Physiological parameters that affect the transfer of radiocaesium to ruminants

Recently there has been a renewed interest in biological scaling relationships between parameters, such as those between, for example, body mass, dry matter intake and biological half-times of radionuclides that are useful in predicting the transfer of radiocaesium to different animal species, particularly to wild animals. However, there is still a considerable unexplained variability in transfer coefficient estimates between individuals of the same species. This paper discusses the physiological parameters that affect the transfer of radiocaesium to ruminants, and it shows how a better understanding of these parameters may help to reduce the within-species variability in radiocaesium transfer coefficients. In light of the improved understanding during the past 10-15 years of the importance of source-dependent bioavailability on absorption of radiocaesium from the gastrointestinal tract, it is concluded that further studies are required on the effects of feed digestibility and physiological factors on absorption and endogenous faecal excretion of radiocaesium to better understand the variability in transfer coefficients.     

Optimization of some variables that affect the synthesis of laccase by Pleurotus ostreatus

The influence of initial pH, concentration of yeast extract, inducer, type of enzyme releaser and buffer system on the composition of a medium for laccase production by Pleurotus ostreatus DM-1513 was investigated. A 2⁵ full factorial experimental design was initially employed to evaluate the effects of these variables on the enzyme synthesis. Data analysis showed that low pH and high yeast extract concentration values, as well as the absence of both an inducer and a buffer system, had positive effects on the secreted enzyme levels, whereas the type of enzyme releaser did not have a significant effect. The highest levels of laccase activity (489–540?U/l) were obtained in optimization experiments using media with initial pH between 6.0 and 6.5 and yeast extract concentrations of 0–0.25%     

Preparation and characterization of magnetic gold nanoparticles to be used as doxorubicin nanocarriers

Magnetic targeted drug delivery (MTD), using magnetic gold nanoparticles (Fe₃O₄@Au NPs) conjugated with an anti-cancer drug is a promise modality for cancer treatment. In this study, Fe₃O₄@Au NPs were prepared and functionalized with thiol-terminated polyethylene glycol (PEG), then loaded with anti-cancer drug doxorubicin (DOX). The physical properties of the prepared NPs were characterized using different techniques. Transmission electron microscopy (TEM) revealed the mono dispersed nature of Fe₃O₄@Au NPs with an average size of 20 nm which was confirmed using Dynamic light scattering (DLS) measurements. Zeta potential measurements along with UV–VIS spectroscopy demonstrated surface DOX loading on Fe₃O₄@Au NPs. Energy Dispersive X-ray Spectroscopy (EDX) assured the existence of both iron and gold elements in the prepared NPs. The paramagnetic properties of the prepared NPs were assessed by vibrating sample magnetometer (VSM). The maximum DOX-loading capacity was 100 μg DOX/mg of Fe₃O₄@Au NPs. It was found that DOX released more readily at acidic pH. In vitro studies on MCF-7 cell line elucidated that DOX loaded Fe₃O₄@Au NPs (Fe₃O₄@Au-PEG-DOX) have more potent therapeutic effect than free DOX. Knowledge gained in this study may open the door to pursue Fe₃O₄@Au NPs as a viable nanocarriers for different molecules delivery in many diagnostic and therapeutic applications.     

Optimization of critical parameters for immobilization of yeast cells to alginate gel matrix

The optimum concentrations of sodium alginate (wt. %), calcium chloride (M) and yeast cells (wt. %), and curing time (h) for enhanced gel stability were obtained employing a full factorial search. The results indicate that the concentrations of sodium alginate and CaCl₂, and the curing time of the beads were found to have a pronounced effect on the stability of the beads. The cell concentration, on the other hand, has an adverse influence either individually or in combination with other variables. The path of steepest ascent method has been used to optimize the variables and the resultant gel beads were evaluated for fermentation ability.     

