Ecology of spontaneous fermentation in one winery during 5 consecutive years

A concentration protocol based on trichloroacetic acid precipitation was evaluated and compared with the reference method using dialysis concentration. Different quantities of purified staphylococcal enterotoxins were added to pasteurized Camembert-type cheeses. Detection of enterotoxins in these cheeses was performed using an automated detection system. Raw goat milk Camembert-type cheeses involved in a staphylococcal food poisoning were also tested. Both enterotoxin extraction methods allowed detection of the lowest enterotoxin concentration level used in this study (0.5 ng g-1). Compared with the dialysis concentration method, TCA precipitation of staphylococcal enterotoxins was 'user-friendly' and less time-consuming. These results suggest that TCA precipitation is a rapid (1 h), simple and reliable method of extracting enterotoxin from food which gives excellent recovery from dairy products.     

Fluorescent labeling of cell-free synthesized proteins with fluorophore-conjugated methionylated tRNA derived from in vitro transcribed tRNA

A simple and practical method for preparing fluorophore-conjugated methionylated tRNA necessary for tRNA-mediated fluorescent labeling of cell-free synthesized proteins was developed. Without complicated chromatographic purification and subsequent concentration, fluorophore-conjugated methionylated tRNA with higher purity and fluorescence concentration could be synthesized from in vitro transcribed tRNA instead of from a total tRNA mixture, which has been routinely used as a tRNA source. Although fluorophore-conjugated methionylated tRNA derived from in vitro transcribed tRNA was purified by simple phenol extraction following alcohol precipitation, it worked well in tRNA-mediated fluorescent labeling, yielding an improved signal-to-noise ratio and higher fluorescence intensity compared to the conventional total tRNA-based method. Based on its simplicity in the preparation of labeling agent with higher purity and fluorescence concentration, the developed method will accelerate the prevalence of fluorescence-based detection of cell-free synthesized proteins.     

Improvements of the Anson assay for measuring proteolytic activities in acidic pH range.

This paper proposes a modification of the classic Anson assay [Anson, M. L. (1939) J. Gen. Physiol.22, 79–89] for proteolytic enzymes which are active in the acidic pH range; it abolishes many of the constraints common to this widely used method. The present procedure allows for: (i) the control of zymogen activation; (ii) the measurement of the hydrolyzed products on a very wide concentration range, due to their extended direct proportionality to both the incubation time and the enzyme concentration; (iii) the possibility to perform the assay at any given pH between 1.3 and 5.0 with minimal interference from ionic strength; (iv) a very simple conversion of the absorbance into proteolytic units, allowed by a better choice of TCA concentration. The method offers good sensitivity and is compared in detail to the original method and its various modifications.     

Virioplankton ‘pegylation’: Use of PEG (polyethylene glycol) to concentrate and purify viruses in pelagic ecosystems

We have described the use of Polyethylene glycol (PEG) for the precipitation of natural communities of aquatic viruses, and its comparison with the usual concentration method based on ultracentrifugation. Experimental samples were obtained from different freshwater ecosystems whose trophic status varied. Based on transmission electron microscope observations and counting of phage-shaped particles, our results showed that the greatest recovery efficiency for all ecosystems was obtained when we used the PEG protocol. On average, this protocol allowed the recovery of > 2-fold more viruses, compared to ultracentrifugation. In addition, the diversity of virioplankton, based on genomic size profiling using pulsed field gel electrophoresis, was higher and better discriminated when we used the PEG method. We conclude that pegylation offers a valid, simple and cheaper alternative method to ultracentrifugation, for the concentration and the purification of pelagic viruses.     

A simple method to mass produce monospores in the thallus of Porphyra yezoensis Ueda

A simple method is presented for obtaining a large number of monospores in the thallus of Porphyra yezoensis. The method implies two principles: induction of monosporangium formation by allantoin, and liberation of monospores from the cell wall by mild homogenization. The induction of monosporangium formation was accomplished by culturing wild thalli in nutrient-enriched seawater with 10 mM of allantoin for approximately 3 weeks. This high concentration (10 mM) of allantoin suppressed the growth of the thalli compared with lower concentrations (0–1 mM). Thalli cultured for 3 weeks were mildly homogenized with a glass homogenizer and the monospore solution was obtained by filtering with a nylon mesh. The monospores grew normally to thalli. This technique of monospore acquisition is a simple and useful method for the propagation and breeding of P. yezoensis thalli.     

