Catalytic plasticity of fatty acid modification enzymes underlying chemical diversity of plant lipids.

Higher plants exhibit extensive diversity in the composition of seed storage fatty acids. This is largely due to the presence of various combinations of double or triple bonds and hydroxyl or epoxy groups, which are synthesized by a family of structurally similar enzymes. As few as four amino acid substitutions can convert an oleate 12-desaturase to a hydroxylase and as few as six result in conversion of a hydroxylase to a desaturase. These results illustrate how catalytic plasticity of these diiron enzymes has contributed to the evolution of the chemical diversity found in higher plants.

Multi-Method Molecular Characterisation of Human Dust-Mite-associated Allergic Asthma.

Asthma is a chronic inflammatory disorder of the airways. Disease presentation varies greatly in terms of cause, development, severity, and response to medication, and thus the condition has been subdivided into a number of asthma phenotypes. There is still an unmet need for the identification of phenotype-specific markers and accompanying molecular tools that facilitate the classification of asthma phenotype. To this end, we utilised a range of molecular tools to characterise a well-defined group of female adults with poorly controlled atopic asthma associated with house dust mite (HDM) allergy, relative to non-asthmatic control subjects. Circulating messenger RNA (mRNA) and microRNA (miRNA) were sequenced and quantified, and a differential expression analysis of the two RNA populations performed to determine how gene expression and regulation varied in the disease state. Further, a number of circulating proteins (IL-4, 5, 10, 13, 17 A, Eotaxin, GM-CSF, IFNy, MCP-1, TARC, TNFα, Total IgE, and Endotoxin) were quantified to determine whether the protein profiles differed significantly dependent on disease state. Finally, we utilised a previously published assessment of the circulating "blood microbiome" performed using 16S rRNA amplification and sequencing. Asthmatic subjects displayed a range of significant alterations to circulating gene expression and regulation, relative to healthy control subjects, that may influence systemic immune activity. Notably, several circulating mRNAs were detected in just the asthma group or just in the control group, and many more were observed to be expressed at significantly different levels in the asthma group compared to the control group. Proteomic analysis revealed increased levels of inflammatory proteins within the serum, and decreased levels of the bacterial endotoxin protein in the asthmatic state. Comparison of blood microbiome composition revealed a significant increase in the Firmicutes phylum with asthma that was associated with a concomitant reduction in the Proteobacteria phylum. This study provides a valuable insight into the systemic changes evident in the HDM-associated asthma, identifies a range of molecules that are present in the circulation in a condition-specific manner (with clear biomarker potential), and highlights a range of hypotheses for further study.

Visual acuity in esotropic cats following occlusion of the non-deviating eye.

The present study examined the possibility that strabismic amblyopia may be explained in terms of binocular competition. This was done by measuring visual acuity in strabismic kittens whose deviating eye was placed at an advantage over the other. Four groups of kittens were studied: Normal/Normal (N/N); Lid-sutured/Normal (LS/N); Strabismic/Normal (S/N); Strabismic/Lid-sutured (S/LS). In conditions 2-4 kittens underwent lateral rectus section and/or unilateral lid suture around the time of natural eye opening. Visual acuity measurements were made using the jumping stand technique and the following results were obtained: all S/N kittens showed an acuity deficit in the strabismic eye; each S/LS kitten had a better acuity in the deviating eye than any S/N kitten; the acuity of the LS/S kittens was slightly below that of the N/N kittens or the normal eye of the LS/N kittens. These data suggest that binocular competition can account for essentially all of the strabismic amblyopia observed in kittens.

In vitro skin corrosivity test using human skin.

The in vitro skin corrosivity test (IVSCT) has been shown to be a reliable alternative for detecting substances that would be classified as corrosive in the standard in vivo test. The assay is based on the observation that corrosive substances produce a significant reduction in the transcutaneous electrical resistance (TER) of ex vivo rat skin. The use in the test of human skin to provide a means of direct assessment of potential corrosive substances in humans has been assessed. The testing of 12 substances allowed a TER threshold for in vitro classification as corrosive to be established. In further testing of 15 surfactants (three classified as corrosive) and eight fatty acid substances (four corrosive), the three surfactants but none of the fatty acids were identified as corrosive in vitro. Thus the fatty acids labelled as corrosive, on the basis of animal data, may be incorrectly classified (i.e. in humans the substances would not lead to a corrosive effect following skin contact). It is envisaged that the IVSCT using human skin will be used together with human patch testing and lead to a reduction in animal testing and a more relevant classification of skin irritation potential to humans.

