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Background: Despite the proven audiological benefits of Percutaneous Bone Anchored Hearing Aids (BAHAs) in paediatric patients with conductive or mixed hearing loss, their adoption has been limited due to concerns over implant failure and associated complications. This paper conducts a systematic review and meta-analysis to assess the prevalence of implant failure in paediatric populations, combined with a case series from our tertiary referral centre., Methods: A comprehensive literature search identified 562 articles, from which 34 were included in the review, covering 1599 implants in 1285 patients. Our retrospective case series included consecutive patients from our tertiary referral centre who underwent percutaneous BAHA implantation from 2003-2019., Results: Meta-analysis revealed an overall implant failure rate of 11%, predominantly attributed to traumatic extrusion. Our retrospective case series comprised 104 implantations in 76 patients, with a 4.8% failure rate., Discussion: Factors contributing to the lower-than-expected failure rates in the case series likely included consistent use of 4 mm fixtures from a single manufacturer and older age at implantation. The study underscores the need for standardised reporting formats in bone conduction implants research, given the systematic review's limitations in study design heterogeneity, especially with the expected rise in the adoption of novel active devices.

Machine learning (ML) models were applied to pharmacovigilance (PV) data in a two-component proof-of-concept study. PV data were partitioned into Training, Validation, and Holdout datasets for model training and selection. During the first component ML models were challenged to identify factors in individual case safety reports (ICSRs) involving spinosad and neurological and ocular clinical signs. The target feature for the models were these clinical signs that were disproportionately reported for spinosad. The endpoints were normalized coefficient values representing the relationship between the target feature and ICSR free text fields. The deployed model accurately identified the risk factors "demodectic," "demodicosis," and "ivomec." In the second component, the ML models were trained to identify high quality and complete ICSRs free of confounders. The deployed model was presented with an external Test dataset of six ICSRs, one that was complete, of high quality, and devoid of confounders, and five that were not. The endpoints were model-generated probabilities for the ICSRs. The deployed ML model accurately identified the ICSR of interest with a greater than 10-fold higher probability score. Although narrow in scope, the study supports further investigation and potential application of ML models to animal health PV data., (© 2023 John Wiley & Sons Ltd.)

Purpose: Patient suitability for cochlear implant (CI) devices compatible with magnetic resonance imaging and CI processor configuration is dependent on their retro-auricular skin flap thickness. This is typically measured intra-operatively using a needle and therefore patients are not guaranteed their implant of choice prior to surgery. We aimed to identify an accurate method to measure skin flap thickness pre-operatively to streamline CI selection and simplify the consent process., Methods: Blinded prospective skin flap thickness measurements for patients undergoing CI surgery were recorded using pre-operative computed tomography (CT) and ultrasound (US), and intraoperative needle measurement., Results: Fifty-six adult patients (36 females, 20 males; mean age 59 years) were included. The mean flap thickness was measured highest by CT (6.9 mm, 95% CI 6.5-7.3 mm), followed by US (6.3 mm, 95% CI 5.9-6.7 mm) and lastly needle (5.5 mm, 95% CI 5.1-5.9 mm) ( p  < 0.0001). A strong positive correlation ( p  < 0.001) was noted between all three modalities: CT vs needle ( r  = 0.869), US vs needle ( r  = 0.865), and CT vs US ( r  = 0.849)., Conclusion: Accurate, non-invasive measurement of skin flap thickness prior to CI surgery can be achieved using CT or US. We recommend the routine use of US in the outpatient clinic to minimise unnecessary radiation exposure.

Background: Neuroblastoma is the most common extracranial solid tumour in children, accounting for 15% of paediatric cancer deaths. Multiple genetic abnormalities have been identified as prognostically significant in neuroblastoma patients. Optical genome mapping (OGM) is a novel cytogenetic technique used to detect structural variants, which has not previously been tested in neuroblastoma. We used OGM to identify copy number and structural variants (SVs) in neuroblastoma which may have been missed by standard cytogenetic techniques., Methods: Five neuroblastoma cell lines (SH-SY5Y, NBLW, GI-ME-N, NB1691 and SK-N-BE2(C)) and two neuroblastoma tumours were analysed using OGM with the Bionano Saphyr ® instrument. The results were analysed using Bionano Access software and compared to previous genetic analyses including G-band karyotyping, FISH (fluorescent in situ hybridisation), single-nucleotide polymorphism (SNP) array and RNA fusion panels for cell lines, and SNP arrays and whole genome sequencing (WGS) for tumours., Results: OGM detected copy number abnormalities found using previous methods and provided estimates for absolute copy numbers of amplified genes. OGM identified novel SVs, including fusion genes in two cell lines of potential clinical significance., Conclusions: OGM can reliably detect clinically significant structural and copy number variations in a single test. OGM may prove to be more time- and cost-effective than current standard cytogenetic techniques for neuroblastoma.

Loss-of-function mutations in the CYP24A1 protein-coding region causing reduced 25 hydroxyvitamin D (25OHD) and 1,25 dihydroxyvitamin D (1,25(OH) 2 D) catabolism have been observed in some cases of infantile hypercalcemia type 1 (HCINF1), which can manifest as nephrocalcinosis, hypercalcemia and adult-onset hypercalciuria, and renal stone formation. Some cases present with apparent CYP24A1 phenotypes but do not exhibit pathogenic mutations. Here, we assessed the molecular mechanisms driving apparent HCINF1 where there was a lack of CYP24A1 mutation. We obtained blood samples from 47 patients with either a single abnormality of no obvious cause or a combination of hypercalcemia, hypercalciuria, and nephrolithiasis as part of our metabolic and stone clinics. We used liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine serum vitamin D metabolites and direct sequencing to confirm CYP24A1 genotype. Six patients presented with profiles characteristic of altered CYP24A1 function but lacked protein-coding mutations in CYP24A1. Analysis upstream and downstream of the coding sequence showed single nucleotide variants (SNVs) in the CYP24A1 3' untranslated region (UTR). Bioinformatics approaches revealed that these 3' UTR abnormalities did not result in microRNA silencing but altered the CYP24A1 messenger RNA (mRNA) secondary structure, which negatively impacted translation. Our experiments showed that mRNA misfolding driven by these 3' UTR sequence-dependent structural elements was associated with normal 25OHD but abnormal 1,25(OH) 2 D catabolism. Using CRISPR-Cas9 gene editing, we developed an in vitro mutant model for future CYP24A1 studies. Our results form a basis for future studies investigating structure-function relationships and novel CYP24A1 mutations producing a semifunctional protein. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)., (© 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).)