Evaluation of inflammatory and angiogenic factors in patients with non-alcoholic fatty liver disease

The liver is a major target of injury in obese patients. Non-alcoholic fatty liver disease (NAFLD) is present in 60-90% of obese Americans and can range from simple steatosis to the more severe non-alcoholic steatohepatitis (NASH). The onset of a chronic inflammatory reaction marks the progression from simple steatosis to NASH and the expansion of adipose tissue is strongly associated with angiogenesis. Therefore, we determined the serum concentration of inflammatory [tumor necrosis factor alpha (TNFα) and interleukin 6 (IL6)] and angiogenic [vascular endothelial growth factor A (VEGF)] cytokines and soluble VEGF receptors 1 and 2 (sVEGFR1, sVEGFR2) in the serum of an obese population with simple steatosis and NASH compared to healthy controls. Moreover, we determined the TNFα, IL6, VEGF, VEGFR1 and VEGFR2 gene expression in the liver of these simple steatosis and NASH patients. The population consisted of 30 obese patients, which were diagnosed with simple steatosis and 32 patients with NASH and compared to 30 age-and-sex matched healthy controls. Mean serum TNFα levels were elevated in the serum of simple steatosis and NASH patients compared to healthy controls, reaching significance in NASH patients. IL6 was significantly increased in simple steatosis and NASH patients compared to the healthy controls. VEGF levels were significantly elevated in patients with simple steatosis and borderline significantly elevated in NASH patients compared to the serum levels of healthy control subjects. The concentration of sVEGFR1 was significantly increased in serum of simple steatosis and NASH patients compared to controls. sVEGFR2 concentration was not significantly different in the three groups. TNFα mRNA expression was higher in NASH patients compared to simple steatosis patients. Hepatic gene expression of VEGF, VEGFR1 and VEGFR2 were slightly decreased in NASH patients compared to simple steatosis patients. These data indicate the involvement of inflammatory (TNFα and IL6), angiogenic (VEGF) cytokines and sVEGFR1 in the pathophysiology of NAFLD.     

Fast determination of ochratoxin A in serum by liquid chromatography: comparison with enzymic spectrofluorimetric method

A rapid high-performance liquid chromatographic method for the determination of low concentrations of ochratoxin A in serum is described. The extraction procedure was simple and short, and liquid chromatographic analysis was carried out isocratically on a reversed-phase C₁₈ column, with methanol—water—acetic acid (30:70:1) as mobile phase and fluorescence detection (excitation at 336 nm, emission at 465 nm). The examined concentration range, 5–50 ng/ml ochratoxin, the recovery method was 87–94%, compared with 62–67% for the enzymic spectrofluorimetric method. The high-performance liquid chromatographic method was faster because the extraction procedure was shorter, and more sensitive so that small sample volumes could be used.     

Virioplankton 'pegylation': use of PEG (polyethylene glycol) to concentrate and purify viruses in pelagic ecosystems

We have described the use of Polyethylene glycol (PEG) for the precipitation of natural communities of aquatic viruses, and its comparison with the usual concentration method based on ultracentrifugation. Experimental samples were obtained from different freshwater ecosystems whose trophic status varied. Based on transmission electron microscope observations and counting of phage-shaped particles, our results showed that the greatest recovery efficiency for all ecosystems was obtained when we used the PEG protocol. On average, this protocol allowed the recovery of >2-fold more viruses, compared to ultracentrifugation. In addition, the diversity of virioplankton, based on genomic size profiling using pulsed field gel electrophoresis, was higher and better discriminated when we used the PEG method. We conclude that pegylation offers a valid, simple and cheaper alternative method to ultracentrifugation, for the concentration and the purification of pelagic viruses.     

