chiral separation for a compound using a chiral selector is an interesting and debatable work. For this purpose, in this study 23 chiral basic drugs with different chemical structures were selected as model solutes and the influence of their chemical structures on the enantioseparation in the presence of maltodextrin (MD) as chiral selector was investigated. For chiral separation, a 100‐mM phosphate buffer solution (pH 3.0) containing 10% (w/v) MD with dextrose equivalent (DE) of 4‐7 as chiral selector at the temperature of 25°C and voltage of 20 kV was used. Under this condition, baseline separation was achieved for nine chiral compounds and partial separation was obtained for another six chiral compounds while no enantioseparation was obtained for the remaining eight compounds. The results showed that the existence of at least two aromatic rings or cycloalkanes and an oxygen or nitrogen atom or –CN group directly bonded to the chiral center are necessary for baseline separation. With the obtained results in this study, chiral separation of a chiral compound can be estimated with MD‐modified capillary electrophoresis before analysis. This prediction will minimize the number of preliminary experiments required to resolve enantiomers and will save time and cost. Chirality 26:620–628, 2014. © 2014 Wiley Periodicals, Inc.     

Selective Divalent Cobalt Ions Detection Using Ag2O3-ZnO Nanocones by ICP-OES Method for Environmental Remediation

Here, we have synthesized Ag₂O₃-ZnO nanocones (NCs) by a wet-chemical route using reducing agents at low temperature. The structural, optical and morphological properties of Ag₂O₃-ZnO NCs were investigated by several conventional techniques such as powder XRD, XPS, FESEM, XEDS, FTIR and UV/vis. spectroscopy. The analytical parameters of prepared NCs were also calculated for a selective detection of divalent cobalt [Co(II)] prior to its determination by inductively coupled plasma-optical emission spectrometry (ICP-OES). The selectivity of NCs toward various metal ions, including Cd(II), Co(II), Cr(III), Cu(II), Fe(III), Ni(II), and Zn(II) was studied. Results of the selectivity study demonstrated that Ag₂O₃-ZnO NC phase was the most selective towards Co(II) ion. The uptake capacity for Co(II) ion was experimentally calculated to be ∼76.69 mgg⁻¹. Moreover, adsorption isotherm data provided that the adsorption process was mainly monolayer on homogeneous adsorbent surfaces of Ag₂O₃-ZnO NCs. Kinetic study revealed that the adsorption of Co(II) on Ag₂O₃-ZnO NCs phase followed the pseudo-second-order kinetic model. In addition, thermodynamic results provided that the adsorption mechanism of Co(II) ions on Ag₂O₃-ZnO NCs was a spontaneous process and thermodynamically favorable. Finally, the proposed method was validated by applying it to real environmental water samples with reasonable results.     

Rapid One-Step Selection Method for Generating Nucleic Acid Aptamers: Development of a DNA Aptamer against α-Bungarotoxin

Background

Nucleic acids based therapeutic approaches have gained significant interest in recent years towards the development of therapeutics against many diseases. Recently, research on aptamers led to the marketing of Macugen®, an inhibitor of vascular endothelial growth factor (VEGF) for the treatment of age related macular degeneration (AMD). Aptamer technology may prove useful as a therapeutic alternative against an array of human maladies. Considering the increased interest in aptamer technology globally that rival antibody mediated therapeutic approaches, a simplified selection, possibly in one-step, technique is required for developing aptamers in limited time period.

Principal Findings

Herein, we present a simple one-step selection of DNA aptamers against α-bungarotoxin. A toxin immobilized glass coverslip was subjected to nucleic acid pool binding and extensive washing followed by PCR enrichment of the selected aptamers. One round of selection successfully identified a DNA aptamer sequence with a binding affinity of 7.58 µM.

Conclusion

We have demonstrated a one-step method for rapid production of nucleic acid aptamers. Although the reported binding affinity is in the low micromolar range, we believe that this could be further improved by using larger targets, increasing the stringency of selection and also by combining a capillary electrophoresis separation prior to the one-step selection. Furthermore, the method presented here is a user-friendly, cheap and an easy way of deriving an aptamer unlike the time consuming conventional SELEX-based approach. The most important application of this method is that chemically-modified nucleic acid libraries can also be used for aptamer selection as it requires only one enzymatic step. This method could equally be suitable for developing RNA aptamers.     

Elimination of inhibitory synapses is a major component of adult ocular dominance plasticity

During development, cortical plasticity is associated with the rearrangement of excitatory connections. While these connections become more stable with age, plasticity can still be induced in the adult cortex. Here we provide evidence that structural plasticity of?inhibitory synapses onto pyramidal neurons is?a major component of plasticity in the adult neocortex. In?vivo two-photon imaging was used to monitor the formation and elimination of fluorescently labeled inhibitory structures on pyramidal neurons. We find that ocular dominance plasticity in the adult visual cortex is associated with rapid inhibitory synapse loss, especially of those present on dendritic spines. This occurs not only with monocular deprivation but also with subsequent restoration of binocular vision. We propose that in the adult visual cortex the experience-induced loss of inhibition may effectively strengthen specific visual inputs with limited need for rearranging the excitatory circuitry.     