Enterotoxigenic Bacteroides fragilis (ETBF) has received significant attention for a possible association with, or causal role in, colorectal cancer (CRC). The goal of this review was to assess the status of the published evidence supporting (i) the association between ETBF and CRC and (ii) the causal role of ETBF in CRC. PubMed and Scopus searches were performed in August 2021 to identify human, animal, and cell studies pertaining to the role of ETBF in CRC. Inclusion criteria included the use of cell lines, mice, exposure to BFT or ETBF, and detection of bft. Review studies were excluded, and studies were limited to the English language. Quality of study design and risk of bias analysis was performed on the cell, animal, and human studies using ToxRTools, SYRCLE, and NOS, respectively. Ninety-five eligible studies were identified, this included 22 human studies, 24 animal studies, 43 cell studies, and 6 studies that included both cells and mice studies. We found that a large majority of studies supported an association or causal role of ETBF in CRC, as well as high levels of study bias was detected in the in vitro and in vivo studies. The high-level heterogeneity in study design and reporting made it difficult to synthesize these findings into a unified conclusion, suggesting that the need for future studies that include improved mechanistic models, longitudinal in vitro and in vivo evidence, and appropriate control of confounding factors will be required to confirm whether ETBF has a direct role in CRC etiopathogenesis., (Copyright © 2022. Published by Elsevier Inc.)

Plant plastidial acyl-acyl carrier protein (ACP) desaturases are a soluble class of diiron-containing enzymes that are distinct from the diiron-containing integral membrane desaturases found in plants and other organisms. The archetype of this class is the stearoyl-ACP desaturase which converts stearoyl-ACP into oleoyl (18:1Δ9cis)-ACP. Several variants expressing distinct regioselectivity have been described including a Δ6-16:0-ACP desaturase from black-eyed Susan vine (Thunbergia alata). We solved a crystal structure of the T. alata desaturase at 2.05 Å resolution. Using molecular dynamics (MD) simulations, we identified a low-energy complex between 16:0-ACP and the desaturase that would position C6 and C7 of the acyl chain adjacent to the diiron active site. The model complex was used to identify mutant variants that could convert the T. alata Δ6 desaturase to Δ9 regioselectivity. Additional modeling between ACP and the mutant variants confirmed the predicted regioselectivity. To validate the in-silico predictions, we synthesized two variants of the T. alata desaturase and analyzed their reaction products using gas chromatography-coupled mass spectrometry. Assay results confirmed that mutants designed to convert T. alata Δ6 to Δ9 selectivity exhibited the predicted changes. In complementary experiments, variants of the castor desaturase designed to convert Δ9 to Δ6 selectivity lost some of their Δ9 desaturation ability and gained the ability to desaturate at the Δ6 position. The computational workflow for revealing the mechanistic understanding of regioselectivity presented herein lays a foundation for designing acyl-ACP desaturases with novel selectivities to increase the diversity of monoenes available for bioproduct applications., (Published by Oxford University Press on behalf of American Society of Plant Biologists 2021. This work is written by US Government employees and is in the public domain in the US.)

Background: Surgical site infection is a major source of morbidity in patients undergoing pancreatic head resection and is often from organisms in intraoperative bile duct cultures. As such, many institutions use prolonged prophylactic antibiotics and tailor based on bile duct cultures. However, standard cultures take days, leaving many patients unnecessarily on prolonged antibiotics. Nanopore sequencing can provide data in hours and, thus, has the potential to improve antibiotic stewardship. The present study investigates the feasibility of nanopore sequencing in intraoperative bile samples., Methods: Patients undergoing pancreatic head resection were included. Intra-operative bile microbial profiles were determined with standard cultures and nanopore sequencing. Antibiotic recommendations were generated, and time-to-results determined for both methods. Organism yields, resistance patterns, antibiotic recommendations, and costs were compared., Results: Out of 42 patients, 22 (52%) had samples resulting in positive standard cultures. All positive standard cultures had microbes detected using nanopore sequencing. All 20 patients with negative standard cultures had negative nanopore sequencing. Nanopore sequencing detected more bacterial species compared to standard cultures (10.5 vs 4.4, p < 0.05) and more resistance genotypes (10.3 vs 2.7, p < 0.05). Antimicrobial recommendations based on nanopore sequencing provided coverage for standard cultures in 27 out of 44 (61%) samples, with broader coverage recommended by nanopore sequencing in 13 out of 27 (48%) of these samples. Nanopore sequencing results were faster (8 vs 98 hours) than standard cultures but had higher associated costs ($165 vs $38.49)., Conclusion: Rapid microbial profiling with nanopore sequencing is feasible with broader organism and resistance profiling compared to standard cultures. Nanopore sequencing has perfect negative predictive value and can potentially improve antibiotic stewardship; thus, a randomized control trial is under development., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Surgical site infections (SSI) are a significant burden to patients and health care systems. We evaluated the use of Nanopore sequencing (NS) to rapidly detect microbial species and antimicrobial resistance (AMR) genes present in intraoperative bile aspirates. Bile aspirates from 42 patients undergoing pancreatic head resection were included. Three methods of DNA extraction using mechanical cell lysis or protease cell lysis were compared to determine the optimum method of DNA extraction. The impact of host DNA depletion, sequence run duration, and use of different AMR gene databases was also assessed. To determine clinical value, NS results were compared to standard culture (SC) results. NS identified microbial species in all culture positive samples. Mechanical lysis improved NS detection of cultured species from 60% to 76%, enabled detection of fungal species, and increased AMR predictions. Host DNA depletion improved detection of streptococcal species and AMR correlation with SC. Selection of AMR database influenced the number of AMR hits and resistance profile of 13 antibiotics. AMR prediction using CARD and ResFinder 4.1 correctly predicted 79% and 81% of the bile antibiogram, respectively. Sequence run duration positively correlated with detection of AMR genes. A minimum of 6 h was required to characterize the biliary microbes, resulting in a turnaround time of 14 h. Rapid identification of microbial species and AMR genes can be achieved by NS. NS results correlated with SC, suggesting that NS may be useful in guiding early antimicrobial therapy postsurgery. IMPORTANCE Surgical site infections (SSI) are a significant burden to patients and health care systems. They increase mortality rates, length of hospital stays, and associated health care costs. To reduce the risk of SSI, surgical patients are administered broad-spectrum antibiotics that are later adapted to target microbial species detected at the site of surgical incision. Use of broad-spectrum antibiotics can be harmful to the patient. We wanted to develop a rapid method of detecting microbial species and their antimicrobial resistance phenotypes. We developed a method of detecting microbial species and predicting resistance phenotypes using Nanopore sequencing. Results generated using Nanopore sequencing were similar to current methods of detection but were obtained in a significantly shorter amount of time. This suggests that Nanopore sequencing could be used to tailor antibiotics in surgical patients and reduce use of broad-spectrum antibiotics.

