Model studies of migration from paper and board into fruit and vegetables and into Tenax as a food simulant.

Four samples of paper and board (P/B) of a type used for packaging dry foods were subjected to migration tests using mushrooms, apples, potatoes and bananas, and using the polymeric powder Tenax as a food simulant. The P/B samples contained only low levels of diisopropylnaphthalene (DiPN) and diisobutyl phthalate (DiBP) and so the experiments were conducted after impregnating the P/B with added model substances. These were o-xylene, acetophenone, dodecane, benzophenone, DiPN and DiBP. Migration levels depended strongly on the nature of the substance and on the nature of the food and much less on the characteristics of the P/B, except insofar as they affected the contact area - flexible papers giving more extensive contact with the food than thick rigid board. Migration into Tenax was at least a factor of 10 higher than migration into the fresh fruit and vegetables. The food samples were placed in contact with the P/B and then overwrapped loosely with aluminium foil and so this correction factor will tend to be conservative compared with a more open storage of the packed foods. Washing, peeling or cooking the fruits and vegetables after contact with the P/B had a surprisingly small effect on contaminant levels in general, and no one processing step was effective in giving a significant reduction of all the types of chemicals studied. This was because either they had penetrated into the food (so resisting peeling), or were not freely water-soluble (so resisting washing) or were not particularly volatile (so resisting loss by evaporation during cooking).

Analytical screening studies on irradiated food packaging.

Foods may be irradiated in their final packaging and this process may affect the composition of the packaging and in turn affect the migration of substances into food. Headspace and liquid injection GC-MS and HPLC with time-of-flight MS have been used to identify and estimate levels of radiolytic products in irradiated finished plastic packaging materials. Fifteen retail packaging materials were studied. Investigations were carried out into the effect of different irradiation types (gamma and electron beam), irradiation doses (1, 3, 7 and 10 kGy) and dose rates (5 kGy s(-1) for electron beam and 0.4 and 1.85 kGy h(-1) for gamma) on the radiolytic products. Any differences seen in comparing the two ionising radiation types were attributed largely to the very different dose rates; for electron beam a 10 kGy dose was delivered in just 2 s whereas using gamma it took 5.4 h. Differences were also seen when comparing the same samples irradiated at different doses. Some substances were not affected by irradiation, others decreased in concentration and others were formed upon increasing doses of irradiation. These results confirm that irradiation-induced changes do occur in substances with the potential to migrate and that the safety of the finished packaging material following irradiation should be assessed.

Methods of analysis for 2-dodecylcyclobutanone and studies to support its role as a unique marker of food irradiation.

Methods of analysis for 2-dodecylcyclobutanone (2-DCB) using gas chromatography with mass spectrometric detection (GC-MS), liquid chromatography with time-of-flight mass spectrometric detection (LC-TOF-MS) and LC with tandem MS (MS/MS) detection have been developed and optimised for maximum sensitivity to allow very low irradiation doses to be detected. The LC-MS/MS method, following derivatisation with 2,4-dinitrophenylhydrazine, was found to be the most sensitive technique and was used to determine the amount of 2-DCB formed from the model compounds palmitic acid, glyceryl tripalmitate and 1,3-dipalmitoyl-2-oleoylglycerol irradiated over a range of doses by two different irradiation sources (gamma and electron beam). The model compounds were also treated with a number of non-irradiation based processing techniques including heating in the presence and absence of oxygen, light, and redox active metal salts, in a conventional oven, microwave oven and pressure cooker. No 2-DCB was detected in any of the processed non-irradiated model compounds, reaffirming the hypothesis that 2-DCB is a unique radiolytic product that can be used as a marker of irradiation in foodstuffs.

Test procedures for obtaining representative extracts suitable for reliable in vitro toxicity assessment of paper and board intended for food contact.