Extracellular matrix components affect the pattern of protein synthesis of endothelial cells responding to hyperthermia

Summary The biosynthetic profile of endothelial cells responding to hyperthermia is altered by extracellular matrix components. The extracellular matrix components influence the quantitative expression of members of the HSP70 family and HSP90. The expression of several HSP70 mRNA species, which are strictly stress inducible, are modulated by extracellular matrix components. Both laminin and collagen type IV decrease the amount of HSP70 protein and mRNA expressed by endothelial cells exposed to hyperthermia relative to control cultures attached to virgin plastic. In contrast, both laminin and collagen type IV increased the amount of HSP90 mRNA constitutively expressed by endothelial cells at 37° C. When endothelial cells were exposed to elevated temperatures, these two extracellular matrix proteins decrease the amount of HSP90 mRNA relative to control cultures attached to virgin plastic. Our observations are consistent with the proposal that the extracellular matrix components regulate gene expression and cell behavior in regard to thermotolerance.     

On the nature of ionic liquids and their effects on lipases that catalyze ester synthesis

Free and immobilized lipases from Candida antarctica (CALA and CALB), Thermomyces lanuginosus (TLL) and Rhizomucor miehei (RML) were used as catalysts in the synthesis of butyl propionate by transesterification in reaction media consisting in nine different ionic liquids. Enzyme activities were clearly dependent on the nature of the ions, the results being improving as the alkyl chain length of the imidazolium cation increased, and as a function of the type of anion ([PF₆], [BF₄] or [ethylsulphate]). The best synthetic activity (655.5 U/mg protein at 40 °C) was obtained when free CALB were assayed in the water-miscible IL cocosalkyl pentaethoxy methyl ammonium methosulfate ([CPMA][MS]), and was clearly related with the water content of the medium. The synthetic activity of free CALB in [CPMA][MS] was enhanced with the increase in temperature, while practically no effect was obtained for TLL. The ability of free CALB to synthesize aliphatic esters of different alkyl chain lengths, using different alkyl vinyl esters and 1-alkanols as substrates, was also studied in [CPMA][MS], the best results (4500 U/mg protein) being obtained for the synthesis of hexyl butyrate.     

Electrospun polylactic acid and polyvinyl alcohol fibers as efficient and stable nanomaterials for immobilization of lipases

Electrospinning was applied to create easy-to-handle and high-surface-area membranes from continuous nanofibers of polyvinyl alcohol (PVA) or polylactic acid (PLA). Lipase PS from Burkholderia cepacia and Lipase B from Candida antarctica (CaLB) could be immobilized effectively by adsorption onto the fibrous material as well as by entrapment within the electrospun nanofibers. The biocatalytic performance of the resulting membrane biocatalysts was evaluated in the kinetic resolution of racemic 1-phenylethanol (rac-1) and 1-phenylethyl acetate (rac-2). Fine dispersion of the enzymes in the polymer matrix and large surface area of the nanofibers resulted in an enormous increase in the activity of the membrane biocatalyst compared to the non-immobilized crude powder forms of the lipases. PLA as fiber-forming polymer for lipase immobilization performed better than PVA in all aspects. Recycling studies with the various forms of electrospun membrane biocatalysts in ten cycles of the acylation and hydrolysis reactions indicated excellent stability of this forms of immobilized lipases. PLA-entrapped lipases could preserve lipase activity and enantiomer selectivity much better than the PVA-entrapped forms. The electrospun membrane forms of CaLB showed high mechanical stability in the repeated acylations and hydrolyses than commercial forms of CaLB immobilized on polyacrylamide beads (Novozyme 435 and IMMCALB-T2-150).     