Background: Schizophrenia is a persistent psychotic disorder often accompanied by severe disability and premature mortality. New pharmacological treatments are urgently needed. Sodium benzoate, a common food preservative holds potential to be an effective, accessible treatment for schizophrenia, though the optimal dosing and mechanism of action of the compound requires further investigation., Methods: Individuals with persistent treatment-refractory schizophrenia (n=52) will be recruited. Patients will be randomised in a 1:1:1:1 ratio to receive treatment of one of three active doses (1000, 2000 or 4000 mg daily) of sodium benzoate or placebo for 6 weeks duration. The primary outcome measurement is change in the Positive and Negative Syndrome Scale (PANSS) total score. Secondary outcome measurements are PANSS subscales, Global Assessment of Function (GAF), Clinical Global Impression (CGI) and Patient Global Impression (PGI-I). Change in concentrations of peripheral amino acids (D-alanine, L-alanine, D-serine, L-serine, glycine and glutamate), plasma sodium benzoate, plasma catalase, 3-nitrotyrosine, malondialdehyde and high-sensitivity C-reactive protein (hs-CRP) will be determined as tertiary measures., Discussion: This trial seeks to build upon previous research indicating potential efficacy of sodium benzoate for reduction of symptoms in individuals with treatment-refractory schizophrenia. The trial aims to improve the understanding of the mechanism of action of the compound., Trial Registration: Australian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12621000327886 . Registered on 23 March 2021., (© 2021. The Author(s).)

Three different functionalities have been incorporated into mesoporous materials by means of a coupling reaction with the siloxanes 3-glycidoxypropyl-trimethoxysilane (GLYMO), 3-methacryloxypropyl-trimethoxysilane (MEMO), and 3-mercaptopropyl-trimethoxysilane (MPTMS). The disposition of the different functional groups, as well as the interaction mechanism, with the mesoporous substrate has been identified. The amount of the antiviral drug acyclovir (ACV) adsorbed depends not only on the available surface area but also on the chemical or physicochemical interactions between functionalities. The drug adsorption isotherm of the materials functionalized with GLYMO and MPTMS follow mechanisms dependent on the different surface coverage and the possibilities to establish physicochemical interactions between the drug molecule and the functionalities. On the contrary, when functionalizing with MEMO, the dominant adsorption mechanism is characteristic of chemically bonded adsorbates. The ACV release kinetics is best fitted to the Weibull model in all the functionalized materials. When the MTPMS is used as a functionalizing agent, the drug diffusion occurs at low kinetics and homogeneously along the mesoporous channels.

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.

Management of congenital adrenal hyperplasia (CAH) requires both glucocorticoid replacement and suppression of adrenal androgen synthesis. It is recommended that children with CAH be treated with hydrocortisone, but the appropriate glucocorticoid regimen in adults is uncertain. In order to review the outcomes of different glucocorticoid regimens in the management of CAH, a systematic search of PubMed/MEDLINE and Web of Science was conducted, including reports published up to 25 February 2019. Studies that compared at least two types of glucocorticoid preparation were included. The following information was extracted from each study: first author, year of publication, number and characteristics of patients and control subjects, types and doses of glucocorticoid regimen used, study design and outcomes [ e.g., biochemical tests, weight, height, body mass index (BMI), bone mineral density (BMD)]. A total of 23 studies were included in the qualitative synthesis, with 19 included in the quantitative synthesis. Dexamethasone was associated with the greatest degree of adrenal suppression; there was no significant difference in 17-hydroxyprogesterone (17OHP) and androstenedione levels between patients treated with hydrocortisone or prednisolone. Patients treated with dexamethasone had the lowest BMD and the highest BMI. Although dexamethasone therapy is associated with significantly lower 17OHP and androstenedione levels, it is also associated with more adverse effects. There do not appear to be significant differences between hydrocortisone and prednisolone therapy, and the choice of agent should be based on individual patient factors.

The term microbiome describes the genetic material encoding the various microbial populations that inhabit our body. Whilst colonization of various body niches (e.g., the gut) by dynamic communities of microorganisms is now universally accepted, the existence of microbial populations in other "classically sterile" locations, including the blood, is a relatively new concept. The presence of bacteria-specific DNA in the blood has been reported in the literature for some time, yet the true origin of this is still the subject of much deliberation. The aim of this study was to investigate the phenomenon of a "blood microbiome" by providing a comprehensive description of bacterially derived nucleic acids using a range of complementary molecular and classical microbiological techniques. For this purpose we utilized a set of plasma samples from healthy subjects ( n = 5) and asthmatic subjects ( n = 5). DNA-level analyses involved the amplification and sequencing of the 16S rRNA gene. RNA-level analyses were based upon the de novo assembly of unmapped mRNA reads and subsequent taxonomic identification. Molecular studies were complemented by viability data from classical aerobic and anaerobic microbial culture experiments. At the phylum level, the blood microbiome was predominated by Proteobacteria , Actinobacteria , Firmicutes , and Bacteroidetes . The key phyla detected were consistent irrespective of molecular method (DNA vs. RNA), and consistent with the results of other published studies. In silico comparison of our data with that of the Human Microbiome Project revealed that members of the blood microbiome were most likely to have originated from the oral or skin communities. To our surprise, aerobic and anaerobic cultures were positive in eight of out the ten donor samples investigated, and we reflect upon their source. Our data provide further evidence of a core blood microbiome, and provide insight into the potential source of the bacterial DNA/RNA detected in the blood. Further, data reveal the importance of robust experimental procedures, and identify areas for future consideration.

