Enumeration of micro-organisms in processed soy products with an automated most probable number method compared with standard plate method

Aim: The automated TEMPO system (bioMerieux) is based on the most probable number (MPN) method for the enumeration of micro‐organisms in foods. In this study, we evaluated the performance of the TEMPO system as a diagnostic tool in comparison with the standard method in processed soy products. Methods and Results: A verification study was conducted using artificially contaminated soy product samples such as soy protein isolate, water‐soluble soy polysaccharides, soy milk and processed soy food. Five types of micro‐organisms were analysed using the automated MPN method (total aerobic bacteria, total coliforms, Enterobacteriaceae, yeast and mould and Staphylococcus aureus) vs the standard plate method. The results from each of the methods were highly correlated (r > 0·95). Naturally contaminated processed soy products on the market were also studied. There were no discrepancies observed between the respective methods. Conclusions: TEMPO methods were equivalent to the corresponding standard plate methods with very good rates of agreement. Significance and Impact of the Study: The automated MPN method is more practical and reliable for in‐house microbiological testing in processed soy products.     

Specific In Situ Visualization of Plasma Cells Producing Antibodies against Porphyromonas gingivalis in Gingival Radicular Cyst: Application of the Enzyme-Labeled Antigen Method

The enzyme-labeled antigen method was applied to visualize plasma cells producing antibodies to Porphyromonas gingivalis, flora of the human oral cavity. Antibodies to P. gingivalis have reportedly been detected in sera of patients with periodontitis. Biotinylated bacterial antigens, Ag53, and four gingipain domains (Arg-pro, Arg-hgp, Lys-pro, and Lys-hgp) were prepared by the cell-free protein synthesis system using the wheat germ extract. In paraformaldehyde-fixed frozen sections of rat lymph nodes experimentally immunized with Ag53-positive and Ag53-negative P. gingivalis, plasma cells were labeled with biotinylated Arg-hgp and Lys-hgp. Antibodies to Ag53 were detected only in the nodes immunized with Ag53-positive bacteria. In two of eight lesions of gingival radicular cyst with inflammatory infiltration, CD138-positive plasma cells in frozen sections were signalized for Arg-hgp and Lys-hgp. An absorption study using unlabeled antigens confirmed the specificity of staining. The AlphaScreen method identified the same-type antibodies in tissue extracts but not in sera. Antibodies to Ag53, Arg-pro, and Lys-pro were undetectable. In two cases, serum antibodies to Arg-hgp and Lys-hgp were AlphaScreen positive, whereas plasma cells were scarcely observed within the lesions. These findings indicate the validity of the enzyme-labeled antigen method. This is the very first application of this novel histochemical technique to human clinical samples.     

AFP-Pred: A random forest approach for predicting antifreeze proteins from sequence-derived properties

Some creatures living in extremely low temperatures can produce some special materials called “antifreeze proteins” (AFPs), which can prevent the cell and body fluids from freezing. AFPs are present in vertebrates, invertebrates, plants, bacteria, fungi, etc. Although AFPs have a common function, they show a high degree of diversity in sequences and structures. Therefore, sequence similarity based search methods often fails to predict AFPs from sequence databases. In this work, we report a random forest approach “AFP-Pred” for the prediction of antifreeze proteins from protein sequence. AFP-Pred was trained on the dataset containing 300 AFPs and 300 non-AFPs and tested on the dataset containing 181 AFPs and 9193 non-AFPs. AFP-Pred achieved 81.33% accuracy from training and 83.38% from testing. The performance of AFP-Pred was compared with BLAST and HMM. High prediction accuracy and successful of prediction of hypothetical proteins suggests that AFP-Pred can be a useful approach to identify antifreeze proteins from sequence information, irrespective of their sequence similarity.     

灰翅麦茎蜂蛹的发育分级研究

室内饲养了灰翅麦茎蜂Cephus fumipennis Eyersmann,对该种蛹的发育进行了研究.结果表明,灰翅麦茎蜂蛹期(30±2.08)d,根据蛹体色的显著变化分为5级(Ⅰ,Ⅱ,Ⅲ,Ⅳ,Ⅴ),其历期依次为:(6.5±0.55)d、(3.9±0.48)d、(4.4±0.5)d、(4.6±0.49)d和(10.5±...     

Improvement on the yield of polyhydroxyalkanotes production from cheese whey by a recombinant Escherichia coli strain using the proton suicide methodology

In this work Escherichia coli strain CML3-1 was engineered through the insertion of Cupriavidus necator P(3HB)-synthesis genes, fused to a lactose-inducible promoter, into the chromosome, via transposition-mediated mechanism. It was shown that polyhydroxyalkanotes (PHAs) production by this strain, using cheese whey, was low due to a significant organic acids (OA) synthesis. The proton suicide method was used as a strategy to obtain an E. coli mutant strain with a reduced OA-producing capacity, aiming at driving bacterial metabolism toward PHAs synthesis.Thirteen E. coli mutant strains were obtained and tested in shake flask assays, using either rich or defined media supplemented with lactose. P8-X8 was selected as the best candidate strain for bioreactor fed-batch tests using cheese whey as the sole carbon source. Although cell growth was considerably slower for this mutant strain, a lower yield of OA on substrate (0.04 Cmol_OA/Cmol_lac) and a higher P(3HB) production (18.88 g_P(3HB)/L) were achieved, comparing to the original recombinant strain (0.11 Cmol_OA/Cmol_lac and 7.8 g_P(3HB)/L, respectively). This methodology showed to be effective on the reduction of OA yield by consequently improving the P(3HB) yield on lactose (0.28 Cmol_P(3HB)/Cmol_lac vs 0.10 Cmol_P(3HB)/Cmol_lac of the original strain).     