This paper describes the use of a suite of extraction procedures applicable to the assessment of the in vitro toxicity of paper/board samples intended for food-contact applications. The sample is extracted with ethanol, water, or exposed to modified polyphenylene oxide (Tenax) for fatty, non-fatty and dry food applications, respectively. The water extracts are directly suitable for safety assessment using in vitro bioassays. The ethanol extracts of the paper/board and of the exposed Tenax require pre-concentration to give acceptable sensitivity. This is because the in vitro bioassays can tolerate only a small percentage of added organic solvent before the solvent itself inhibits. The extraction procedures have been selected such that they mimic the foreseeable conditions of use with foods and that they are also fully compatible with the battery of in vitro biological assays for the safety assessment of the total migrate. The application of the extraction protocols is illustrated by the results for one of the many paper/board samples provided by the BIOSAFEPAPER project industrial platform members. The assessment indicated that this sample should not be considered as suitable for use with fatty foodstuffs but was suitable for dry and non-fatty foods. Information subsequently received from the manufacturer revealed that this was a non-food-grade product included in the project to test the capabilities of the bioassay procedures. The selection criteria for the test conditions and the suite of methods developed have been prepared in Comité Européen de Normalisation (CEN) format and is currently being progressed by CEN/TC172 as a European Standard.

[Specific aspects of vaccine manufacturing]. / Aspects spécifiques de la production dans le domaine des vaccins.

The industrial development of vaccine manufacturing expanded during the second half of the 20th century. Vaccines are medicines which target healthy people. The active principles of vaccines arise from live organisms and can be distinguished from standard pharmaceutical compounds by their extreme complexity. The properties of vaccines depend mainly of the manufacturing process and it common to state that "the process makes the product". The manufacturing is done in confined rooms because of the pathogenic characters of the organisms involved. Raw materials are subjected to rigorous controls to guarantee the integrity of products towards non-conventional agents like prion. The vaccine industry is characterized by the length of the manufacturing cycle. The complexity of the products implies heavy quality controls, which represent 75% of the total duration of the manufacture cycle. As a key element of public health, the vaccine production can be subjected to variations and adaptations, in particular in the context of outbreaks or bioterrorism. The vaccine industry must integrate the regulatory requirements, which are more and more pressing. The spectacular development of the quality assurance and quality control departments these last 15 years testifies to this evolution.

The BIOSAFEPAPER project for in vitro toxicity assessments: preparation, detailed chemical characterisation and testing of extracts from paper and board samples.

Nineteen food contact papers and boards and one non-food contact board were extracted following test protocols developed within European Union funded project BIOSAFEPAPER. The extraction media were either hot or cold water, 95% ethanol or Tenax, according to the end use of the sample. The extractable dry matter content of the samples varied from 1200 to 11,800 mg/kg (0.8-35.5 mg/dm2). According to GC-MS the main substances extracted into water were pulp-derived natural products such as fatty acids, resin acids, natural wood sterols and alkanols. Substances extracted into ethanol particularly, were diisopropylnaphthalenes, alkanes and phthalic acid esters. The non-food contact board showed the greatest number and highest concentrations of GC-MS detectable compounds. The extracts were subjected to a battery of in vitro toxicity tests measuring both acute and sublethal cytotoxicity and genotoxic effects. None of the water or Tenax extracts was positive in cytotoxicity or genotoxicity assays. The ethanol extract of the non-food contact board gave a positive response in the genotoxicity assays, and all four ethanol extracts gave positive response(s) in the cytotoxicity assays to some extent. These responses could not be pinpointed to any specific compound, although there appeared a correlation between the total amount of extractables and toxicity.

Survey of the migration of melamine and formaldehyde from melamine food contact articles available on the UK market.

