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Development of in vivo confocal Raman spectroscopy (ICRS) methodology over the last 20 years has enabled previously unavailable capability to acquire molecular concentration gradients across the stratum corneum (SC), at the micron level and in a clinical setting. Professor Tony Rawlings has been a driving force in SC research for over 30 years, working with a wide range of teams across the world. Because a detailed knowledge of skin biochemistry was key to interpreting ICRS-acquired molecular concentration gradients, the authors formed a close working relationship with Professor Rawlings during the development of ICRS. This article, therefore, presents a summary of this process and how challenges raised by application of ICRS were tackled, towards the goal of validating the technique for clinical skin measurement., (© 2024 Society of Cosmetic Scientists and Societe Francaise de Cosmetologie.)

Objective: While there are a wide range of approaches for the assessment of skin hydration, it is not always clear how data from them relate to one another or to the skin itself. With the development of in vivo Confocal Raman Spectroscopy (ICRS), it has become possible to measure water concentration as a function of protein/depth within the stratum corneum (SC). This article reports a comparison between electrical skin hydration measures/visual/optical grading and water concentration profiles measured using ICRS, to better understand the relationship between these approaches., Methods: SC hydration of lower-leg skin with varying degrees of dryness was assessed using visual grading (live and from digital images), Corneometer®, Visioscan and ICRS. In addition, a custom fingerprint sensor was used to image surface capacitance (as a surrogate of SC hydration), and SC barrier function was assessed using evaporimetry (to measure trans-epidermal water loss; TEWL)., Results: Significant correlations were observed between a number of different skin grading/measurement approaches and ICRS data. ICRS hydration profiles also revealed a region near the SC surface with a relatively flat water profile in dry skin subjects., Conclusions: The advent of quantitative in vivo analytical techniques such as ICRS, which can be used in a clinical setting, has enabled greater insight into more conventional approaches for assessing skin dryness. While traditional skin grading and biophysical methods for measuring skin hydration have varying degrees of correlation with one another, they also provide comparatively unique information about different regions within the SC. This should enable a more informed approach to product development in the future., (© 2024 Society of Cosmetic Scientists and Societe Francaise de Cosmetologie.)

The properties of milk proteins differ between mammalian species. β-Lactoglobulin (βlg) proteins from caprine and bovine milk are sequentially and structurally highly similar, yet their physicochemical properties differ, particularly in response to pH. To resolve this conundrum, we compared the dynamics of both the monomeric and dimeric states for each homologue at pH 6.9 and 7.5 using hydrogen/deuterium exchange experiments. At pH 7.5, the rate of exchange is similar across both homologues, but at pH 6.9 the dimeric states of the bovine βlg B variant homologue have significantly more conformational flexibility compared with caprine βlg. Molecular dynamics simulations provide a mechanistic rationale for the experimental observations, revealing that variant-specific substitutions encode different conformational ensembles with different dynamic properties consistent with the hydrogen/deuterium exchange experiments. Understanding the dynamic differences across βlg homologues is essential to understand the different responses of these milks to processing, human digestion, and differences in immunogenicity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Objective: Microscopy is widely used during the development and testing of topical formulations; however, it often lacks the ability to be chemically specific with regard to what is being imaged. This article describes how moving outside of the visible light region and into different parts of the ultraviolet (UV) spectrum enables differently UV absorbing components in topical emulsions to be directly visualized using optical transmission microscopy., Methods: Optical transmission microscopy of different sunscreen emulsions was carried out using a custom-built microscope, imaging in the UVB (313 nm), UVA (365 nm) and visible light (546 nm) and with different magnifications., Results: By using light of different wavelengths, direct visualization of different UV absorbing ingredients within the product emulsion using optical transmission microscopy has been performed and the locations of the UV absorbing actives in the formulations imaged., Conclusions: Microscopy has long been a valuable tool for the skin researcher, providing structural information about the products and how they perform. By moving outside of the spectral region of visible light and into the UV, it has been possible for the first time to directly image different SPF ingredients within topical formulations using optical microscopy., (© 2022 Society of Cosmetic Scientists and Societe Francaise de Cosmetologie.)