A simple colorimetric method for determination of hydrogen peroxide in plant tissues

A simple colorimetric method for determination of hydrogen peroxide in plant materials is described. The method is based on hydrogen peroxide producing a stable red product in reaction with 4-aminoantipyrine and phenol in the presence of peroxidase. Plant tissues was ground with trichloroacetic acid (5% w/v) and extracts were adjusted to pH 8.4 with ammonia solution. Activated charcoal was added to the homogenate to remove pigments, antioxidants and other interfering substances. The colorimetric reagent (pH 5.6) consisted of 4-aminoantipyrine, phenol, and peroxidase. With this method, we have determined the hydrogen peroxide concentration in leaves of eight species which ranged from 0.2 to 0.8 μmol g⁻¹ FW. Changes in hydrogen peroxide concentration of Stylosanthes guianensis in response to heat stress are also analyzed using this method.     

A fast and simple turbidimetric method for the determination of sulfate in sulfate-reducing bacterial cultures

A standard turbidimetric assay for the determination of sulfate in water was modified with the objective of achieving a quick and simple method for monitoring the decrease of sulfate in cultures of sulfate-reducing bacteria. The effects of sulfate concentration, mixing time and the ratio of sample to conditioning reagent were optimized using a central composite face-centered response surface model design. The results suggested that a mixing time of 30 s resulted in smaller absorbance variance, the variance in absorbance measurements tended to increase with concentration of sulfate and that the ratio between the amount of conditioning reagent and sample had no significant influence on the absorbance variance. The modified assay thus developed is simple and quick, and covers a comparatively large sulfate concentration range (0-5 mM) compared to the standard turbidimetric assay.     

A simple innovative spectrofluorometric method for the determination of alendronate in bulk and in pharmaceutical tablets

Sodium alendronate is the first in a pharmacological class known as bisphosphonates, used for treatment of various bone diseases. Assay of bisphosphonates by a spectroscopic technique is very challenging due to the fact that they lack chromophores and none of them are fluorescent. In this work, a simple method is presented for determination of alendronate in bulk and in pharmaceutical tablets using spectrofluorometry by exploiting the ability of alendronate to displace salicylate from the iron(III)–salicylate chelate, forming a non‐fluorescent colorless iron(III)–alendronate complex. The liberated salicylate is fluorescent and is equivalent to the mount of alendronate added. The response was linear over the concentration range 20–90 μM and the proposed method was validated according to the guidelines of the International Conference on Harmonization. The correlation coefficient was found to be 0.995 and the limit of detection was 7.5 μM. The method was successfully applied for determination of alendronate in the commercially available Osteonate® tablets. The average percent recovery ± percent relative standard deviation was found to be 102.118 ± 2.033 which is congruent with the label claim of the dosage form. The results were also compared to a reported method using t‐test and F‐test at 95% confidence level; no significant differences were observed. The presented method is simple, fast, easy, cost‐effective and suitable for routine pharmaceutical analysis.     

表面张力曲线法测定生物表面活性剂的浓度

介绍一种利用表面张力曲线测定生物表面活性剂发酵液浓度的方法,通过测定不同浓度鼠李糖脂标准溶液的表面张力,制作浓度和表面张力的x-y散点图,再根据希斯科夫斯基经验公式利用Origin软件做x-y散点图的拟合曲线,得到该公式的相关参数,然后利用该经验公式,通过测定稀释后待测发酵液的表面张力,可求得原发酵液的鼠李糖脂浓度。和常用的方法相比,该方法具有快速、简单、准确和测定成本低的优点。     

On numerical integration of the Hodgkin and Huxley equations for a membrane action potential