Background: Psychological problems are frequently comorbid with diabetes and can be complex and time consuming to manage in general practice. They can also complicate diabetes management., Objective: This pre-post, mixed-methods study examined whether a diabetes-specific online module, integrated into an existing e-mental health program, is acceptable to patients and helps to improve psychological wellbeing., Methods: The study was conducted in three stages. First, qualitative information was obtained from consumers and practitioners to inform module content. Second, clinical content for the module was developed, including psychoeducational material and clinical content. In the final stage, acceptability and feasibility was evaluated in a group of patients with diabetes., Results: Patients' satisfaction with and acceptance of the module was high. Significant improvement in mental health and diabetes outcomes was also observed., Discussion: Preliminary evidence suggests that the online diabetes module may be a useful psychological support for patients with diabetes. Further controlled investigation is warranted.

Objectives: Treatment of acute severe colitis (ASC) has been associated with high morbidity and high colectomy rate in children. In the prebiologics era, our centre used short-term high-dose intravenous corticosteroids (IVCS) at 2 to 30 mg · kg · day. We conducted a retrospective review to compare efficacy of different dosing regimes of IVCS., Methods: Thirty-four children treated with IVCS for ASC were included over 8 years. Patients were studied as 2 groups with similar pretreatment patient characteristics. Group 1 (standard dose) received IVCS at 2 mg · kg · day and group 2 (high dose) received IVCS at 10 to 30 mg · kg · day. Safety, efficacy, and follow-up of the entire cohort for >1 year were studied. The median IVCS dose in the standard- and high-dose cohort was 1.5 mg · kg · day (maximum 60 mg · kg · day) and 24.8 mg · kg · day (maximum 1000 mg · kg · day), respectively., Results: Pediatric Ulcerative Colitis Activity Index scores at day 5 were significantly lower in high-dose (15, interquartile range 8.5-20) than in standard-dose IVCS (30, interquartile range 20-30). IVCS side effects were minor and reversible. Overall, medical salvage therapy was required in 5.8% (2 children) before discharge, and in 17% (6 children) at follow-up after 1 year. The colectomy rate of the entire cohort was remarkably low with 0% during admission and 11% (4 children) after 1 year, with a trend of less colectomies in high-dose (4.8%-1 child) than in standard-dose (23%-3 children)., Conclusions: Our data show that in paediatric ASC, the short-term use of high-dose IVCS is safe and effective. Prospective studies are needed to define the role of IVCS within salvage therapy protocols.

Mimicry illustrates the power of selection to produce phenotypic convergence in biology [1]. A striking example is the imitation of female insects by plants that are pollinated by sexual deception of males of the same insect species [2-4]. This involves mimicry of visual, tactile, and chemical signals of females [2-7], especially their sex pheromones [8-11]. The Mediterranean orchid Ophrys exaltata employs chemical mimicry of cuticular hydrocarbons, particularly the 7-alkenes, in an insect sex pheromone to attract and elicit mating behavior in its pollinators, males of the cellophane bee Colletes cunicularius [11-13]. A difference in alkene double-bond positions is responsible for reproductive isolation between O. exaltata and closely related species, such as O. sphegodes [13-16]. We show that these 7-alkenes are likely determined by the action of the stearoyl-acyl-carrier-protein desaturase (SAD) homolog SAD5. After gene duplication, changes in subcellular localization relative to the ancestral housekeeping desaturase may have allowed proto-SAD5's reaction products to undergo further biosynthesis to both 7- and 9-alkenes. Such ancestral coproduction of two alkene classes may have led to pollinator-mediated deleterious pleiotropy. Despite possible evolutionary intermediates with reduced activity, amino acid changes at the bottom of the substrate-binding cavity have conferred enzyme specificity for 7-alkene biosynthesis by preventing the binding of longer-chained fatty acid (FA) precursors by the enzyme. This change in desaturase function enabled the orchid to perfect its chemical mimicry of pollinator sex pheromones by escape from deleterious pleiotropy, supporting a role of pleiotropy in determining the possible trajectories of adaptive evolution., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objectives: Men are almost two times more likely to die by suicide than women, yet little research has focused on what is required to prevent suicide among men. This paper aims to investigate what factors interrupt suicidal behaviour in men, and to examine differences according to known suicide risk factors., Setting: Australia., Participants: 251 Australian men aged 18 years and over who had made a suicide attempt 6-18 months prior to completing the survey., Outcomes: The survey canvassed the language men use to describe their depression and suicidality, warning signs, barriers to accessing help and what is needed to interrupt a suicide attempt. ORs and χ(2) were used to test for differences by age, geographic location and current depression severity., Results: Of 299 men screened and eligible to participate, 251 completed all or part of the survey. Participants identified different words and warning signs for depression compared with suicidality. The most commonly endorsed barriers to accessing help were not wanting to burden others (66%) and having isolated themselves (63%). Men overwhelmingly endorsed 'I thought about the consequences for my family' as the factor which stopped a suicide attempt (67%). 'I need support from someone I really trust and respect' was also strongly endorsed. There were few differences by age, region or depression severity., Conclusions: Participants were able to identify signs, albeit often subtle ones, that they were becoming depressed or suicidal. Similarly, most were able to identify active strategies to interrupt this downward spiral. Men wanted others to notice changes in their behaviour, and to approach them without judgement., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Despite higher rates of suicide in men, there is a dearth of research examining the perspectives and experiences of males at risk of suicide, particularly in terms of understanding how interventions can be tailored to men's specific needs. The current study aimed to examine factors assisting, complicating or inhibiting interventions for men at risk, as well as outlining the roles of family, friends and others in male suicide prevention. Thirty-five male suicide survivors completed one-to-one interviews, and forty-seven family and friends of male suicide survivors participated in eight focus groups. Thematic analysis revealed five major themes: (1) development of suicidal behaviours tends to follow a common path associated with specific types of risk factors (disrupted mood, unhelpful stoic beliefs and values, avoidant coping strategies, stressors), (2) men at risk of suicide tend to systematically misinterpret changes in their behaviour and thinking, (3) understanding mood and behavioural changes in men enables identification of opportunities to interrupt suicide progression, (4) distraction, provision of practical and emotional supports, along with professional intervention may effectively interrupt acute risk of harm, and (5) suicidal ideation may be reduced through provision of practical help to manage crises, and helping men to focus on obligations and their role within families. Findings suggest that interventions for men at risk of suicidal behaviours need to be tailored to specific risk indicators, developmental factors, care needs and individuals' preferences. To our knowledge this is the first qualitative study to explore the experiences of both suicidal men and their family/friends after a suicide attempt, with the view to improve understanding of the processes which are effective in interrupting suicide and better inform interventions for men at risk.