The assay design used for measurement of therapeutic antibody concentrations can affect pharmacokinetic parameters

To interpret pharmacokinetic (PK) data of biotherapeutics, it is critical to understand which drug species is being measured by the PK assay. For therapeutic antibodies, it is generally accepted that “free” circulating antibodies are the pharmacologically active form needed to determine the PK/ pharmacodynamic (PD) relationship, safety margin calculations, and dose projections from animals to humans and the eventual characterization of the exposure in the clinic. However, “total” drug may be important in evaluating the dynamic interaction between the drug and the target, as well as the total drug exposure. In the absence of or with low amounts of soluble ligand /shed receptor, total and free drug species are often equivalent and their detection is less sensitive to assay formats or reagent choices. In contrast, in the presence of a significant amount of ligand, assay design and characterization of assay reagents are critical to understanding the PK profiles. Here, we present case studies where different assay formats affected measured PK profiles and data interpretation. The results from reagent characterizations provide a potential explanation for the observed discrepancies and highlight the importance of reagent characterization in understanding which drug species are being measured to accurately interpret PK parameters.     

Feasibility of polyacrylamide as a matrix for immobilizing yeast cells (Saccharomyces cerevisiae) for inversion of sucrose syrups

Summary Polyacrylamide containing yeast cells is capable of hydrolysing concentrated sucrose syrups. The optimum conditions for sucrose inversion have been standardized. The matrix has good operational stability and capability to retain high cell loading (20%). However, because of operational problems, polyacrylamide may have limitations for the inversion of concentrated solution of sucrose.     

Characterization of a methanogenic sludge to be used as inoculum for a high-rate reactor

The sludge of an anaerobic lagoon treating the wastewater from a factory producing baker's yeast was evaluated as inoculum for anaerobic digestion. Specific methanogenic activity tests failed to give a good estimation of the trophic groups that were evidenced by enumerations involving Most Probable Number estimations. This failure was ascribed to the toxic effects of either the acetate concentrations used or to the ammonia content of the sludge.     

Investigation of parameters that affect the success rate of microarray-based allele-specific hybridization assays

Background

The development of microarray-based genetic tests for diseases that are caused by known mutations is becoming increasingly important. The key obstacle to developing functional genotyping assays is that such mutations need to be genotyped regardless of their location in genomic regions. These regions include large variations in G+C content, and structural features like hairpins.

Methods/Findings

We describe a rational, stable method for screening and combining assay conditions for the genetic analysis of 42 Phenylketonuria-associated mutations in the phenylalanine hydroxylase gene. The mutations are located in regions with large variations in G+C content (20–75%). Custom-made microarrays with different lengths of complementary probe sequences and spacers were hybridized with pooled PCR products of 12 exons from each of 38 individual patient DNA samples. The arrays were washed with eight buffers with different stringencies in a custom-made microfluidic system. The data were used to assess which parameters play significant roles in assay development.

Conclusions

Several assay development methods found suitable probes and assay conditions for a functional test for all investigated mutation sites. Probe length, probe spacer length, and assay stringency sufficed as variable parameters in the search for a functional multiplex assay. We discuss the optimal assay development methods for several different scenarios.     

Methods for the immobilization of lipases and their use for ester synthesis

The lipase from Pseudomonas fluorescens was immobilized onto five different carriers: celite, octyl-silica, aminopropyl-silica, gluterdialdehyde-activated silica and Eupergit C250L. Activities and operational stabilities of the prepared catalysts were compared using the enantioselective acylation of (R,S)-1-phenylethanol by vinyl acetate as acyl donor and t-butylmethyl ether with variable water content (0.038-0.97% v/v) as reaction medium. The above carriers provide catalysts with widely different specific activities ranging from excellent 25 mmol/h mg protein (celite) to 0.07 mmol/h mg protein (glutardialdehyde-activated silica) on the lower end. The lipase immobilized onto Eupergit C250L exhibited the best operational stability among the catalysts studied. It retained 30% of its initial activity after 11 cycles of application, each with a duration between 2 and 6 h.     