Several methods of numerical integration of the Hodgkin and Huxley equations (6·3°C) for a membrane action potential have been compared for speed and accuracy of computations. Conversational languages make it possible to write compact programs for these equations which will run on minicomputers with only 4000 words of core memory.The shape of the computed action potential was found to be rather method-invariant but the latency (for a given stimulus) was found to depend on the method of integration and the size of the time increment. For stimuli near threshold, the computed response was found to be especially sensitive to these two variables. A new threshold value independent of integration method, has been determined to be ? 6·50790 mV by linear extrapolations of threshold-time step relations to a zero step size.The simple Euler method was found to be fast but gave large latency errors compared to the true solution. The Runge-Kutta (RK) method (in the sequential mode, defined in the section on numerical integration methods) was slower but gave smaller latency errors. The Adams corrector-predictor method was still slower, gave less stable solutions, and had large latency errors. A hybrid program which combined a RK numerical integration for the membrane potential with analytic expressions for the variables m, n, and h gave errors indistinguishable from the RK method, but ran much faster. Modified Euler methods run in the parallel mode gave very accurate solutions with moderate computation times. Solutions run with the RK method in the parallel mode took about twice as long but gave no appreciable improvement in accuracy.We were able to develop a simple method for correcting the Euler integration, resulting in quite accurate solutions which were essentially independent of step size and stimuli. Because this method is not only accurate but also runs about as fast as the simple Euler, it appears to be the method of choice for most purposes.     

An automated continuous assay of membrane-bound and solube ATPases and related enzymes.

A simple, rapid, and precise method is described for the continuous automated determination of the activity of membrane-bound enzymes which deliberate inorganic phosphate, e.g., ATPases and phosphatases. The characteristics of this method, which is based on the determination of liberated phosphate in the presence of nucleotides, are: (A) the enzyme reaction can be followed continuously during a certain period, thus providing a higher precision, as compared to other methods in which the enzyme reaction is measured by few distinct determinations; (B) the enzyme protein and other (membrane) proteins of the enzyme preparation have not to be removed during the continuous determination of enzyme activity because they remain solubilized after denaturation; and (C) low or moderate concentration of nonionic detergents do not disturb the reading of the absorbancy.     

A simple gravimetric technique for estimating honeydew or nectar production

We describe a simple gravimetric technique for measuring the standing crop or production of carbohydrate-rich solutions such as honeydew or nectar. Simulated honeydew was sampled by absorbing droplets of solutions of known concentration and volume with dried and weighed pieces of filter paper. The change in mass of the paper after redrying provides an estimate of the total solution carbohydrates. This method was compared with a widely-used technique, whereby the volume and concentration of droplets is measured with microcapillary tubes and a sugar refractometer. A factor was derived to convert gravimetric refractometer readings (g sucrose 100 g^-1 solution) to volumetric carbohydrate concentration (g carbohydrate 100 ml^-1 solution) for the simulated honeydew solutions. There was no difference in the ratio of measured-to-expected carbohydrate mass between the two techniques, showing that the quick, easy, and accurate filter-paper method is appropriate for measuring carbohydrate-rich solutions.     

Spectrofluorimetric determination of dopamine receptor antagonist LE 300 in mice plasma

A simple, rapid and highly sensitive spectrofluorimetric method was developed for determination of a novel type of dopamine receptor antagonist LE300 in mouse plasma. The method is based on measuring the native fluorescence of LE‐300 in methanol at 343 nm after excitation at 280 nm. The fluorescence concentration plot was rectilinear over the range of 3.5–100 ng/mL with a lower detection limit of 1.0 ng/mL and quantification limit of 3.5 ng/mL. The method was statistically validated for linearity, accuracy, precision and selectivity according to the International Conference on Harmonization guidelines. The accuracy and precision results was expressed as % recovery and relative standard deviation (RSD). The accuracy for LE‐300 was in the range 95.5–103.6% and RSD values were in the range of 0.21–1.55% of the theoretical value. The method was successfully applied to the analysis of LE‐300 in mice plasma. The results were compared statistically with those obtained by the reported method and were found to be in good agreement, which could be applied in a pharmacokinetic study. Copyright © 2015 John Wiley & Sons, Ltd.     

Rapid high-performance liquid chromatographic determination of vancomycin in human plasma

A rapid simple and robust reversed-phase HPLC method was developed for rapid screening in bioavailability studies or comparative bioequivalence studies. The method is specific for vancomycin as no interference from acetylsalicylic acid, paracetamol and caffeine was observed. The mean intra-day precision was from 11.7% (low concentration) to 0.3% (high concentration) and the within-day precision from 15.0 to 0.3%, determined on spiked samples. The accuracy of the method was 106.4–99.8% (intra-day) and 103.5–100.2% (inter-day).     