Plants store triacylglycerides in organelles called oil bodies, which are important fuel sources for germination. Oil bodies consist of a lipid core surrounded by an interfacial single layer membrane of phospholipids and proteins. Oleosins are highly conserved plant proteins that are important for oil body formation, solubilising the triacylglycerides, stabilising oil bodies, and playing a role in mobilising the fuel during the germination process. The domain structure of oleosins is well established, with N- and C-terminal domains that are hydrophilic flanking a long hydrophobic domain that is proposed to protrude into the triacylglyceride core of the oil body. However, beyond this general understanding, little molecular level detail on the structure is available and what is known is disputed. This lack of knowledge limits our understanding of oleosin function and concomitantly our ability to engineer them. Here, we review the state of play in the literature regarding oleosin structure and function, and provide some examples of how oleosins can be used in commercial settings., Competing Interests: Competing interestsThe authors declare no competing interests., (© International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany, part of Springer Nature 2022.)

Objective: Humectants perform essential roles in the formulation of topical moisturizing products in terms of delivery of active ingredients, consumer experience and biophysical behaviour. How they retain and release water is key to understanding their behaviour., Methods: Dynamic vapour sorption (DVS) was used to monitor the dehydration kinetics of three humectants widely used in topical formulations-glycerine, dexpanthenol and urea. Model aqueous solutions with concentrations of 20% w/w were tested and compared against pure deionized water., Results: The three humectants varied in their ability to retain water during the dehydration process. Dexpanthenol was able to retain water most efficiently during the latter stages of dehydration. Urea demonstrated evidence of crystallization during the final stage of water loss, which was not shown by glycerine or dexpanthenol., Conclusions: Humectants perform vital roles in the formulation of consumer acceptable topical products including the delivery of actives to the skin. Their ability to influence water movement in the skin is also essential for the maintenance of stratum corneum flexibility. DVS assessment of aqueous solutions has demonstrated how the behaviour of three commonly used humectants differs. Knowledge of the mechanisms by which these humectants operate enables the formulator to develop topical products optimized for the roles for which they are intended., (© 2021 Society of Cosmetic Scientists and the Société Française de Cosmétologie.)

Angiotensinogen fine-tunes the tightly controlled activity of the renin-angiotensin system by modulating the release of angiotensin peptides that control blood pressure. One mechanism by which this modulation is achieved is via angiotensinogen's Cys18-Cys138 disulfide bond that acts as a redox switch. Molecular dynamics simulations of each redox state of angiotensinogen reveal subtle dynamic differences between the reduced and oxidised forms, particularly at the N-terminus. Surface plasmon resonance data demonstrate that the two redox forms of angiotensinogen display different binding kinetics to an immobilised anti-angiotensinogen monoclonal antibody. Mass spectrometry mapped the epitope for the antibody to the N-terminal region of angiotensinogen. We therefore provide evidence that the different redox forms of angiotensinogen can be detected by an antibody-based detection method., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Background: Topical emollient therapy with sunflower seed oil (SSO) reduces risk of sepsis and mortality in very preterm infants in low- or middle-income countries (LMICs). Proposed mechanisms include modulation of skin and possibly gut barrier function. The skin and gut microbiota play important roles in regulating barrier function, but the effects of emollient therapy on these microbiotas are poorly understood., Methods: We characterised microbiota structure and diversity with 16S rRNA gene amplicon sequence data and ecological statistics in 20 children with severe acute malnutrition (SAM) aged 2-24 months, at four skin sites and in stool, during a randomised, controlled trial of emollient therapy with SSO in Bangladesh. Microbes associated with therapy were identified with tree-based sparse discriminant analysis., Results: The skin microbiota of Bangladeshi children with SAM was highly diverse and displayed significant variation in structure as a function of physical distance between sites. Microbiota structure differed between the study groups ( P  = 0.005), was more diverse in emollient-treated subjects-including on the forehead which did not receive direct treatment-and changed with each day ( P  = 0.005) at all skin sites. Overall, Prevotellaceae were the most differentially affected by emollient treatment; several genera within this family became more abundant in the emollient group than in the controls across several skin sites. Gut microbiota structure was associated with sample day ( P  = 0.045) and subject age ( P  = 0.045), but was not significantly affected by emollient treatment ( P  = 0.060)., Conclusions: Emollient therapy altered the skin microbiota in a consistent and temporally coherent manner. We speculate that therapy with SSO enhances skin barrier function in part through alterations in the microbiota, and through systemic mechanisms. Strategies to strengthen skin and gut barrier function in populations at risk, such as children in LMICs like Bangladesh, might include deliberate manipulation of their skin microbiota., Trial Registration: ClinicalTrials.gov: NCT02616289., Competing Interests: Competing interests: The following authors are current or former employees and shareholders of GlaxoSmithKline: Lindsay Kendall, Jonathan Crowther and Rachel Gibson. The authors have completed the ICMJE competing interests form (available upon request from the corresponding author), and declare no further conflicts of interest., (Copyright © 2021 by the Journal of Global Health. All rights reserved.)

