The fluorescent protein iLOV as a reporter for screening of high-yield production of antimicrobial peptides in Pichia pastoris.

The methylotrophic yeast Pichia pastoris is commonly used for the production of recombinant proteins at scale. The identification of an optimally overexpressing strain following transformation can be time and reagent consuming. Fluorescent reporters like GFP have been used to assist identification of superior producers, but their relatively big size, maturation requirements and narrow temperature range restrict their applications. Here, we introduce the use of iLOV, a flavin-based fluorescent protein, as a fluorescent marker to identify P. pastoris high-yielding strains easily and rapidly. The use of this fluorescent protein as a fusion partner is exemplified by the production of the antimicrobial peptide NI01, a difficult target to overexpress in its native form. iLOV fluorescence correlated well with protein expression level and copy number of the chromosomally integrated gene. An easy and simple medium-throughput plate-based screen directly following transformation is demonstrated for low complexity screening, while a high-throughput method using fluorescence-activated cell sorting (FACS) allowed for comprehensive library screening. Both codon optimization of the iLOV_NI01 fusion cassettes and different integration strategies into the P. pastoris genome were tested to produce and isolate a high-yielding strain. Checking the genetic stability, process reproducibility and following the purification of the active native peptide are eased by visualization of and efficient cleavage from the iLOV reporter. We show that this system can be used for expression and screening of several different antimicrobial peptides recombinantly produced in P. pastoris.

Perception of Breakfast Ingestion Enhances High-Intensity Cycling Performance.

PURPOSE: To examine the effect on short-duration, high-intensity cycling time-trial (TT) performance when a semisolid breakfast containing carbohydrate (CHO) or a taste- and texture-matched placebo is ingested 90 min preexercise compared with a water (WAT) control. METHODS: A total of 13 well-trained cyclists (mean [SD]: age = 25 [8] y, body mass = 71.1 [5.9] kg, height = 1.76 [0.04] m, maximum power output = 383 [46] W, and peak oxygen uptake = 4.42 [0.53] L·min-1) performed 3 experimental trials examining breakfast ingestion 90 min before a 10-min steady-state cycle (60% maximum power output) and an ∼20-min TT (to complete a workload target of 376 [36] kJ). Subjects consumed either WAT, a semisolid CHO breakfast (2 g carbohydrate CHO·kg-1 body mass), or a taste- and texture-matched placebo (PLA). Blood lactate and glucose concentrations were measured periodically throughout the rest and exercise periods. RESULTS: The TT was completed more quickly in CHO (1120 [69] s; P = .006) and PLA (1112 [50] s; P = .030) compared with WAT (1146 [74] s). Ingestion of CHO caused an increase in blood glucose concentration throughout the rest period in CHO (peak at 30-min rest = 7.37 [1.10] mmol·L-1; P < .0001) before dropping below baseline levels after the steady-state cycling. CONCLUSION: A short-duration cycling TT was completed more quickly when subjects perceived that they had consumed breakfast (PLA or CHO) 90 min prior to the start of the exercise. The improvement in performance is likely attributable to a psychological rather than physiological effect.

Effective management tools for participants at Codex Committee on Residues of Veterinary Drugs meetings.

The Codex Committee on Residues of Veterinary Drugs in Food (CCRVDF) fulfils a number of functions revolving around standard setting. The core activities of the CCRVDF include agreeing priorities for assessing veterinary drug residues, recommending maximum residue limits for veterinary drugs in foods of animal origin, considering methods of sampling and analyses, and developing codes of practice. Draft standards are developed and progress through an agreed series of steps common to all Codex Alimentarius Commission Committees. Meetings of the CCRVDF are held at approximately 18-month intervals. To ensure effective progress is made with meetings at this frequency, the CCRVDF makes use of a number of management tools. These include circular letters to interested parties, physical and electronic drafting groups between plenary sessions, meetings of interested parties immediately prior to sessions, as well as break out groups within sessions and detailed discussions within the CCRVDF plenary sessions. A range of these approaches is required to assist advances within the standards setting process and can be applied to other Codex areas and international standard setting more generally. Copyright © 2016 John Wiley & Sons, Ltd.

Can the unauthorised use of ceftiofur be detected in poultry?