Optimization of treatment planning parameters used in tomotherapy for prostate cancer patients

Background and purposeTomotherapy treatment planning depends on parameters that are not used conventionally such as: field width (FW), pitch factor (PF) and modulation factor (MF). The aim of this study is to analyze the relationship between these parameters and their influence on the quality of treatment plans and beam-on time.Material and methodsTen prostate cancer patients were included in the study. For each patient, two cases of irradiation were considered depending on the target volume: PTV1 included the prostate gland, seminal vesicles, pelvic lymph nodes and a 1 cm margin, whereas PTV2 included only the prostate gland with a 1 cm margin. For each patient and each case of irradiation (PTV1 and PTV2) 8 treatment plans were created – all consisted of a different combination of planning parameters (FW = 1.05, 2.5, 5 cm; PF = 0.107, 0.215, 0.43; MF = 1.5, 2.5, 3.5). Default values used in this study were FW = 2.5 cm, PF = 0.215 and MF = 2.5. Hence, for plans with different FWs, parameters of PF and MF were 0.215 and 2.5, respectively; for different PFs, FW and MF were 2.5 and 2.5, respectively; finally for different MFs, FW and PF were 2.5 and 0.215, respectively. The reference plan was optimized for FW = 1.05 cm, PF = 0.107 and MF = 3.5, which was assumed to result in the best dose distribution and the longest treatment time. As a result, 160 plans were created. Each plan was analyzed for dose distribution and execution time.Results and conclusion: Treatment plans with FW of 5 cm resulted in the shortest execution time compromising the dose distribution. Moreover, the dose fall off in the longitudinal direction was not sharp. FW of 1.05 cm and PF of 0.107 were not recommended for routine prostate plans due to long execution time, which was 3 times longer than for plans with FW = 5 cm. There was no substantial decrease of irradiation time when PF was increased from 0.215 to 0.43 for both cases (PTV1 and PTV2); however, the dose distribution was slightly compromised. Finally, decreasing MF from 2.5 to 1.5 was useless because it did not change the beam-on time; however, it did remarkably decrease the dose distribution. Nevertheless, increasing MF up to 3.5 could be considered. The lowest EUD for the rectum and intestines, could be observed for PF = 0.107. For the other plans the differences were rather small (the EUD was almost the same). By reducing PF from 0.43 to 0.107 or FW from 5 to 1.05 the EUD for bladder (in PTV1 case) decreased by 3.13% and 2.60%. When PTV2 was a target volume, the EUD for bladder decreased by 4.54% and 3.43% when FW was changed from 5 to 1.05 and MF from 1.5 to 3.5, respectively. For optimal balance between beam-on time and dose distribution in OARs for routine patients, the authors would suggest to use: FW = 2.5, PF = 0.215 and MF = 2.5.     

Genomic DNA can be used with cationic methods for highly efficient transformation of maize protoplasts

Summary Efficient delivery of genomic DNA fragments to maize protoplasts was obtained by new methods using the polycation Polybrene or Lipofectin cationic liposomes. Stable kanamycin-resistant secondary transformants were recovered after transfection with genomic DNA from a maize cell line that had previously been tagged with the bacterial gene neomycin phosphotransferase (nptII) in a first-round transformation. The frequency of secondary transformants with nptII-homologous DNA sequences was 3% or 6% of all randomly picked microcalli after Polybrene or Lipofectin-mediated transfection, respectively. Transformation with genomic DNA by these methods may allow easy transfer of uncloned genes encoding desirable characteristics to crop species that can be regenerated from protoplasts.     

Does trifluoroethanol affect folding pathways and can it be used as a probe of structure in transition states?