Role of the angiotensin II AT2 receptor in inflammation and oxidative stress: opposing effects in lean and obese Zucker rats

Inflammation and oxidative stress are believed to contribute to hypertension in obesity/diabetes. Recently, we reported a role for the AT(2) receptor in blood pressure control in obese Zucker rats. However, the role of AT(2) receptors in inflammation and oxidative stress in obesity is not known. Therefore, in the present study, we tested the effects of the AT(2) receptor agonist CGP-42112A on inflammation and oxidative stress in obese Zucker rats and compared them in their lean counterparts. Rats were systemically treated with either vehicle (control) or CGP-42112A (1 μg·kg(-1)·min(-1); osmotic pump) for 2 wk. Markers of inflammation (CRP, MCP-1, TNF-α, and IL-6) and oxidative stress (HO-1, gp-91(phox)) as well as an antioxidant (SOD) were determined. Control obese rats had higher plasma levels of CRP, MCP-1, TNF-α, IL-6, and HO-1 compared with control lean rats. Conversely, plasma SOD activity was lower in control obese than in control lean rats. Furthermore, the protein levels of TNF-α and gp-91(phox) were higher in the kidney cortex of control obese rats. Interestingly, CGP-42112A treatment in obese rats reduced the plasma and kidney cortex inflammatory (TNF-α, IL-6) and oxidative stress (gp-91(phox)) markers and increased plasma SOD activity to the levels seen in lean control rats. However, CGP-42112A treatment in lean rats increased inflammatory (TNF-α, IL-6) and oxidative stress (gp-91(phox)) markers in the plasma and kidney cortex. Our present studies suggest anti-inflammatory and antioxidative functions of AT(2) receptor in obese Zucker rats but proinflammatory and prooxidative functions in lean Zucker rats.     

Analysis of Genes Involved in Body Weight Regulation by Targeted Re-Sequencing

Introduction

Genes involved in body weight regulation that were previously investigated in genome-wide association studies (GWAS) and in animal models were target-enriched followed by massive parallel next generation sequencing.

Methods

We enriched and re-sequenced continuous genomic regions comprising FTO, MC4R, TMEM18, SDCCAG8, TKNS, MSRA and TBC1D1 in a screening sample of 196 extremely obese children and adolescents with age and sex specific body mass index (BMI) ≥ 99^th percentile and 176 lean adults (BMI ≤ 15^th percentile). 22 variants were confirmed by Sanger sequencing. Genotyping was performed in up to 705 independent obesity trios (extremely obese child and both parents), 243 extremely obese cases and 261 lean adults.

Results and Conclusion

We detected 20 different non-synonymous variants, one frame shift and one nonsense mutation in the 7 continuous genomic regions in study groups of different weight extremes. For SNP Arg695Cys (rs58983546) in TBC1D1 we detected nominal association with obesity (p_TDT = 0.03 in 705 trios). Eleven of the variants were rare, thus were only detected heterozygously in up to ten individual(s) of the complete screening sample of 372 individuals. Two of them (in FTO and MSRA) were found in lean individuals, nine in extremely obese. In silico analyses of the 11 variants did not reveal functional implications for the mutations. Concordant with our hypothesis we detected a rare variant that potentially leads to loss of FTO function in a lean individual. For TBC1D1, in contrary to our hypothesis, the loss of function variant (Arg443Stop) was found in an obese individual. Functional in vitro studies are warranted.     

The Prooxidant Action of Dietary Antioxidants Leading to Cellular DNA Breakage and Anticancer Effects: Implications for Chemotherapeutic Action Against Cancer

Plant-derived dietary antioxidants have attracted considerable interest in recent past for their ability to induce apoptosis and regression of tumors in animal models. While it is believed that the antioxidant properties of these agents may contribute to lowering the risk of cancer induction by impeding oxidative injury to DNA, it could not account for apoptosis induction and chemotherapeutic observations. In this article, we show that dietary antioxidants can alternatively switch to a prooxidant action in the presence of transition metals such as copper. Such a prooxidant action leads to strand breaks in cellular DNA and growth inhibition in cancer cells. Further, the cellular DNA breakage and anticancer effects were found to be significantly enhanced in the presence of copper ions. Moreover, inhibition of antioxidant-induced DNA strand breaks and oxidative stress by Cu(I)-specific chelators bathocuproine and neocuproine demonstrated the role of endogenous copper in the induction of the prooxidant mechanism. Since it is well established that tissue, cellular, and serum copper levels are considerably elevated in various malignancies, such a prooxidant cytotoxic mechanism better explains the anticancer activity of dietary antioxidants against cancer cells.     

Detection of new transposable element families in Drosophila melanogaster and Anopheles gambiae genomes

The techniques that are usually used to detect transposable elements (TEs) in nucleic acid sequences rely on sequence similarity with previously characterized elements. However, these methods are likely to miss many elements in various organisms. We tested two strategies for the detection of unknown elements. The first, which we call "TBLASTX strategy," searches for TE sequences by comparing the six-frame translations of the nucleic acid sequences of known TEs with the genomic sequence of interest. The second, "repeat-based strategy," searches genomic sequences for long repeats and clusters them in groups of similar sequences. TE copies from a given family are expected to cluster together. We tested the Drosophila melanogaster genomic sequence and the recently sequenced Anopheles gambiae genome in which most TEs remain unknown. We showed that the "TBLASTX strategy" is very efficient as it detected at least 332 new TE families in D. melanogaster and 400 in A. gambiae. This was unexpected in Drosophila as TEs of this organism have been extensively studied. The "repeat-based strategy" appeared to be very inefficient because of two problems: (i) TE copies are heavily deleted and few copies share homologous regions, and (ii) segmental duplications are frequent and it is not easy to distinguish them from TE copies.