The migration of melamine and formaldehyde, monomers used in the production of melamine-ware food contact articles, has been determined from 50 retail articles purchased in the UK. The food simulant 3% aqueous acetic acid was used as this is the most aggressive simulant towards melamine plastics. The test conditions used were repeated exposure to the simulant for 2 hours at 70 degrees C, since the articles were all intended for general use including contact with hot foods and beverages. Melamine migrated from 43 of the 50 samples tested and formaldehyde migrated from all 50 samples. Directive 2002/72/EC specifies migration limits for both of these monomers in foods and food simulants. Melamine is restricted by a specific migration limit (SML) of 30 mg/kg (equivalent to 5 mg/dm(2)) and formaldehyde, along with hexamethylenetetramine expressed as formaldehyde, is restricted by a total (T) SML(T) of 15 mg/kg (equivalent to 2.5 mg/dm(2)). In all cases the migration of melamine was much lower than the SML for this monomer. The migration of formaldehyde exceeded the SML(T) for 5 of the 50 samples tested. The failure to comply with the SML(T) was accompanied by a number of visible surface effects including discolouration and/or pitting of the simulant contact surface and cracking of the articles. Similar surface effects were observed when one of the samples was exposed to fruit juice which confirmed the suitability of the exposure conditions and 3% acetic acid as a simulant for the articles tested. The ratio of specific migration to overall migration was consistent with, but did not prove, the hypothesis that high formaldehyde migration could be due to the use of excessive hexamethylenetetramine in the polymer formulation. All illegal products were voluntarily removed from the market by the product suppliers.

Method of test and survey of caprolactam migration into foods packaged in nylon-6.

An analytical method for the determination of the nylon-6 monomer caprolactam in foods is described. The foodstuff was extracted with ethanol: water (1:2) containing capryllactam as internal standard and the extract was defatted using hexane. The extract was analysed by liquid chromatography coupled with mass spectrometry. The test method was calibrated down to 0.7 mg kg(-1). The repeatability of the method was good, with a relative standard deviation of 9% at the 15 mg kg(-1) level. The method was demonstrated to be accurate in an independent external check sample exercise. The new method was applied to the analysis of 50 retail foodstuffs packaged in nylon-6. Caprolactam was detected and confirmed in nine of the 50 food samples, in the range 2.8-13 mg kg(-1). The presence of caprolactam was indicated in a further 15 samples, in the range 0.8-11 mg kg(-1), but these samples did not meet all of the five confirmation criteria applied. All migration levels (both confirmed and unconfirmed) were below the European specific migration limit for caprolactam, which is 15 mg kg(-1). The average migration for all 50 samples, setting non-detectables at half the limit of detection, was 2.6 mg kg(-1) with a standard deviation of 3.1 mg kg(-1) (n = 50). All samples found to contain detectable levels of caprolactam migration were for applications involving heating the food in the packaging. They were packs of, for example, sausage meat for which the food would have been heat processed in the nylon casing, or they were nylon pouches for heating foods by boiling, microwaving or roasting.

Laryngeal and diaphragmatic muscle activities after central nervous system lesions in cats.

AIM: To examine the central control and coordination of respiratory pump muscles and laryngeal valve muscles by systematic decerebration (DECER), cerebellectomy (CBELL), pontine respiratory group lesioning (PRG) and pontomedullary section (PMED). METHODS: Activities of posterior cricoarytenoid (PCA), thyroarytenoid (TA) and diaphragm (D) muscles and their responses to inspiratory (I) and expiratory (E) total occlusions were determined in 10 adult cats. RESULTS: INTACT anesthetized cats (n = 6) exhibited inspiratory PCA (PCA(I)) and D activities. Expiratory PCA (PCA(E)) was present but TA activity was absent. It was found that successive DECER, CBELL and PRG lesions attenuated PCA(E), the intact pattern being noted in 7/10, 4/10 and 0/6 cats, respectively. After PMED, variable PCA, TA and continuous D activities occurred only with blood gas abnormalities. Augmented PCA and D responses to I- and E-loads occurred after PRG lesions: the I-load PCA(I) and D responses resembled apneusis and the E-load PCA(E) and D responses resembled central apnea. CONCLUSION: The decreasing PCA(E) activity observed with successive DECER, CBELL and PRG lesions suggests that these areas influence laryngeal abductor control of glottic size. The synchronous activities after PMED transection suggest a role for more rostral structures in coordinating laryngeal and diaphragmatic muscle activities.