Background: Children with severe acute malnutrition (SAM) have inadequate levels of fatty acids (FAs) and limited capacity for enteral nutritional rehabilitation. We hypothesized that topical high-linoleate sunflower seed oil (SSO) would be effective adjunctive treatment for children with SAM., Methods: This study tested a prespecified secondary endpoint of a randomized, controlled, unblinded clinical trial with 212 children with SAM aged 2 to 24 months in two strata (2 to < 6 months, 6 to 24 months in a 1:2 ratio) at Dhaka Hospital of icddr,b, Bangladesh between January 2016 and December 2017. All children received standard-of-care management of SAM. Children randomized to the emollient group also received whole-body applications of 3 g/kg SSO three times daily for 10 days. We applied difference-in-difference analysis and unsupervised clustering analysis using t-distributed stochastic neighbor embedding (t-SNE) to visualize changes in FA levels in blood from day 0 to day 10 of children with SAM treated with emollient compared to no-emollient., Results: Emollient therapy led to systematically higher increases in 26 of 29 FAs over time compared to the control. These effects were driven primarily by changes in younger subjects (27 of 29 FAs). Several FAs, especially those most abundant in SSO showed high-magnitude but non-significant incremental increases from day 0 to day 10 in the emollient group vs. the no-emollient group; for linoleic acid, a 237 μg/mL increase was attributable to enteral feeding and an incremental 98 μg/mL increase (41%) was due to emollient therapy. Behenic acid (22:0), gamma-linolenic acid (18:3n6), and eicosapentaenoic acid (20:5n3) were significantly increased in the younger age stratum; minimal changes were seen in the older children., Conclusions: SSO therapy for SAM augmented the impact of enteral feeding in increasing levels of several FAs in young children. Further research is warranted into optimizing this novel approach for nutritional rehabilitation of children with SAM, especially those < 6 months., Trial Registration: ClinicalTrials.gov : NCT02616289 .

Objective: The development of dry skin is a complex process, with a wide variety of factors each playing different roles in its evolution. Given this, it is important when designing a formulation to tackle dry skin that these varied aspects of skin behaviour are addressed. Presented here are the results of a 3-week moisturization study carried out on dry legs. A wide range of traditional and more recently developed biophysical measurement methods have been combined with visual assessment of skin condition to enable multiple aspects of skin function to be determined. The observed changes in the skin are discussed in terms of the ingredients used in the moisturizing formulation., Methods: A range of novel and traditional skin assessment methods and techniques were used to assess the effects of an oil in water-based moisturizing product compared to an untreated site during a 3-week in vivo study on dry lower leg skin., Results: Statistically significant improvements were observed in a range of skin parameters as a result of product usage. Skin hydration assessed using Corneometer®, Epsilon® and visual dry skin grading all increased after 3 weeks of use. Skin barrier function measured using transepidermal water loss also improved. Levels of cholesterol, free fatty acids and Ceramide NH increased, as well as the average length of the stratum corneum (SC) lipid lamella bilayers, and the ratio of lipid to protein increased (measured using Lipbarvis® and in vivo Confocal Raman Spectroscopy). Increases in the levels of Ceramide EOS and NP were also observed, along with an improvement in corneocyte maturity, although these were not statistically significant., Conclusions: Using a variety of traditional and novel skin assessment techniques, a wide range of factors associated with the evolution of dry skin have been assessed upon treatment with a new topical moisturizer. Product usage resulted in significant improvements to skin hydration and barrier function, the levels and morphology of SC barrier lipids, and overall epidermal differentiation. As a result there was a significant reduction in the characteristics associated with the development of dry skin after use of the test product., (©.)