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., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Acoustic sensitivity of the vestibular apparatus is well-established, but the contribution of vestibular receptors to the late auditory evoked potentials of cortical origin is unknown. Evoked potentials from 500 Hz tone pips were recorded using 70 channel EEG at several intensities below and above the vestibular acoustic threshold, as determined by vestibular evoked myogenic potentials (VEMPs). In healthy subjects both auditory mid- and long-latency auditory evoked potentials (AEPs), consisting of Na, Pa, N1 and P2 waves, were observed in the sub-threshold conditions. However, in passing through the vestibular threshold, systematic changes were observed in the morphology of the potentials and in the intensity dependence of their amplitude and latency. These changes were absent in a patient without functioning vestibular receptors. In particular, for the healthy subjects there was a fronto-central negativity, which appeared at about 42 ms, referred to as an N42, prior to the AEP N1. Source analysis of both the N42 and N1 indicated involvement of cingulate cortex, as well as bilateral superior temporal cortex. Our findings are best explained by vestibular receptors contributing to what were hitherto considered as purely auditory evoked potentials and in addition tentatively identify a new component that appears to be primarily of vestibular origin., (Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.)

Prenatal alcohol exposure may have adverse effects on the developing foetus resulting in significant growth restriction, characteristic craniofacial features, and central nervous system dysfunction. The toxic effects of alcohol on the developing brain are well recognised. However, little is known about the effects of alcohol on the developing gastrointestinal tract or their mechanism. There are few case reports showing an association between foetal alcohol syndrome and gastrointestinal neuropathy. We report a rare association between foetal alcohol syndrome and severe gastrooesophageal reflux disease in an infant who ultimately required fundoplication to optimise her growth and nutrition. The child had failed to respond to maximal medical treatment (domperidone and omeprazole), high calorie feeds, PEG feeding, or total parenteral nutrition. The effect of alcohol on the developing foetus is not limited to the central nervous system but also can have varied and devastating effects on the gastrointestinal tract.

Regiospecific desaturation of long-chain saturated fatty acids has been described as approaching the limits of the discriminatory power of enzymes because the substrate entirely lacks distinguishing features close to the site of dehydrogenation. To identify the elusive mechanism underlying regioselectivity, we have determined two crystal structures of the archetypal Δ9 desaturase from castor in complex with acyl carrier protein (ACP), which show the bound ACP ideally situated to position C9 and C10 of the acyl chain adjacent to the diiron active site for Δ9 desaturation. Analysis of the structures and modeling of the complex between the highly homologous ivy Δ4 desaturase and ACP, identified a residue located at the entrance to the binding cavity, Asp280 in the castor desaturase (Lys275 in the ivy desaturase), which is strictly conserved within Δ9 and Δ4 enzymes but differs between them. We hypothesized that interaction between Lys275 and the phosphate of the pantetheine, seen in the ivy model, is key to positioning C4 and C5 adjacent to the diiron center for Δ4 desaturation. Mutating castor Asp280 to Lys resulted in a major shift from Δ9 to Δ4 desaturation. Thus, interaction between desaturase side-chain 280 and phospho-serine 38 of ACP, approximately 27 Å from the site of double-bond formation, predisposes ACP binding that favors either Δ9 or Δ4 desaturation via repulsion (acidic side chain) or attraction (positively charged side chain), respectively. Understanding the mechanism underlying remote control of regioselectivity provides the foundation for reengineering desaturase enzymes to create designer chemical feedstocks that would provide alternatives to those currently obtained from petrochemicals.

The orchids Ophrys sphegodes and O. exaltata are reproductively isolated from each other by the attraction of two different, highly specific pollinator species. For pollinator attraction, flowers chemically mimic the pollinators' sex pheromones, the key components of which are alkenes with different double-bond positions. This study identifies genes likely involved in alkene biosynthesis, encoding stearoyl-acyl carrier protein (ACP) desaturase (SAD) homologs. The expression of two isoforms, SAD1 and SAD2, is flower-specific and broadly parallels alkene production during flower development. SAD2 shows a significant association with alkene production, and in vitro assays show that O. sphegodes SAD2 has activity both as an 18:0-ACP Δ(9) and a 16:0-ACP Δ(4) desaturase. Downstream metabolism of the SAD2 reaction products would give rise to alkenes with double-bonds at position 9 or position 12, matching double-bond positions observed in alkenes in the odor bouquet of O. sphegodes. SAD1 and SAD2 show evidence of purifying selection before, and positive or relaxed purifying selection after gene duplication. By contributing to the production of species-specific alkene bouquets, SAD2 is suggested to contribute to differential pollinator attraction and reproductive isolation among these species. Taken together, these data are consistent with the hypothesis that SAD2 is a florally expressed barrier gene of large phenotypic effect and, possibly, a genic target of pollinator-mediated selection.

Plant oils containing ω-7 fatty acids (FAs; palmitoleic 16:1Δ(9) and cis-vaccenic 18:1Δ(11)) have potential as sustainable feedstocks for producing industrially important octene via metathesis chemistry. Engineering plants to produce seeds that accumulate high levels of any unusual FA has been an elusive goal. We achieved high levels of ω-7 FA accumulation by systematic metabolic engineering of Arabidopsis (Arabidopsis thaliana). A plastidial 16:0-ACP desaturase has been engineered to convert 16:0 to 16:1Δ(9) with specificity >100-fold than that of naturally occurring paralogs, such as that from cat's claw vine (Doxantha unguis-cati). Expressing this engineered enzyme (Com25) in seeds increased ω-7 FA accumulation from <2% to 14%. Reducing competition for 16:0-ACP by down-regulating the β-ketoacyl-ACP synthase II 16:0 elongase further increased accumulation of ω-7 FA to 56%. The level of 16:0 exiting the plastid without desaturation also increased to 21%. Coexpression of a pair of fungal 16:0 desaturases in the cytosol reduced the 16:0 level to 11% and increased ω-7 FA to as much as 71%, equivalent to levels found in Doxantha seeds.