Ceftiofur is a third-generation cephalosporin antibiotic used to treat cattle and swine for bacterial infection of the respiratory tract. It is not authorised for use in poultry within the European Union. Due to the complexity of the chemistry and metabolism of ceftiofur, maximum residue limits (MRLs) are based on desfuroylceftiofur (DFC) equivalents after chemical conversion of all compounds that have an intact ß-lactam ring. In practice the DFC is usually stabilised as the acetamide (desfuroylceftiofur acetamide - DFCA) for analysis. Because of recent evidence of off-label use in the European Union, a policy need emerged to develop a cost-effective method for the detection of ceftiofur residues in poultry tissues. One-day-old chicks were each dosed subcutaneously with ceftiofur and samples taken from day 1 to day 44 post-dosing. Residues of ceftiofur parent compound were detected in whole chicks, wing feathers and faeces. On the basis of this finding it was decided to evaluate ceftiofur parent, as the marker, instead of proceeding with the time-consuming conversion to DFCA. Expected metabolites, DFC and desfuroylceftiofur cysteine disulfide (DCCD), were not detected in whole chicks, muscle or liver, but DFC was found in wing feathers. These results indicate that determination of ceftiofur parent compound in either whole chicks or possibly wing feathers and faeces may allow the detection of the misuse of ceftiofur.

Investigation of the fate of trifluralin in shrimp.

Juvenile Pacific white shrimp (Litopenaeus vannamei) were exposed to trifluralin at 0.1 and 0.01 mg L(-1) for 72 h under controlled conditions. Samples of shrimp and tank water were collected at intervals up to 48 days after exposure. Analysis of the shrimp tissues by gas chromatography-mass spectrometry (GC-MS) and ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-qToF-MS) in combination with profiling and metabolite identification software (Agilent MET-ID and Mass Profiler Professional) detected the presence of parent trifluralin together with two main transformation products (TPs), 2-ethyl-7-nitro-5-(trifluoromethyl)benzimidazole (TP1) and 2-amino-6-nitro-4-(trifluoromethyl)phenyl)propylamine (TP2). The highest concentration of trifluralin, determined by GC-MS, was 120 µg kg(-1) at 0 day withdrawal. Residues of trifluralin (CCα = 0.25 µg kg(-1), CCß = 0.42 µg kg(-1)) were detectable for up to 7 days after exposure. Similarly, the highest concentrations of TP1 and TP 2, determined by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), were 14 and 18 µg kg(-1), respectively. Residues of TP1 (CCα = 0.05 µg kg(-1), CCß = 0.09 µg kg(-1)) and TP2 (CCα = 0.1 µg kg(-1), CCß = 0.17 µg kg(-1)) were detectable for up to 4 and 24 withdrawal days, respectively.

Combining ISO/IEC 17025:2005 and European Commission Decision 2002/657 audit requirements: a practical way forward.

Laboratories involved in the analyses of veterinary drug residues are under increasing pressure to demonstrate that they produce meaningful and reliable data. Quality assurance and quality control systems are implemented in laboratories to provide evidence of this and these are subject to external assessment to ensure that they are effective. Audits to ISO/IEC 17025:2005, an internationally accepted standard, and subsequent accreditation provide laboratories and their customers with a degree of assurance that the laboratories are operating in control and the data they report can be relied on. However, national or regional authorities may place additional requirements on laboratories to ensure quality data are reported. For example, in the European Union, all official control laboratories involved in veterinary drug residue analyses must also meet the requirements of European Commission Decision 2002/657/EC which sets performance criteria for analytical methods used in this area and these are subject to additional audits by national or regional authorities. All audits place considerable time and resource demands on laboratories and this paper discusses the burden audits place on laboratories and describes a UK initiative to combine these audits to the benefit of both the regulatory authority and the laboratory.

Investigation into the experimental protocols required to determine maximum residue limits (MRLs) in honey.

There is current debate within the EU, and internationally, on how withdrawal periods and maximum residue limits (MRLs) may be set for honey production. Whilst comprehensive EU guidelines exist for calculating the withdrawal times of veterinary medicines in most food-producing species, the analytical variables to be studied for bees/honey are not well defined. The objective of this study was therefore to investigate and understand the factors, for example sampling variability, that is important in the development of a harmonized protocol that can be used to generate the robust scientific data necessary to assist risk assessors in proposing MRLs for honey. Ten bee colonies were treated in the spring with a model compound (ciprofloxacin). One hive was used to study intra-hive variation in residue concentrations and the other nine were used in an inter-hive study over a 41-week sampling period. All samples were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The highest mean concentration from nine hives used in the inter-hive study was 4627 µg/kg eight days (D8) after treatment. The concentration of ciprofloxacin declined to an average concentration of 1756 µg/kg at D30 and 733 µg/kg at D283 (over-winter sample). A generalized additive model was used to fit a smooth curve for trend estimation. For some individual hives the concentration of ciprofloxacin increased slightly at the later sampling time-points. Consequently it was not possible to interpolate, with confidence, a finite withdrawal period for ciprofloxacin at theoretical MRLs between 25 and 500 µg/kg. The observed variation in concentration of ciprofloxacin between hives indicates that the validity of the EU guideline for bees/honey, which requires five samples from five hives to calculate a withdrawal period, may require revision.

