Backcross-breeding and doubled-haploid facilitated introgression of stripe rust resistance in bread wheat

Stripe rust caused by the fungus Puccinia striiformis f. sp. tritici (Pst) may decrease wheat yield significantly in severe outbreaks. The most cost-effective and environmentally friendly approach to reduce yield losses due to rust diseases is deployment of effective resistant genes in wheat cultivars. The causal agents evolve and may break existing resistant sources as well. Therefore, long-term conventional breeding strategies and the ongoing evolution of pathogen populations in the region would put the success of breeding programmes at risk so that there is always a need for speeding up the process of germplasm enhancement through production of doubled-haploid breeding materials. In this study, we aimed at introgression of stripe rust resistance trait from three genotypes (Flanders, Martonvasar-17 (MV17) and Bersee) into a widely adapted cultivar “Ghods”. Positively selected F2BC² progenies of three backcrossing schemas, i.e. (i) Flanders/3^*Ghods; (ii) Ghods^*3/MV17; and (iii) Hybride-de-bersee/3^*Ghods, were used to produce three small-size doubled-haploid populations via wheat × Maize pollination methodology. The doubled-haploid populations were examined against two predominantly isolates of P. striiformis f. sp. tritici (Pst) i.e. 6E134A⁺ and 6E2A⁺Yr27⁺ and the screening revealed that 44 and 52 of the progenies are resistant to the above-mentioned isolates, respectively. Field data have shown that the stripe rust resistance doubled-haploid germplasm are comparable to local check cultivars in yield and earliness.     

Evidence of Online Performance Deterioration in User Sessions on Reddit

This article presents evidence of performance deterioration in online user sessions quantified by studying a massive dataset containing over 55 million comments posted on Reddit in April 2015. After segmenting the sessions (i.e., periods of activity without a prolonged break) depending on their intensity (i.e., how many posts users produced during sessions), we observe a general decrease in the quality of comments produced by users over the course of sessions. We propose mixed-effects models that capture the impact of session intensity on comments, including their length, quality, and the responses they generate from the community. Our findings suggest performance deterioration: Sessions of increasing intensity are associated with the production of shorter, progressively less complex comments, which receive declining quality scores (as rated by other users), and are less and less engaging (i.e., they attract fewer responses). Our contribution evokes a connection between cognitive and attention dynamics and the usage of online social peer production platforms, specifically the effects of deterioration of user performance.     

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) ^1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells ³, and cell-free in vitro translation systems ^2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins ^5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein ^5,10.In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO₂ incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.     

High-level production of extracellular lipase by Yarrowia lipolytica mutants from methyl oleate

The yeast Yarrowia lipolytica degrades efficiently low-cost hydrophobic substrates for the production of various added-value products such as lipases. To obtain yeast strains producing high levels of extracellular lipase, Y. lipolytica DSM3286 was subjected to mutation using ethyl methanesulfonate (EMS) and ultraviolet (UV) light. Twenty mutants were selected out of 1600 mutants of Y. lipolytica treated with EMS and UV based on lipase production ability on selective medium. A new industrial medium containing methyl oleate was optimized for lipase production. In the 20 L bioreactor containing new industrial medium, one UV mutant (U6) produced 356 U/mL of lipase after 24h, which is about 10.5-fold higher than that produced by the wild type strain. The properties of the mutant lipase were the same as those of the wild type: molecular weight 38 kDa, optimum temperature 37°C and optimum pH 7. Furthermore, the nucleotide sequences of extracellular lipase gene (LIP2) in wild type and mutant strains were determined. Only two silent substitutions at 362 and 385 positions were observed in the ORF region of LIP2. Two single substitutions and two duplications of the T nucleotide were also detected in the promoter region. LIP2 sequence comparison of the Y. lipolytica DSM3286 and U6 strains shows good targets to effective DNA recombinant for extracellular lipase of Y. lipolytica.     

Intercalation of manganese-mefenamic acid complex into double stranded of calf thymus DNA

Abstract

The interaction of the [Mn(mef)₂(phen)H₂O] complex in which mef is mefenamic acid drug and phen is 1,10 phenanthrolin ligand with calf thymus DNA (ct-DNA) was studied by using different spectroscopic methods, molecular docking and viscometery. The competitive fluorescence and UV–Vis absorption spectroscopy indicated that the complex interacted with ctDNA via intercalating binding mode with the binding constant of 1.16?×?10⁴ Lmol^?1. The thermodynamic studies showed that the reaction between the complex and ctDNA is exothermic. Furthermore, the complex induced changes in DNA viscosity. Circular dichroism spectroscopy (CD) was employed to measure the conformational changes of ctDNA in the presence of the complex and verified intercalation binding mode. The molecular modeling results illustrated that the complex interacted via intercalation by relative binding energy of ?28.45?kJ mol^?1.     

Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages

The positive role of fatty acids in the prevention and alleviation of non-human and human diseases have been and continue to be extensively documented. These roles include influences on infectious and non-infectious diseases including prevention of inflammation as well as mucosal immunity to infectious diseases. Cholera is an acute intestinal illness caused by the bacterium Vibrio cholerae. It occurs in developing nations and if left untreated, can result in death. While vaccines for cholera exist, they are not always effective and other preventative methods are needed. We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin using mouse BALB/C macrophages and human intestinal epithelial cells, respectively. We solubilized the above fatty acids and used cell proliferation assays to determine the concentration ranges and specific concentrations of the fatty acids that are not detrimental to human intestinal epithelial cell viability. We solubilized cholera toxin and used it in an assay to determine the concentration ranges and specific concentrations of cholera toxin that do not statistically decrease cell viability in BALB/C macrophages. We found the optimum fatty acid concentrations to be between 1-5 ng/μl, and that for cholera toxin to be < 30 ng per treatment. This data may aid future studies that aim to find a protective mucosal role for fatty acids in prevention or alleviation of cholera infections.     

Rapid Prototyping of a High Sensitivity Graphene Based Glucose Sensor Strip

A rapid prototyping of an inexpensive, disposable graphene and copper nanocomposite sensor strip using polymeric flexible substrate for highly sensitive and selective nonenzymatic glucose detection has been developed and tested for direct oxidization of glucose. The CuNPs were electrochemically deposited on to the graphene sheets to improve electron transfer rates and to enhance electrocatalytic activity toward glucose. The graphene based electrode with CuNPs demonstrated a high degree of sensitivity (1101.3±56 μA/mM.cm²), excellent selectivity (without an interference with Ascorbic Acid, Uric Acid, Dopamine, and Acetaminophen), good stability with a linear response to glucose ranging from 0.1 mM to 0.6 mM concentration, and detection limits of 0.025 mM to 0.9 mM. Characterization of the electrodes was performed by scanning electron microscopy (FESEM and SEM). The electrochemical properties of the modified graphene electrodes were inspected by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and amperometry.     

A Molecular Dynamics Simulation Study of Nanomechanical Properties of Asymmetric Lipid Bilayer

A very important part of the living cells of biological systems is the lipid membrane. The mechanical properties of this membrane play an important role in biophysical studies. Investigation as to how the insertion of additional phospholipids in one leaflet of a bilayer affects the physical properties of the obtained asymmetric lipid membrane is of recent practical interest. In this work a coarse-grained molecular dynamics simulation was carried out in order to compute the pressure tensor, the lateral pressure, the surface tension and the first moment of lateral pressure in each leaflet of such a bilayer. Our simulations indicate that adding more phospholipids into one monolayer results in asymmetrical changes in the lateral pressure of the individual bilayer leaflets. Interestingly, it has been observed that a change in phospholipid density in one leaflet affects the physical properties of unperturbed leaflet as well. The asymmetric behavior of the physical properties of the two leaflets as a result of a change in the contribution of the various intermolecular forces in the presence of additional phospholipids may be expressed formally.     

New immunological investigations on Helicobacter pylori‐induced gastric ulcer in patients

Although Helicobacter pylori (Hp) plays an important role in the pathogenesis of chronic gastritis and gastric ulcer, little is known about the probable mechanisms of these types of gastrointestinal damage. To determine the precise mechanisms involved in ulcer formation, immune responses in patients with gastric ulcer (GUP) caused by Hp infection (Hp⁺) were compared with those of other gastritis patients (GP). The sensitivity and proliferation of peripheral blood mononuclear cells (PBMNCs) obtained from patients were evaluated by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay against exposure with complex Hp crude antigen (HPCA) and mitogen (phytohemagglutinin, PHA). Production of inflammatory cytokines, including interleukin (IL)‐1β and IL‐8, in serum and supernatants of PBMNCs were then measured by ELISA. It was found that, after stimulation with PHA, both IL‐8 and IL‐1β concentrations in sera and supernatants as well as proliferation and sensitivity were statistically greater in GUP Hp⁺ than GP Hp^?. Furthermore, HPCA inhibited the proliferation of PBMNCs dose‐dependently; however, it stimulated IL‐8 and IL‐1β production in supernatants of mononuclear cells. Therefore, the up‐regulated concentrations of IL‐8 and IL‐1β may have been caused by increase in the size of mononuclear cell subpopulations or in their cytokine secretory activity, indicating the greatest cell responsiveness in GUP Hp⁺ patients. These results suggest that tissue damage and ulcers occur in patients who produce more IL‐8 and IL‐1β than patients who do not develop ulcers; the former consequently have more activated immune cells at the site of infection. Therefore, both host responses and Hp virulence factors may be involved in the development of gastric ulcers.

     

Purification of plasmid DNA with polymer-salt aqueous two-phase system: optimization using response surface methodology

An experimental design was used to optimize plasmid purification from an alkaline lysate of Escherichia coli cells using PEG-sodium citrate aqueous two-phase systems (ATPS), and to evaluate the influence of pH, PEG molecular weight, tie line length, phase volume ratio, and lysate load. To build the mathematical model and minimize the number of experiments for the design parameters, response surface methodology (RMS) with an orthogonal rotatable central composite design was defined based on the conditions found for the highest purification by preliminary tests. The adequacy of the calculated models for the plasmid recovery and remaining RNA were confirmed by means of variance analysis and additional experiments. Analysis of contours of constant response as a function of pH, PEG molecular weight, tie line length, and cell lysate load for three different phase volume ratios revealed different effects of these five factors on the studied parameters. Plasmid recovery of 99% was predicted for a system with PEG 400, pH 6.9, tie line length of 38.7%, phase volume ratio of 1.5, and lysate load of 10% (v/v). Under these conditions the predicted RNA removal was 68%.