The stereochemistry of castor stearoyl-ACP Delta(9) desaturase-mediated 10-sulfoxidation has been determined. This was accomplished by (19)F NMR analysis of a fluorine-tagged product, 18-fluoro-10-thiastearoyl ACP S-oxide, in combination with a chiral solvating agent, (R)-AMA. Sulfoxidation proceeds with the same stereoselectivity as hydrogen removal from the parent stearoyl substrate. These data validate the use of thia probes to determine the stereochemistry and cryptoregiochemistry of desaturase-mediated oxidations.

The multifunctional acyl-acyl carrier protein (ACP) desaturase from Hedera helix (English ivy) catalyzes the Delta(4) desaturation of 16:0-ACP and the Delta(9) desaturation of 18:0-ACP and further desaturates Delta(9)-16:1 or Delta(9)-18:1 to the corresponding Delta(4,9) dienes. The crystal structure of the enzyme has been solved to 1.95 A resolution, and both the iron-iron distance of approximately 3.2A and the presence of a mu-oxo bridge reveal this to be the only reported structure of a desaturase in the oxidized FeIII-FeIII form. Significant differences are seen between the oxidized active site and the reduced active site of the Ricinus communis (castor) desaturase; His(227) coordination to Fe2 is lost, and the side chain of Glu(224), which bridges the two iron ions in the reduced structure, does not interact with either iron. Although carboxylate shifts have been observed on oxidation of other diiron proteins, this is the first example of the residue moving beyond the coordination range of both iron ions. Comparison of the ivy and castor structures reveal surface amino acids close to the annulus of the substrate-binding cavity and others lining the lower portion of the cavity that are potential determinants of their distinct substrate specificities. We propose a hypothesis that differences in side chain packing explains the apparent paradox that several residues lining the lower portion of the cavity in the ivy desaturase are bulkier than their equivalents in the castor enzyme despite the necessity for the ivy enzyme to accommodate three more carbons beyond the diiron site.

The stereochemistry of palmitoyl-ACP Delta(4) desaturase-mediated dehydrogenation has been examined by tracking the fate of deuterium atoms located on stereospecifically monodeuterated substrates-(4S)- and (4R)-[4-(2)H(1)]-palmitoyl-ACP and (5S)- and (5R)-[5-(2)H(1)]-palmitoyl-ACP. It was found that the introduction of the (Z)-double bond between C-4 and C-5 of a palmitoyl substrate occurs with pro-R enantioselectivity-a result which matches that obtained for a closely related homolog-castor stearoyl-ACP Delta(9) desaturase. These data show that despite the difference in regioselectivity between the two enzymes, the stereochemistry of hydrogen removal is conserved.

In plants, changes in the levels of oleic acid (18:1), a major monounsaturated fatty acid (FA), results in the alteration of salicylic acid (SA)- and jasmonic acid (JA)-mediated defense responses. This is evident in the Arabidopsis ssi2/fab2 mutant, which encodes a defective stearoyl-acyl carrier protein-desaturase (S-ACP-DES) and consequently accumulates high levels of stearic acid (18:0) and low levels of 18:1. In addition to SSI2, the Arabidopsis genome encodes six S-ACP-DES-like enzymes, the native expression levels of which are unable to compensate for a loss-of-function mutation in ssi2. The presence of low levels of 18:1 in the fab2 null mutant indicates that one or more S-ACP-DES isozymes contribute to the 18:1 pool. Biochemical assays show that in addition to SSI2, four other isozymes are capable of desaturating 18:0-ACP but with greatly reduced specific activities, which likely explains the inability of these SSI2 isozymes to substitute for a defective ssi2. Lines containing T-DNA insertions in S-ACP-DES1 and S-ACP-DES4 show that they are altered in their lipid profile but contain normal 18:1 levels. However, overexpression of the S-ACP-DES1 isoform in ssi2 plants results in restoration of 18:1 levels and thereby rescues all ssi2-associated phenotypes. Thus, high expression of a low specific activity S-ACP-DES is required to compensate for a mutation in ssi2. Transcript level of S-ACP-DES isoforms is reduced in high 18:1-containing plants. Enzyme activities of the desaturase isoforms in a 5-fold excess of 18:1-ACP show product inhibition of up to 73%. Together these data indicate that 18:1 levels are regulated at both transcriptional and post-translational levels.

Sequence analysis of the diiron cluster-containing soluble desaturases suggests they are unrelated to other diiron enzymes; however, structural alignment of the core four-helix bundle of desaturases to other diiron enzymes reveals a conserved iron binding motif with similar spacing in all enzymes of this structural class, implying a common evolutionary ancestry. Detailed structural comparison of the castor desaturase with that of a peroxidase, rubrerythrin, shows remarkable conservation of both identity and geometry of residues surrounding the diiron center, with the exception of residue 199. Position 199 is occupied by a threonine in the castor desaturase, but the equivalent position in rubrerythrin contains a glutamic acid. We previously hypothesized that a carboxylate in this location facilitates oxidase chemistry in rubrerythrin by the close apposition of a residue capable of facilitating proton transfer to the activated oxygen (in a hydrophobic cavity adjacent to the diiron center based on the crystal structure of the oxygen-binding mimic azide). Here we report that desaturase mutant T199D binds substrate but its desaturase activity decreases by approximately 2 x 10(3)-fold. However, it shows a >31-fold increase in peroxide-dependent oxidase activity with respect to WT desaturase, as monitored by single-turnover stopped-flow spectrometry. A 2.65-A crystal structure of T199D reveals active-site geometry remarkably similar to that of rubrerythrin, consistent with its enhanced function as an oxidase enzyme. That a single amino acid substitution can switch reactivity from desaturation to oxidation provides experimental support for the hypothesis that the desaturase evolved from an ancestral oxidase enzyme.

1H-decoupled 19F NMR has been used to monitor the highly regioselective oxidation of a fluorine-tagged thia-fatty acid derivative by castor stearoyl-ACP delta9 desaturase. The major enzymatic product, after reductive work-up, was identified as 9-fluoro-1-nonanol. This compound could be easily distinguished from substrate and a 9-sulfoxy by-product on the basis of its 19F NMR chemical shift and spiking experiments using authentic standards. Structural assignment of the cleavage product was confirmed by GC-MS analysis of the enzymatic products., (2006 John Wiley & Sons, Ltd.)