The in vitro skin corrosivity test. Development of method using human skin.

To meet the EEC regulations for classification, packaging, labelling and transportation, many new substances have to be tested for their potential cutaneous toxicity. At present, this is assessed in an in vivo rabbit dermal test. The in vitro skin corrosivity test has been developed to identify those substances which would cause a corrosive response in an in vivo test, thereby alleviating the need to assess corrosive substances in an in vivo test. The method, which uses ex vivo rat skin, is based on the observation that corrosive substances cause a significant reduction in the electrical resistance of skin. The present study investigated the possibility of using human skin in the in vitro skin corrosivity test to provide a means of direct assessment of effect in humans. 12 substances were assessed in this model. The transcutaneous electrical resistance (TER) following treatment with distilled water is essentially the same as that for untreated skin. Of the six preparations that would be formally classified as corrosive by the EEC, on the basis of a 4-hr rabbit covered patch test, all caused a sharp fall in the TER for each specimen of human skin on which they were tested. On the basis of these data, the TER threshold for determination of potential corrosivity was judged to be 11.0 kohms/disc of skin. For the five preparations which would be classified as irritant or would not require a label, the reduction was usually much less marked. Continuing work in this laboratory is directed towards an assessment of a much wider range of chemicals using the human skin in vitro model, ultimately with a view to its use as a method of detecting those substances which may be corrosive to the skin of humans, leading to their classification and appropriate labelling.

The in vitro skin corrosivity test. Comparison of in vitro human skin with in vivo data.

Regulations require that many substances and chemicals are tested for their cutaneous toxicity potential. At present this is assessed in an in vivo rabbit dermal test. A rapid, robust and reliable alternative for testing the corrosive potential of substances is the in vitro skin corrosivity test, which alleviates the need to assess corrosive substances in an in vivo test. The method is based on the observation that corrosive substances cause a significant reduction in the electrical resistance of skin. It was anticipated that the more resistant nature of human skin would lead to a number of substances being classified as non-corrosive, contrary to historical in vivo animal data. In the present study 15 surfactants and eight fatty acid substances were tested on human skin in the assay. Three surfactants and four fatty acids were classified as corrosive in vivo. In the assay, the three surfactants were identified as corrosive, but all the fatty acid substances were found to be non-corrosive. It is suggested that the fatty acid substances labelled as corrosive on the basis of animal data are over-labelled, that is, in humans the substances would not lead to a corrosive effect. It is envisaged that the in vitro human skin corrosivity test will be used in conjunction with human patch tests, currently being developed. This approach should allow a reduction in animal testing and a more relevant classification of skin irritation potential to humans.

Skin corrosivity potential of fatty acids: In vitro rat and human skin testing and QSAR studies.

The corrosive potential of a series of fatty acids-propanoic acid (C3), butanoic acid (C4), hexanoic acid (C6), octanoic acid (C8), decanoic acid (C10) and dodecanoic acid (C12)-was investigated in the in vitro skin corrosivity test (IVSCT) using both rat skin and human skin. All the fatty acids with alkyl chain lengths up to and including C8 were found to be corrosive to rat skin. When human skin was used, the corrosive/non-corrosive threshold was shifted to around the C6 fatty acid. The results are discussed in the context of a QSAR for the corrosivity of organic acids, with the putative mechanism that corrosivity is a function of the ability of the chemical to permeate the skin together with its cytotoxicity, expressed in this case as acidity (pK(a)). This mechanistic interpretation is consistent with the known differences in barrier properties between rat and human skin.

The skin corrosivity test in vitro. Results of an inter-laboratory trial.

The collaborative study reported here was performed to evaluate the reliability of the skin corrosivity test in vitro when performed in independent laboratories. Twenty substances were examined in each of three participating laboratories and the results were compared with existing data from standard assays in vivo. The skin corrosivity test is based on the assumption that corrosive substances destroy the skin's natural outer protective barrier, the stratum corneum. Corrosive action in vitro is measured by a fall in the transcutaneous electrical resistance (TER) below a predetermined threshold. A refined test using a MgSO(4) electrolyte solution for TER measurements has recently been shown to reduce the number of false positive results in the test, while maintaining excellent predictive value for skin corrosive substances. Although in the present study there was some variation between laboratories in terms of the absolute mean TER values obtained, all 6 substances corrosive in vivo were correctly predicted by the three laboratories. The other 14 substances ranged from being non-irritant to severe skin irritants in vivo, but the test was unable to discriminate between these different categories in any of the three laboratories. However, these inter-laboratory comparisons demonstrate that the refined skin corrosivity test is a robust and reliable method in vitro for identifying potential skin corrosive substances.