Exploring the multiscale signaling behavior of phototropin1 from Chlamydomonas reinhardtii using a full‐residue space kinetic Monte Carlo molecular dynamics technique

Devising analysis tools for elucidating the regulatory mechanism of complex enzymes has been a challenging task for many decades. It generally requires the determination of the structural‐dynamical information of protein solvent systems far from equilibrium over multiple length and time scales, which is still difficult both theoretically and experimentally. To cope with the problem, we introduce a full‐residue space multiscale simulation method based on a combination of the kinetic Monte Carlo and molecular dynamics techniques, in which the rates of the rate‐determining processes are evaluated from a biomolecular forcefield on the fly during the simulation run by taking into account the full space of residues. To demonstrate its reliability and efficiency, we explore the light‐induced functional behavior of the full‐length phototropin1 from Chlamydomonas reinhardtii (Cr‐phot1) and its various subdomains. Our results demonstrate that in the dark state the light oxygen voltage‐2‐Jα (LOV2‐Jα) photoswitch inhibits the enzymatic activity of the kinase, whereas the LOV1‐Jα photoswitch controls the dimerization with the LOV2 domain. This leads to the repulsion of the LOV1‐LOV2 linker out of the interface region between both LOV domains, which results in a positively charged surface suitable for cell–membrane interaction. By contrast, in the light state, we observe that the distance between both LOV domains is increased and the LOV1‐LOV2 linker forms a helix–turn–helix (HTH) motif, which enables gene control through nucleotide binding. Finally, we find that the kinase is activated through the disruption of the Jα‐helix from the LOV2 domain, which is followed by a stretching of the activation loop (A‐loop) and broadening of the catalytic cleft of the kinase. Proteins 2014; 82:2018–2040. © 2014 Wiley Periodicals, Inc.     

The impact of humic and fulvic acids on the dynamic properties of liposome membranes: the ESR method

This paper presents the results of research on the influence of two fractions of humic substances (HS): fulvic acids (FA) and humic acids (HA), as a function of concentration, on the liposome membranes formed from egg yolk lecithin (EYL). The concentration of HS in relation to EYL changed from 0% to 10% by weight. The influence of HS on various areas of membranes: interphase water-lipid, in the lipid layer just below the polar part of the membrane and in the middle of the lipid bilayer, was investigated by different spin labels (TEMPO, DOXYL 5, DOXYL 16). The study showed that HA slightly decreased the fluidity of the analyzed membranes on the surface layer, while FA significantly liquidated the center of the lipid bilayer. The strong effect of both fractions of HS on the concentration of free radicals as a function of time was also described.     

Chemical aspects of hydrogen sulfide measurements in physiological samples

Background

Owing to recent discoveries of many hydrogen sulfide-mediated physiological processes, sulfide biology is in the focus of scientific research. However, the promiscuous chemical properties of sulfide pose complications for biological studies, which led to accumulation of controversial observations in the literature.

Scope of review

We intend to provide an overview of fundamental thermodynamic and kinetic features of sulfide redox- and coordination-chemical reactions and protonation equilibria in relation to its biological functions. In light of these chemical properties we review the strengths and limitations of the most commonly used sulfide detection methods and recently developed fluorescent probes. We also give a personal perspective on blood and tissue sulfide measurements based on proposed biomolecule–sulfide interactions and point out important chemical aspects of handling sulfide reagent solutions.

Major conclusions

The diverse chemistries of sulfide detection methods resulted in orders of magnitude differences in measured physiological sulfide levels. Investigations that were aimed to dissect the underlying molecular reasons responsible for these controversies made the important recognition that there are large sulfide reserves in biological systems. These sulfide pools are tightly regulated in a dynamic manner and they are likely to play a major role in regulation of endogenous-sulfide-mediated biological functions and avoiding toxic side effects.

General significance

Working with sulfide is challenging, because it requires considerable amounts of chemical knowledge to adequately handle reagent sulfide solutions and interpret biological observations. Therefore, we propose that a rigorous chemical approach could aid the reconciliation of the increasing number of controversies in sulfide biology. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Laser-induced hyperthermia of nanoshell mediated vascularized tissue – A numerical study

Laser-induced hyperthermia treatment of tumor in a 2-D axisymmetric tissue embedded with moderate size (100–150 µm) blood vessels is studied. Laser absorption is enhanced by embedding gold–silica nanoshells in the tumor. Heat transfer in the tissue is modeled using Weinbaum–Jiji bioheat transfer equation. With laser irradiation, the volumetric radiation is accounted in the governing bioheat equation. Radiative information needed in the bioheat equation is calculated using the discrete ordinate method, and the coupled bioheat-radiation equation is solved using the finite volume method. Effects of power density, laser exposure time, beam radius, diameter of blood vessel and volume fractions of nanoshells on temperature spread in the tissue are analyzed.