Nonaqueous co-solvents, particularly 2,2,2-trifluoroethanol (TFE), have been used as tools to study protein folding. By analyzing FKBP12, an alpha/beta-protein that folds with two-state kinetics, we have been able to address three key questions concerning the use of TFE. First, does TFE perturb the folding pathway? Second, can the observed changes in the rate of folding and unfolding in TFE be attributed to a change in free energy of a single state? Finally, can TFE be used to infer information on secondary structure formation in the transition state? Protein engineering experiments on FKBP12, coupled with folding and unfolding experiments in 0% and 9.6% TFE, conclusively show that TFE does not perturb the folding pathway of this protein. Our results also suggest that the changes in folding and unfolding rates observed in 9.6% TFE are due to a global effect of TFE on the protein, rather than the stabilization of any elements of secondary structure in the transition state. Thus, studies with TFE and other co-solvents can be accurately interpreted only when combined with other techniques.     

A biofilm growth protocol and the design of a magnetic field exposure setup to be used in the study of magnetic fields as a means of controlling bacterial biofilms

The use of prosthetic implants is increasing both in the United States and around the world and there is a concomitant rise in cases of biofilm‐based, persistent infections that are quite serious and virtually impervious to antibiotic treatment. The development of alternate therapies that do not involve long term use of high levels of antibiotics or surgical intervention is needed. Based on the success of using electric or magnetic fields to alter certain physiological processes, it is hypothesized that relatively low level magnetic fields, in conjunction with the appropriate antibiotic, may be able to help control and eventually clear bacterial biofilms on a prosthetic. In order to test this hypothesis, it is necessary to first develop a means of growing laboratory grade biofilms on specific materials in a way that is repeatable between experiments and that can be reproduced by other laboratories. Secondly, a means of applying controlled magnetic fields to the surfaces supporting the biofilms at a defined temperature must be developed. This article addresses both of these points. Bioelectromagnetics 31:56–63, 2010. © 2009 Wiley‐Liss, Inc.     

The reductase that catalyzes mycolic motif synthesis is required for efficient attachment of mycolic acids to arabinogalactan

Mycolic acids are essential components of the cell walls of bacteria belonging to the suborder Corynebacterineae, including the important human pathogens Mycobacterium tuberculosis and Mycobacterium leprae. Mycolic acid biosynthesis is complex and the target of several frontline antimycobacterial drugs. The condensation of two fatty acids to form a 2-alkyl-3-keto mycolate precursor and the subsequent reduction of this precursor represent two key and highly conserved steps in this pathway. Although the enzyme catalyzing the condensation step has recently been identified, little is known about the putative reductase. Using an extensive bioinformatic comparison of the genomes of M. tuberculosis and Corynebacterium glutamicum, we identified NCgl2385, the orthologue of Rv2509 in M. tuberculosis, as a potential reductase candidate. Deletion of the gene in C. glutamicum resulted in a slow growing strain that was deficient in arabinogalactan-linked mycolates and synthesized abnormal forms of the mycolate-containing glycolipids trehalose dicorynomycolate and trehalose monocorynomycolate. Analysis of the native and acetylated trehalose glycolipids by MALDI-TOF mass spectrometry indicated that these novel glycolipids contained an unreduced beta-keto ester. This was confirmed by analysis of sodium borodeuteride-reduced mycolic acids by gas chromatography mass spectrometry. Reintroduction of the NCgl2385 gene into the mutant restored the transfer of mature mycolic acids to both the trehalose glycolipids and cell wall arabinogalactan. These data indicate that NCgl2385, which we have designated CmrA, is essential for the production of mature trehalose mycolates and subsequent covalent attachment of mycolic acids onto the cell wall, thus representing a focus for future structural and pathogenicity studies.     

Selection criteria for lactic acid bacteria to be used as starter cultures for various food commodities

Abstract: Lactic acid bacteria (LAB) are the most important bacteria used in food fermentations. Apart from general demands for starter cultures from the view of safety, technological effectiveness and economics, numerous specific aspects have to be considered when selecting strains for the different food fermentations. Therefore selection criteria for LAB depend on the type and the desired characteristics of the final product, the desired metabolic activities, the characteristics of the raw materials and the applied technology. Special selection criteria for LAB for use in the fermentation of sausages and vegetables as well as for the malolactic fermentation in wine are discussed.