Identification of irritation and corrosion hazards to skin: an alternative strategy to animal testing.

To date the use of in vitro/alternative tests to achieve formal classification in the EEC of the toxic properties of new substances is very limited. An opportunity exists in the area of skin irritation/corrosivity to adopt a strategic approach which will limit the need to use animals. The approach would involve use of human skin in vitro to identify corrosive materials followed by ethically approved human patch testing. In the patch test, the irritation potential of the substance or preparation would be judged against a suitable positive irritant control. In addition to the avoidance of the use of animals, a further benefit would be that use of human skin should lead to a more relevant classification of skin irritation/corrosion hazard for humans.

The identification and classification of skin irritation hazard by a human patch test.

There exist various regulatory instruments the purpose of which is to ensure that the intrinsic toxic hazards associated with substances and preparations are identified. In the context of identification of skin irritation potential, the method is normally the Draize test. Guidance notes provided by the OECD and the EEC expect that corrosive substances will have been screened out by a variety of methods. Substances or preparations which cause a sufficient degree of skin irritation will be classified as skin irritants. The primary motivation behind the present work was to introduce the concept that it is possible to assess the hazard potential of a substance or preparation to produce skin irritation in a human study. In the example presented here, 20% sodium lauryl sulfate (SLS) has been chosen as the positive control. With the protocol currently devised, occluded patch treatment with 20% SLS for up to 4 hr produces an irritant response in just over half of the panel. An irritant response is taken as a clinically evident and significant increase in erythema, oedema or dryness--a minimum of a+ reaction on the ICDRG scale. At such a level of response with the positive control (both in terms of intensity and in proportion of the panel), it is then possible to judge and/or to determine statistically, whether the test material has produced a level of skin irritation which is similar to, greater, or lower than the positive control. In this way a human patch test protocol can form a fundamental component of a strategy for the replacement of animals in determination of skin irritation and corrosion potential. By use of a careful and progressive protocol and by comparison of test data against a positive control it is both possible and practical to classify substances and preparations in terms of their skin irritation potential using that endpoint in the species of concern, man.

Fate of ethanol topically applied to skin.

Ethanol is a major component of many aerosol sprays and consumer products that are designed to contact the skin. It is theoretically possible that small amounts of ethanol from alcohol-based sprays can be absorbed across the skin or inhaled during spraying. In order to assess the potential systemic dose, three parameters were measured: the evaporation of [14C]ethanol from the skin surface, the in vitro penetration of [14C]ethanol through excised pig skin and the ethanol concentration in the blood of human volunteers following simulated use of an alcohol based deodorant spray. The rate of evaporation from Benchkote and whole pig skin was similar (t(1/2)=13.6 sec and 11.7 sec, respectively) while that from glass was longer (t(1/2)=24.8 sec). Ethanol penetration through pig skin in vitro was greater in occluded cells than in non-occluded cells (2.19 mg/cm(2) and 0.10 mg/cm(2) in 24 hours, respectively). At the maximum flux seen in this experiment under occlusion, the amount of ethanol penetrating from a 1 m(2) area of skin would give a blood alcohol level of about 4 mg% in a 70-kg man. In the human use study, none of the blood samples taken from 16 human volunteers exhibited a detectable level of alcohol. These studies provide evidence that a systemic dose of ethanol is likely to be very low after the use of formulations delivering ethanol to the skin.

The classification of skin irritants by human patch test.

The human 4 hour patch test provides an opportunity to identify substances with significant skin irritation potential without recourse to the use of animals. The protocol is designed to avoid the production of more than mild irritant reactions and meets the highest ethical standards. This paper provides the background to the development of the method and comments on its performance in the light of recent intra- and inter-laboratory investigations. In particular, the value of the method in providing 'gold standard' data for the identification of those substances (or preparations) which should, or should not, be classified as irritant to skin in European legislation is discussed. On the basis of the published data and supplementary investigations, recommendations are made on both the conduct and interpretation of the human 4 hour patch test. Finally, the lack of any necessity for formal validation of this assay is addressed.