An RNA-aptamer-based assay for the detection and analysis of malachite green and leucomalachite green residues in fish tissue.

A robust screening assay employing solid phase extraction (SPE) followed by a novel aptamer-based procedure is presented for the rapid detection and semiquantitation of the triphenylmethane dye, Malachite Green (MG) and its primary metabolite Leucomalachite Green (LMG) in fish tissue. To the authors' knowledge, this is the first reported use of an RNA aptamer for the development of a diagnostic assay for the detection of chemical residues in food. The aptamer based screening assay is found to be highly specific for MG; but has negligible affinity for the LMG metabolite. However, because the LMG metabolite is lipophilic and known to be highly persistent in tissues, an oxidation step has been incorporated within the sample cleanup procedure to ensure that all LMG residues are converted to MG prior to measurement. This article provides evidence that an oligonucleotide aptamer can be used as an alternative recognition element to conventional antibodies with application to the detection of residues in food. Furthermore, this finding has the future potential to reduce the number of animals currently being used in the production of antibodies for immunodiagnostic kits.

Vibrational frequencies and structural determinations of divinyl sulfoxide.

We present a detailed analysis of the structure and infrared spectra of divinyl sulfoxide. The vibrational frequencies of the divinyl sulfoxide molecule were analyzed using standard quantum chemical techniques. Frequencies were calculated at the MP2 and DFT levels of theory using the standard 6-311G* basis set. The molecule exists normally in a C(s) configuration. High-energy forms of divinyl sulfoxide with C(S) and C(1) symmetries also exist.

Correlation of structure and vibrational spectra of the zwitterion L-alanine in the presence of water: an experimental and density functional analysis.

Infrared vibrational spectra were collected along with the vibrational circular dichroism (VCD) spectra for the zwitterions alpha-D-alanine, alpha-L-alanine, alpha-D-mannose and alpha-L-mannose as potassium bromide (KBr) pressed samples. VCD for D- and L-alanine dissolved in water was also measured and compared against the spectra resulting from KBr pressed samples. The experimental data were compared against the ab initio B3LYP/6-31G* optimized geometry. The zwitterion structure of alpha-L-alanine was stabilized by the addition of water molecules. Computationally, beta-L-mannose was studied and resulting expected VCD bands assigned. We present the molecular structures resulting VCD spectra and infrared vibrational spectra from experimentation as compared with the computed results.

Vibrational frequencies and structural determinations of di-vinyl sulfone.

We present a detailed analysis of the structure and infrared spectra of di-vinyl sulfone. The vibrational frequencies of the di-vinyl sulfone molecule were analyzed using standard quantum chemical techniques. Frequencies were calculated at the MP2 and DFT levels of theory using the standard 6-311G* basis set. The structural transformation of the chemical agent bis(2-chloroehtyl) sulfide (HD, mustard gas) and the related symmetry to a previously study compounds [Spectrochim. Acta Part A 55 (1999) 121; Spectrochim. Acta Part A 57 (2001) 2417] makes the symmetry of the di-vinyl sulfone molecule an interesting candidate for study. The molecule exists normally in a C(2) configuration. High-energy forms of di-vinyl sulfone with C(S) and C(1) symmetries also exist.

An International Marine-Atmospheric 222Rn Measurement Intercomparison in Bermuda Part II: Results for the Participating Laboratories.

As part of an international measurement intercomparison of instruments used to measure atmospheric 222Rn, four participating laboratories made nearly simultaneous measurements of 222Rn activity concentration in commonly sampled, ambient air over approximately a 2 week period, and three of these four laboratories participated in the measurement comparison of 14 introduced samples with known, but undisclosed ("blind") 222Rn activity concentration. The exercise was conducted in Bermuda in October 1991. The 222Rn activity concentrations in ambient Bermudian air over the course of the intercomparison ranged from a few hundredths of a Bq · m-3 to about 2 Bq · m-3, while the standardized sample additions covered a range from approximately 2.5 Bq · m-3 to 35 Bq · m-3. The overall uncertainty in the latter concentrations was in the general range of 10 %, approximating a 3 standard deviation uncertainty interval. The results of the intercomparison indicated that two of the laboratories were within very good agreement with the standard additions, and almost within expected statistical variations. These same two laboratories, however, at lower ambient concentrations, exhibited a systematic difference with an averaged offset of roughly 0.3 Bq · m-3. The third laboratory participating in the measurement of standardized sample additions was systematically low by about 65 % to 70 %, with respect to the standard addition which was also confirmed in their ambient air concentration measurements. The fourth laboratory, participating in only the ambient measurement part of the intercomparison, was also systematically low by at least 40 % with respect to the first two laboratories.