Background: Topical moisturizing products are widely used to alleviate the problems associated with xerotic skin. Their use affects many properties of the stratum corneum (SC) in a complex and interrelated manner. The range of measurement techniques available to the researcher has increased in recent years. However, few studies have looked for correlations between the different techniques for assessing how aspects of xerotic skin change over time as a result of topical moisturizer usage., Objectives: A 3-week in vivo study using an oil-in-water based moisturizing product and an untreated site was conducted to determine the clinical significance of and any correlations between a range of different approaches for the measurement of skin lipid content and also skin hydration and visual grading of dry skin., Methods: A range of traditional and more recently developed skin measurement techniques have been used to examine a variety of SC properties in normal and xerotic skin during topical moisturizer usage., Results: In vivo confocal Raman spectroscopy and analysis of SC lipids from tape strips both showed an increase in SC lipid level and organization after 3 weeks of moisturizer usage on xerotic skin. Hydration, measured both optically and electrically, also increased and skin barrier function improved, with strong correlations between the different measures of dryness being observed., Conclusions: Strong correlations were observed between the skin measurements for lipid assessment and skin hydration with regard to the assessment of xerotic skin, providing valuable new information for future in vivo clinical research into dry and atopic skin. Keywords biophysical assessment, skin barrier, skin hydration, topical moisturizers, Xerosis., Competing Interests: H. Stettler and R. de Salvo are employees of Bayer Consumer Care AG. P. Blenkiron and B. Lu are employees of Bayer Healthcare SAS. J. M. Crowther and A. Boxshall are consultants who have worked with Bayer Consumer Care AG and Bayer Healthcare SAS. M. Brandt, S. Laing, N. Hennighausen, and S. Bielfeldt are employees of proDERM GmbH where the study was carried out., (© 2021 Bayer Consumer Care AG. Skin Health and Disease published by John Wiley & Sons Ltd on behalf of British Association of Dermatologists.)

Peroxiredoxin 2 (Prdx2) is a thiol peroxidase with an active site Cys (C52) that reacts rapidly with H 2 O 2 and other peroxides. The sulfenic acid product condenses with the resolving Cys (C172) to form a disulfide which is recycled by thioredoxin or GSH via mixed disulfide intermediates or undergoes hyperoxidation to the sulfinic acid. C172 lies near the C terminus, outside the active site. It is not established whether structural changes in this region, such as mixed disulfide formation, affect H 2 O 2 reactivity. To investigate, we designed mutants to cause minimal (C172S) or substantial (C172D and C172W) structural disruption. Stopped flow kinetics and mass spectrometry showed that mutation to Ser had minimal effect on rates of oxidation and hyperoxidation, whereas Asp and Trp decreased both by ∼100-fold. To relate to structural changes, we solved the crystal structures of reduced WT and C172S Prdx2. The WT structure is highly similar to that of the published hyperoxidized form. C172S is closely related but more flexible and as demonstrated by size exclusion chromatography and analytical ultracentrifugation, a weaker decamer. Size exclusion chromatography and analytical ultracentrifugation showed that the C172D and C172W mutants are also weaker decamers than WT, and small-angle X-ray scattering analysis indicated greater flexibility with partially unstructured regions consistent with C-terminal unfolding. We propose that these structural changes around C172 negatively impact the active site geometry to decrease reactivity with H 2 O 2 . This is relevant for Prdx turnover as intermediate mixed disulfides with C172 would also be disruptive and could potentially react with peroxides before resolution is complete., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

β-Lactoglobulin is the most abundant protein in the whey fraction of ruminant milks, yet is absent in human milk. It has been studied intensively due to its impact on the processing and allergenic properties of ruminant milk products. However, the physiological function of β-lactoglobulin remains unclear. Using the fluorescence-detection system within the analytical ultracentrifuge, we observed an interaction involving fluorescently labelled β-lactoglobulin in its native environment, i.e. cow and goat milk, for the first time. Co-elution experiments support that these β-lactoglobulin interactions occur naturally in milk and provide evidence that the interacting partners are immunoglobulins, while further sedimentation velocity experiments confirm that an interaction occurs between these molecules. The identification of these interactions, made possible through the use of fluorescence-detected analytical ultracentrifugation, provides possible clues to the long debated physiological function of this abundant milk protein.