A desaturase with 83% sequence identity to the coriander delta(4)-16:0-ACP desaturase was isolated from developing seeds of Hedera helix (English ivy). Expression of the ivy desaturase in Arabidopsis resulted in the accumulation of 16:1delta(4) and its expected elongation product 18:1delta(6) (petroselinic acid). Expression in Escherichia coli resulted in the accumulation of soluble, active protein that was purified to apparent homogeneity. In vitro assays confirmed delta(4) desaturation with 16:0-ACP; however, with 18:0-acyl acyl carrier protein (ACP) desaturation occurred at the delta(9) position. The ivy desaturase also converted 16:1delta(9)-ACP and 18:1delta(9)-ACP to the corresponding delta(4,9) dienes. These data suggest at least two distinct substrate binding modes, one placing C4 at the diiron active site and the other placing C9 at the active site. In the latter case, 18:0 would likely bind in an extended conformation as described for the castor desaturase with 9-carbons accommodated in the cavity beyond the dirron site. However, delta(4) desaturation would require the accommodation of 12 carbons for C16 substrates or 14 carbons for C18 substrates. The amino acids lining the substrate binding cavity of ivy and castor desaturases are conserved except for T117R and P179I (castor/ivy). Paradoxically, both substitutions, when introduced into the castor desaturase, favored the binding of shorter acyl chains. Thus, it seems likely that delta(4) desaturation would require a non-extended, perhaps U-shaped, substrate conformation. A cis double bond may facilitate the initiation of such a non-extended conformation in the monounsaturated substrates. The multifunctional properties of the ivy desaturase make it well suited for further dissection of the determinants of regiospecificity.

Subjective effects such as stinging, itching and burning commonly occur in the absence of any visible irritation and give rise to discomfort, which may be enough to deter an individual from using even the most effective of skin care products. The purpose of this study was to evaluate the sensitivity of different anatomical regions of the face to determine which region displayed the most intense stinging response to the application of lactic acid. The effect of occlusion on the level of response was also investigated. 45 volunteers were treated with 10% lactic acid on the nasolabial fold, forehead, chin and cheek, occluded and unoccluded for 8 min. Sensory reactions were recorded at 2.5, 5 and 8 min. The response levels on the occluded sites were always significantly lower than on the unoccluded sites, despite the dose per unit area being comparable. Females showed a trend towards being more sensitive to the subjective effects elicited by lactic acid than males, but these results were not conclusive. Interestingly, there was not a complete correlation between individuals who reacted on the nasolabial fold and the other sites, particularly the forehead. A positive stinging response on the nasolabial fold may not necessarily predict subjective responses to a product when used on other areas of the face.

Pseudomonas oleovorans alkane omega-hydroxylase (AlkB) is an integral membrane diiron enzyme that shares a requirement for iron and oxygen for activity in a manner similar to that of the non-heme integral membrane desaturases, epoxidases, acetylenases, conjugases, ketolases, decarbonylase and methyl oxidases. No overall sequence similarity is detected between AlkB and these desaturase-like enzymes by computer algorithms; however, they do contain a series of histidine residues in a similar relative positioning with respect to hydrophobic regions thought to be transmembrane domains. To test whether these conserved histidine residues are functionally equivalent to those of the desaturase-like enzymes we used scanning alanine mutagenesis to test if they are essential for activity of AlkB. These experiments show that alanine substitution of any of the eight conserved histidines results in complete inactivation, whereas replacement of three non-conserved histidines in close proximity to the conserved residues, results in only partial inactivation. These data provide the first experimental support for the hypotheses: (i) that the histidine motif in AlkB is equivalent to that in the desaturase-like enzymes and (ii) that the conserved histidine residues play a vital role such as coordinating the Fe ions comprising the diiron active site.

Exchanging the identity of amino acids at four key locations within the Arabidopsis thaliana oleate desaturase (FAD2) and the Lesquerella fendleri hydroxylase/desaturase (LFAH) was shown to influence partitioning between desaturation and hydroxylation (Broun, P., Shanklin, J., Whittle, E., and Somerville, C. (1998) Science 282, 1315-1317). We report that four analogous substitutions in the FAD2 sequence by their equivalents from the castor oleate hydroxylase result in hydroxy fatty acid accumulation in A. thaliana to the same levels as for the wild-type castor hydroxylase. We also describe the relative contribution of these substitutions, both individually and in combination, by analyzing the products resulting from their expression in A. thaliana and/or Saccharomyces cerevisiae. Yeast expression showed that M324V, a change reachable by a single point mutation, altered the product distribution approximately 49-fold, and that residue 148 is also a predominant determinant of reaction outcome. Comparison of residues at position 148 of FAD2, LFAH, and the Ricinus oleate hydroxylase prompted us to rationally engineer LFAH-N149I, a variant with approximately 1.9-fold increase in hydroxylation specificity compared with that of wild-type LFAH. Control experiments showed that the wild-type Arabidopsis thaliana FAD2 desaturase has inherent, low level, hydroxylation activity. Further, fatty acid desaturases from different kingdoms and with different regiospecificities exhibit similar intrinsic hydroxylase activity, underscoring fundamental mechanistic similarities between desaturation and hydroxylation. For LFAH mutants the hydroxylation:desaturation ratio is 5-9-fold higher for 18-carbon versus 16-carbon substrates, supporting our hypothesis that substrate positioning in the active site plays a key role in the partitioning of catalytic specificity.

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.

There is a widespread, but largely unsubstantiated, view that certain skin types may be more susceptible to the effect of skin irritants than others. One expression of this would be that certain ethnic groups may also be more likely to experience skin irritation. As a consequence, when evaluating the skin compatibility of substances/preparations, these differences may need to be taken into account. However, other evidence indicates that, within any particular group, inter-individual variation is likely to be much larger than the differences between means for distinct groups. In this study, we have investigated 2 carefully matched panels of Caucasian and Japanese women volunteers to determine their topical irritant reaction, both acute and cumulative, to a range of materials. The results indicated that the acute irritant response tended to be greater in the Japanese panel and this reached statistical significance with the stronger irritants. Cumulative irritation was investigated only with the weaker irritants and, although again the trend was to a higher response in Japanese compared to Caucasian panelists, this rarely reached significance. Nevertheless, where risk assessment for skin irritation was critical, then at the population level, these differences might be relevant, both for safety in use of substances and products and for skin acceptability.