Experimental analysis of gene assembly with TopDown one-step real-time gene synthesis

Herein we present a simple, cost-effective TopDown (TD) gene synthesis method that eliminates the interference between the polymerase chain reactions (PCR) assembly and amplification in one-step gene synthesis. The method involves two key steps: (i) design of outer primers and assembly oligonucleotide set with a melting temperature difference of >10°C and (ii) utilization of annealing temperatures to selectively control the efficiencies of oligonucleotide assembly and full-length template amplification. In addition, we have combined the proposed method with real-time PCR to analyze the step-wise efficiency and the kinetics of the gene synthesis process. Gel electrophoresis results are compared with real-time fluorescence signals to investigate the effects of oligonucleotide concentration, outer primer concentration, stringency of annealing temperature, and number of PCR cycles. Analysis of the experimental results has led to insights into the gene synthesis process. We further discuss the conditions for preventing the formation of spurious DNA products. The TD real-time gene synthesis method provides a simple and efficient method for assembling fairly long DNA sequence, and aids in optimizing gene synthesis conditions. To our knowledge, this is the first report that utilizes real-time PCR for gene synthesis.     

Artificial chaperone-assisted refolding in a microchannel

Protein refolding using a simple dilution method in a microchannel often led to the formation of protein aggregates, which bound to the microchannel wall, resulting in low refolding yields. To inhibit aggregation and improve refolding yields, an artificial chaperone-assisted (ACA) refolding, which employed detergents and β-cyclodextrin was used. Model proteins, hen egg white lysozyme and yeast α-glucosidase, were successfully refolded in a microchannel. The microscopic observation showed that the ACA method suppressed protein aggregation and facilitated the refolding of lysozyme, whereas significant aggregation was observed when a simple dilution method was employed. The ACA method increased the lysozyme refolding yield by 40% over the simple dilution approach. Similarly, for α-glucosidase, the refolding yield using the ACA method (ca. 50%) was approximately three times compared with the simple dilution method. The ACA refolding method is a suitable approach to use in the refolding of proteins using a microfluidic system.     

A Response Surface Analysis of the Effects of Cyclohexanecarboxylic Acid and 2,4-Dichlorophenoxy Acetic Acid on Nitrogen Metabolism in Phaseolus vulgaris L.

Primary leaves of 2-week-old bush bean plants (Phaseolus vulgariscv ‘Top Crop’) were treated with one of 36 combinationsof cyclohexanecarboxylic acid (CHCA) and 2,4-dichlorophenoxyacetic acid (2,4-D), each at six equally spaced concentrationsFive, 7, 9 and 11 days after treatment, one plant from eachcombination was harvested, and primary leaves were monitoredfor nitrate reductase activity (NRA), nitrate nitrogen (N) concentration,organic N concentration, and d wt NRA was assayed by an in vivoprocedure Data from the resulting 6 x 6 x 4 factorial configuration foreach of the four responses were analysed using the method oforthogonal polynomials, which is described in detail This method,based on equally spaced levels, produced independent polynomialexpressions in CHCA concentration, 2,4-D concentration, and/ortime-to-harvest after treatment, which were then fitted to thedata on each response by simple linear regression analyses.A graphical procedure was used to compare predicted responsesfrom the best fit equations with the actual data. Examination of computer-generated plots of the three-dimensionalresponse surfaces revealed that NRA, organic N concentration,and d wt decreased with increasing 2,4-D concentration, whereasnitrate N content increased Increasing CHCA concentration generallyhad the opposite effect on NRA and nitrate N content, althoughinteraction between the two chemicals was evident in the latterresponse Organic N content remained unchanged as CHCA concentrationincreased, while d wt first increased then decreased Possibleexplanations for these results are discussed, as are the advantagesof using factorial designs with equally-spaced levels in plantgrowth studies. Phaseolus vulgaris L, bush bean, nitrogen metabolism, plant growth regulators, cyclohexanecarboxylic acid, 2,4-dichlorophenoxy acetic acid, regression analysis, orthogonal polynomials, response surfaces analysis