Non-animal testing strategies for assessment of the skin corrosion and skin irritation potential of ingredients and finished products.

The dermatotoxicologist today is faced with a dilemma. Protection of workers and consumers from skin toxicities (irritation and allergy) associated with exposure to products, and the ingredients they contain, requires toxicological skin testing prior to manufacture, transport, or marketing. Testing for skin corrosion or irritation has traditionally been conducted in animals, particularly in rabbits via the long established Draize test method. However, this procedure, among others, has been subject to criticism, both for its limited predictive capacity for human toxicity, as well as for its use of animals. In fact, legislation is pending in the European Union which would ban the sale of cosmetic products, the ingredients of which have been tested in animals. These considerations, and advancements in both in vitro skin biology and clinical testing, have helped drive an intensive effort among skin scientists to develop alternative test methods based either on in vitro test systems (e.g. using rat, pig or human skin ex vivo, or reconstructed human skin models) or ethical clinical approaches (human volunteer studies). Tools are now in place today to enable a thorough skin corrosion and irritation assessment of new ingredients and products without the need to test in animals. Herein, we describe general testing strategies and new test methods for the assessment of skin corrosion and irritation. The methods described, and utilized within industry today, provide a framework for the practicing toxicologist to support new product development initiatives through the use of reliable skin safety testing and risk assessment tools and strategies.

Source analysis of short and long latency vestibular-evoked potentials (VsEPs) produced by left vs. right ear air-conducted 500 Hz tone pips.

Todd et al. (2014) have recently demonstrated the presence of vestibular dependent changes both in the morphology and in the intensity dependence of auditory evoked potentials (AEPs) when passing through the vestibular threshold as determined by vestibular evoked myogenic potentials (VEMPs). In this paper we extend this work by comparing left vs. right ear stimulation and by conducting a source analysis of the resulting evoked potentials of short and long latency. Ten healthy, right-handed subjects were recruited and evoked potentials were recorded to both left- and right-ear sound stimulation, above and below vestibular threshold. Below VEMP threshold, typical AEPs were recorded, consisting of mid-latency (MLR) waves Na and Pa followed by long latency AEPs (LAEPs) N1 and P2. In the supra-threshold condition, the expected changes in morphology were observed, consisting of: (1) short-latency vestibular evoked potentials (VsEPs) which have no auditory correlate, i.e. the ocular VEMP (OVEMP) and inion response related potentials; (2) a later deflection, labelled N42/P52, followed by the LAEPs N1 and P2. Statistical analysis of the vestibular dependent responses indicated a contralateral effect for inion related short-latency responses and a left-ear/right-hemisphere advantage for the long-latency responses. Source analysis indicated that the short-latency effects may be mediated by a contralateral projection to left cerebellum, while the long-latency effects were mediated by a contralateral projection to right cingulate cortex. In addition we found evidence of a possible vestibular contribution to the auditory T-complex in radial temporal lobe sources. These last results raise the possibility that acoustic activation of the otolith organs could potentially contribute to auditory processing.

Engineering delta 9-16:0-acyl carrier protein (ACP) desaturase specificity based on combinatorial saturation mutagenesis and logical redesign of the castor delta 9-18:0-ACP desaturase.

Six amino acid locations in the soluble castor Delta(9)-18:0-acyl carrier protein (ACP) desaturase were identified that can affect substrate specificity. Combinatorial saturation mutagenesis of these six amino acids, in conjunction with selection, using an unsaturated fatty acid auxotroph system, led to the isolation of variants with up to 15-fold increased specific activity toward 16-carbon substrates. The most improved mutant, com2, contained two substitutions (T117R/G188L) common to five of the 19 complementing variants subjected to further analysis. These changes, when engineered into otherwise wild-type 18:0-ACP desaturase to make mutant 5.2, produced a 35-fold increase in specific activity with respect to 16-carbon substrates. Kinetic analysis revealed changes in both k(cat) and K(m) that result in an 82-fold improvement in specificity factor for 16-carbon substrate compared with wild-type enzyme. Improved substrate orientation apparently compensated for loss of binding energy that results from the loss of desolvation energy for 16-carbon substrates. Mutant 5.2 had specific activity for 16-carbon substrates 2 orders of magnitude higher than those of known natural 16-carbon specific desaturases. These data support the hypothesis that it should be possible to reengineer archetypal enzymes to achieve substrate specificities characteristic of recently evolved enzymes while retaining the desired stability and/or turnover characteristics of a parental paralog.