Development and application of an LC-MS method to determine possible migration of mercaptobenzothiazole, benzothiazole and related vulcanization residues from rubber used in contact with food and drink.

A method was developed for the analysis of food and drink for residues of specific vulcanization accelerators used to cross-link rubber. The method was applied to the analysis of 236 samples of selected retail foodstuffs that may have been in contact with rubber during their manufacture, transport and storage. The method of analysis involved extraction of the food using acidified solvent and analysis by liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC-APcI-MS). The detection limit depended on the sample type and was in the range 0.005-0.043 mg kg(-1) for 2-mercaptobenzothiazole (MBT) and benzothiazole (BT). The average analytical recovery rate was 82% for MBT and 87% for BT. The analytical method was validated using a blind check sample exercise. For MBT and BT at seven different concentrations in the range 0.1-0.2 mg kg(-1), the laboratory found a mean of 91 and 90% of the expected concentrations, respectively. No trace of MBT or BT was found in any of the retail samples. It is also concluded that no sample contained significant 2-mercaptobenzothiazyl disulphide (MBTS) or N- cyclohexyl-2-benzothiazole sulphenamide (CBS). Both MBTS and CBS are important accelerators used to vulcanize rubber and they break down in foodstuffs to form MBT and BT. The absence of MBT and BT in the foodstuffs therefore also provides proof of the absence of MBTS and CBS.

Verification of the findings of acrylamide in heated foods.

We report here the first confirmation of the recent Swedish findings of acrylamide in heated foods. The verification exercise used an LC-MS/MS method developed for the purpose as well as an established GCMS method for acrylamide analysis. LC-MS/MS was suitable for the direct determination of acrylamide in aqueous extracts of foods by isotope dilution mass spectrometry (IDMS) using triply deuterated acrylamide. Some food matrices were not suited to the new method and mixed-mode solid-phase extraction (SPE) was used to clean these extracts. The foods tested included UK versions of some of the key food groups analysed in Sweden. Also tested were some foods heated under home-cooking conditions. There was good agreement between the LC-MS/MS results and the GC-MS results and the levels of acrylamide found here were similar to those reported for the corresponding foods analysed in the Swedish study. The analyses confirmed that acrylamide is absent from the raw or boiled foods but present at significant levels in fried, grilled, baked and toasted foods. The highest result was 12000 microg kg(-1) acrylamide in overcooked oil-fried chips.

Evidence for medullary projections of the intercostal nerve-elicited inspiratory termination.

This study hypothesized that the ICN-elicited inspiratory termination reflex required synaptic activation in two distinct regions of the ventral respiratory group (VRG): (1) transitional (tVRG), and (2) pre-Bötzinger complex (pre-BötC). Data from adult cats indicate that axons of passage associated with the ICN-elicited termination reflex traverse tVRG, but that relevant synaptic processing does not occur in this region. Furthermore, data indicate that neither synaptic nor axonal transmission within the pre-BötC is required for the SLN- or ICN-elicited termination reflex.

Differential effects of excitatory amino acid receptor antagonism in the ventral respiratory group.

This study tested the hypothesis that selective antagonism of excitatory amino acid (EAA) receptors within the ventral respiratory group (VRG) would induce changes in both respiratory rhythm and pattern. In the paralyzed, decerebrate, vagotomized and ventilated cat, baseline values for respiratory (Ttot), inspiratory (Ti), and expiratory (Te) durations and peak integrated phrenic nerve (integralPN) amplitude were established. Microinjection of the non-NMDA (N-methyl-d-aspartate) receptor antagonist NBQX (2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline) into rostral/inspiratory-modulated (iVRG) or caudal/expiratory-modulated VRG elicited an immediate apnea. When PN activity resumed, Ttot was significantly decreased, and integralPN amplitude was attenuated. NMDA receptor antagonism with microinjections of AP5 (2-amino-5-phosphonopentanoic acid) into iVRG decreased Te for more than 30 min. NMDA receptor antagonism in inspiratory/expiratory-modulated VRG (level of obex, transitional VRG) yielded either apnea or a significant reduction in Ttot, Ti and integralPN amplitude. Our data suggest that endogenous EAA receptor-mediated neurotransmission throughout the VRG is active in the determination of both respiratory timing and pattern. Our data further suggest that tVRG serves a unique function within the respiratory network.