Salicylic acid (SA) plays an important role in activating various plant defense responses, including expression of the pathogenesis-related (PR) genes and systemic acquired resistance. A critical positive regulator of the SA signaling pathway in Arabidopsis is encoded by the NPR1 gene. However, there is growing evidence that NPR1-independent pathways can also activate PR expression and disease resistance. To elucidate the components associated with NPR1-independent defense signaling, we isolated a suppressor of the npr1-5 allele, designated ssi2. The recessive ssi2 mutation confers constitutive PR gene expression, spontaneous lesion formation, and enhanced resistance to Peronospora parasitica. In contrast, a subset of defense responses regulated by the jasmonic acid (JA) signaling pathway, including expression of the defensin gene PDF1.2 and resistance to Botrytis cinerea, is impaired in ssi2 plants. With the use of a map-based approach, the SSI2 gene was cloned and shown to encode a stearoyl-ACP desaturase (S-ACP DES). S-ACP DES is an archetypical member of a family of soluble fatty acid (FA) desaturases; these enzymes play an important role in regulating the overall level of desaturated FAs in the cell. The activity of mutant S-ACP DES enzyme was reduced 10-fold, resulting in elevation of the 18:0 FA content in ssi2 plants. Because reduced S-ACP DES activity leads to the induction of certain defense responses and the inhibition of others, we propose that a FA-derived signal modulates crosstalk between different defense signaling pathways.

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.

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.

When benzalkonium chloride (BKC), a cationic surfactant, is added to sodium dodecyl sulfate (SDS), an anionic surfactant, and used in patch testing, on the basis of their known physicochemical interaction, it is possible to predict that there will be a tendency towards a reduction in the expected irritant response when compared to SDS alone. The aim of this study was to investigate whether BKC could reduce the irritant response to SDS when applied after the SDS exposure. 54 non-atopic adult volunteers were recruited for the study. 20% SDS was applied for 2 h under occlusion. 1% BKC was then applied to the same site. Various controls, including SDS application followed by water for 2 h, were included. The irritant reaction was assessed at 24 h and 48 h. 40 of the 54 subjects had some reaction when SDS was applied for 2 h followed by either benzalkonium chloride or water control under occlusion. In comparison to water control, where BKC was applied after SDS, 20 of the 40 responders had a weaker reaction but only 4 had a stronger response. This study shows that BKC applied to skin exposed to SDS attenuates the resulting irritant reaction.

Background: A human 4-hour patch test has been developed for evaluating the acute irritation potential of chemicals. This method was developed for comparative irritation assessments. Although skin irritation responses in human subjects can be quite variable, this test method has proven robust in both intra- and interlaboratory tests. However, the previous interlaboratory studies were not optimal in that slightly differing protocols were used and the studies were not controlled for time of year or source of test chemicals. As a result, some variation in acute irritation responses were seen that might have been reduced somewhat had these variables been controlled to a greater extent., Objective: The purpose of the current study was to examine interlaboratory reproducibility of the 4-hour patch test when conducted under as identical as set of test conditions as possible., Methods: Two laboratories conducted a direct comparison study of the acute irritation potential of three structurally related, undiluted fatty acids (octanoic acid, decanoic acid, and dodecanoic acid) in comparison to a benchmark positive control chemical (20% sodium dodecyl sulfate [SDS]). The studies were run within a 4-month period using the same commercial source of test chemicals. Test subjects were treated with each chemical under occluded patch conditions for gradually increasing exposure duration up to 4 hours. The results were then evaluated in terms of total cumulative incidence of positive responses and time response patterns., Results: Using statistical comparisons of the proportion of the subjects with a positive irritant reaction to each substance, the rank order of irritation potential was decanoic acid >/= octanoic acid > SDS >> and dodecanoic acid. The statistical comparisons and the time-response patterns for each chemical were nearly identical at the two laboratories. There were also very similar, and intriguing, variations in the interchemical response patterns seen in the two studies., Conclusion: When conducted under as controlled a set of test conditions as was reasonably possible, this acute irritation protocol shows remarkably high consistency across independent test laboratories, further supporting its continued development and acceptance as a valid and more predictive tool for assessing skin irritation potential.

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.

In the risk assessment of the ability of a substance or preparation to cause skin irritation, a patch test involving human volunteers is often seen as providing definitive information. However, the skin exposure conditions found in an occluded patch may be far removed from those in a particular use situation, not least for such reasons as the high dose per unit area, the duration of exposure and the maceration of the skin. Whilst these factors may enhance sensitivity in the identification of intrinsic hazard, they may do little to ensure accurate safety evaluation for real use. In this paper, we report data from a series of studies with an unmarketed facial skin cosmetic product. Whilst the product was unexpectedly highly irritating in a standard patch test in 30 volunteers, subsequent use tests, including a 6x daily open application to the elbow for 3 weeks and 2x daily application in a half-face test lasting 3 to 4 weeks and involving 52 volunteers failed to show any evidence of skin irritation. It is concluded that the most meaningful results for skin irritation risk assessment are likely to come from studies which involve relevant patterns of exposure.

Adverse skin reactions cover many types of response: toxic, irritant, allergic, urticarial, sensory, etc. The relationships between an individual's tendency to develop different types of skin response are not well-described. We examined whether those who perceive stinging might be more likely to experience urticarial, sensory and irritation reactions in skin. A panel of 86 volunteers was tested with 10% lactic acid in the nasolabial fold to assess their ability to perceive stinging. At the same time, their capacity to develop non-immunologic contact urticaria was evaluated using chemicals of different structural type and urticant ability: methyl nicotinate, benzoic acid, cinnamic acid, cinnamaldehyde and dimethyl sulfoxide (DMSO). DMSO was also used to assess sensory effects and skin irritation. 44 were classes as "stingers" and 42 as "non-stingers". The pattern of urticant reactivity in the stingers and non-stingers was essentially the same, with neat DMSO generating the strongest reactions in both groups. Sensory reactions to DMSO (stinging, itching, tingling or burning) were similar in stingers and non-stingers; although the former may have reacted more quickly, a smaller proportion reacted (64% versus 76%). The skin irritation response to DMSO was also identical in stingers and non-stingers and the intensity of the urticant response in an individual did not correlate with the intensity of their subsequent irritant reaction. In conclusion, this study demonstrated that an individual's ability to perceive skin stinging does not give a general indication of their susceptibility to other types of non-immunologic skin response. Indeed, there appeared to be little evidence of correlations between any of the skin effects studied.