Performance of a prototype for a large-aperture multipass Nd:glass laser for inertial confinement fusion.

The Beamlet is a single-beam prototype of future multibeam megajoule-class Nd:glass laser drivers for inertial confinement fusion. It uses a multipass main amplifier, adaptive optics, and efficient, high-fluence frequency conversion to the third harmonic. The Beamlet amplifier contains Brewster-angle glass slabs with a clear aperture of 39 cm x 39 cm and a full-aperture plasma-electrode Pockels cell switch. It has been successfully tested over a range of pulse lengths from 1-10 ns up to energies at 1.053 mum of 5.8 kJ at 1 ns and 17.3 kJ at 10 ns. A 39-actuator deformable mirror corrects the beam quality to a Strehl ratio of as much as 0.4. The 1.053-mum output has been converted to the third harmonic at efficiencies as high as 80% and fluences as high as 8.7 J/cm(2) for 3-ns pulses.

Reduced space sequence alignment.

MOTIVATION: Sequence alignment is the problem of finding the optimal character-by-character correspondence between two sequences. It can be readily solved in O(n2) time and O(n2) space on a serial machine, or in O(n) time with O(n) space per O(n) processing elements on a parallel machine. Hirschberg's divide-and-conquer approach for finding the single best path reduces space use by a factor of n while inducing only a small constant slowdown to the serial version. RESULTS: This paper presents a family of methods for computing sequence alignments with reduced memory that are well suited to serial or parallel implementation. Unlike the divide-and-conquer approach, they can be used in the forward-backward (Baum-Welch) training of linear hidden Markov models, and they avoid data-dependent repartitioning, making them easier to parallelize. The algorithms feature, for an arbitrary integer L, a factor proportional to L slowdown in exchange for reducing space requirement from O(n2) to O(n1 square root of n). A single best path member of this algorithm family matches the quadratic time and linear space of the divide-and-conquer algorithm. Experimentally, the O(n1.5)-space member of the family is 15-40% faster than the O(n)-space divide-and-conquer algorithm.

Production of lipidated meningococcal transferrin binding protein 2 in Escherichia coli.

Neisseria meningitidis strains grown under iron starvation conditions produce transferrin binding proteins (Tbp1 and Tbp2) which have been shown to play a major role in iron acquisition. Recent studies performed with Tbp2 purified from N. meningitidis suggest that this surface protein is a potential vaccine component. In order to further evaluate the immunogenicity of Tbp2, it was essential to develop a heterologous expression system to generate high amounts of purified protein. Tbp2 is produced in Neisseria as a precursor with a signal peptide whose cleavage follows a lipidation step on a cysteine residue which is the first amino acid in the mature protein. When produced in Escherichia coli with its natural signal peptide, a high amount of Tbp2 (about 10% of total cell proteins) was detected. However, most of the protein was nonlipidated precursor and only a small fraction was mature Tbp2. In order to optimize the maturation of the precursor, the natural signal sequence was replaced by several E. coli lipoprotein signal peptides. Expression levels and maturation of the precursor were highly variable depending on the signal peptide used. With one of these, an efficient maturation and a high amount of mature lipidated Tbp2 were obtained (about 3% of total cell proteins). A large-scale production process was then established for this E. coli-produced Tbp2.

Short-latency excitation of phrenic motor output mediated by non-NMDA receptors.

This study tested the hypothesis that the short-latency excitation of the phrenic motor output elicited by superior laryngeal nerve (SLN) stimulation requires non-NMDA receptor-mediated neurotransmission in the region of the dorsal respiratory group (DRG) of the adult cat. Injection of the non-NMDA receptor antagonist NBQX into the DRG severely attenuated or abolished the short-latency excitation, indicating that the short-latency excitation requires non-NMDA receptor-mediated neurotransmission within the DRG.

Parallel sequence alignment in limited space.

Sequence comparison with affine gap costs is a problem that is readily parallelizable on simple single-instruction, multiple-data stream (SIMD) parallel processors using only constant space per processing element. Unfortunately, the twin problem of sequence alignment, finding the optimal character-by-character correspondence between two sequences, is more complicated. While the innovative O(n2)-time and O(n)-space serial algorithm has been parallelized for multiple-instruction, multiple-data stream (MIMD) computers with only a communication-time slowdown, typically O(log n), it is not suitable for hardware-efficient SIMD parallel processors with only local communication. This paper proposes several methods of computing sequence alignments with limited memory per processing element. The algorithms are also well-suited to serial implementation. The simpler algorithms feature, for an arbitrary integer L, a factor of L slowdown in exchange for reducing space requirements from O(n) to O(L square root of n) per processing element. Using this result, we describe an O(n log n) parallel time algorithm that requires O(log n) space per processing element on O(n) SIMD processing elements with only a mesh or linear interconnection network.

Recombinant cholera toxin B subunit in Escherichia coli: high-level secretion, purification, and characterization.

The gene coding for cholera toxin subunit B (CT-B) was fused to a modified ompA signal sequence and subsequently cloned into a high expression vector based on the regulatory signals of the arabinose operon of Salmonella typhimurium. Upon induction of gene expression in Escherichia coli, a product of the expected size for CT-B monomer was detected at a level of approximately 60% of total periplasmic protein. At pilot scale, batch cultivation in a 20-liter bioreactor allowed a production level of 1 g/liter of recombinant CT-B (rCT-B), the majority of which was released into the culture medium. The latter phenomenon was dependent on the medium selected for cultivation. A simple and inexpensive purification scheme was developed which enabled the recovery of 81% of rCT-B from the culture supernatant. Comparing amino acid composition, amino-terminal sequence, mass spectrum, pentamerisation, and GM1-binding, rCT-B is indistinguishable from natural CT-B produced by Vibrio cholerae. This rCT-B overproducing E. coli strain represents an interesting alternative to overexpressing systems developed in V. cholerae.

Role of N-methyl-D-aspartate receptors in the pontine pneumotaxic mechanism in the cat.

Systemic injection of MK-801, an N-methyl-D-aspartate (NMDA) receptor-associated channel blocker, induces an apneusis in vagotomized cats similar to that produced by pontine respiratory group (PRG) lesions, suggesting the possible involvement of NMDA receptors in the pontine pneumotaxic mechanism. Previous results from our laboratory indicate that the efferent limb of the pontine pneumotaxic mechanism is unlikely to require NMDA receptor-mediated neurotransmission. Therefore, the present study examined the potential involvement of PRG NMDA receptors in the pontine pneumotaxic mechanism. Experiments were conducted in decerebrate, paralyzed, and ventilated adult cats. The effects on inspiratory time (TI) of MK-801 microinjection into PRG were tested in 12 cats. Pressure microinjection of MK-801 (15 mM, 80-3,000 nl) significantly prolonged TI in all animals when lung inflation was withheld. TI progressively increased in most animals for > or = 30 min. After this period, partial recovery of the effect occurred in eight cats as TI shortened toward predrug levels. In three animals, microinjection of MK-801 induced a complete apneusis in the absence of lung inflation from which there was no detectable recovery. Microinjections into regions approximately 2 mm distant from PRG produced little or no effect. These results provide evidence that NMDA receptors located in the region of PRG play an important functional role in the control of the breathing cycle.