While humans lack the biosynthetic pathways for meso -diaminopimelate and l-lysine, they are essential for bacterial survival and are therefore attractive targets for antibiotics. It was recently discovered that members of the Chlamydia family utilize a rare aminotransferase route of the l-lysine biosynthetic pathway, thus offering a new enzymatic drug target. Here we characterize diaminopimelate aminotransferase from Verrucomicrobium spinosum ( Vs DapL), a nonpathogenic model bacterium for Chlamydia trachomatis. Complementation experiments verify that the V. spinosum dapL gene encodes a bona fide diaminopimelate aminotransferase, because the gene rescues an Escherichia coli strain that is auxotrophic for meso -diaminopimelate. Kinetic studies show that Vs DapL follows a Michaelis-Menten mechanism, with a K M app of 4.0 mM toward its substrate l,l-diaminopimelate. The k cat (0.46 s -1 ) and the k cat / K M (115 s -1 M -1 ) are somewhat lower than values for other diaminopimelate aminotransferases. Moreover, whereas other studied DapL orthologs are dimeric, sedimentation velocity experiments demonstrate that Vs DapL exists in a monomer-dimer self-association, with a K D 2-1 of 7.4 μM. The 2.25 Å resolution crystal structure presents the canonical dimer of chalice-shaped monomers, and small-angle X-ray scattering experiments confirm the dimer in solution. Sequence and structural alignments reveal that active site residues important for activity are conserved in Vs DapL, despite the lower activity compared to those of other DapL homologues. Although the dimer interface buries 18% of the total surface area, several loops that contribute to the interface and active site, notably the L1, L2, and L5 loops, are highly mobile, perhaps explaining the unstable dimer and lower catalytic activity. Our kinetic, biophysical, and structural characterization can be used to inform the development of antibiotics.

Background: Topical emollient therapy can improve neonatal health and growth and potentially provides an additional avenue for augmenting the provision of nutrition to children with severe acute malnutrition (SAM). We hypothesised that topical treatment of hospitalised children with SAM using sunflower seed oil (SSO), in addition to standard-of-care for SAM, would improve skin barrier function and weight gain, reduce risk of infection, and accelerate clinical recovery., Methods: We conducted a randomised, two-arm, controlled, unblinded clinical trial in 212 subjects aged 2 to 24 months who were admitted for care of SAM at the 'Dhaka Hospital' of icddr,b during January 2016 to November 2017. Enrollment was age-stratified into 2 to <6 months and 6 to 24 months age groups in a 1:2 ratio. All children received SAM standard-of-care, and the SSO group was also treated with 3 g of SSO per kg body weight three times daily for 10 days. Primary outcome was rate of weight gain over the 10-day study period. Secondary endpoints included rate of nosocomial infection, time to recovery from acute illness, skin condition score, rate of transepidermal water loss (TEWL) and C-reactive protein (CRP) level., Results: Rate of weight gain was higher in the SSO than the control group (adjusted mean difference, AMD = 0.90 g/kg/d, 95% confidence interval (CI) = -1.22 to 3.03 in the younger age stratum), but did not reach statistical significance. Nosocomial infection rate was significantly lower in the SSO group in the older age stratum (adjusted odds ratio (OR) = 0.41, 95% CI = 0.19 to 0.85; P  = 0.017), but was comparable in the younger age stratum and overall. Skin condition score improved (AMD = -14.88, 95% CI = -24.12 to -5.65, P  = 0.002) and TEWL was reduced overall (AMD = -2.59, 95% CI = -3.86 to -1.31, P  < 0.001) in the SSO group. Reduction in CRP level was significantly greater in the SSO group (median: -0.28) than the control group (median 0.00) ( P  = 0.019) in the younger age stratum., Conclusions: Topical therapy with SSO was beneficial for children with SAM when applied as adjunctive therapy. A community-based trial with a longer intervention period is recommended to validate these results., Trial Registration: ClinicalTrials.gov: NCT02616289., Competing Interests: Competing interests: All authors have completed the ICMJE uniform disclosure form (available upon request from the corresponding author) and declare: The following authors are current or former employees and shareholders of GlaxoSmithKline: Lindsay Kendall, Xinyi Zhu, Krishan Singh, Jonathan M. Crowther, Sunita Singh, Rachel A Gibson. All authors, external and internal, had full access to all of the data (including statistical reports and tables) in the study and take responsibility for the integrity of the data and the accuracy of the data analysis., (Copyright © 2020 by the Journal of Global Health. All rights reserved.)

Objective: Photography can be a powerful tool for researching the skin. Moving outside the visible spectrum and into the ultra violet (UV) presents a unique set of challenges to the skin photographer because of the restrictions imposed by the equipment being used. This article discusses these challenges in relation to camera sensitivity, lens and filter transmission and lighting spectrum, with the aim of demystifying what is actually being captured when imaging skin., Methods: In addition to a discussion of existing data on the subject of camera sensor sensitivity, filter transmission and flash spectral analysis, transmission in the UV of a variety of camera lenses using a new method has been carried out., Results: Using the described approach, lens transmission between 280 and 420 nm of a range of lenses has been measured. Combining this with camera sensor sensitivity data and filter and light source characteristics, it has been possible to determine an overall, harmonized, spectral sensitivity curve for what is being imaged with a given setup., Conclusions: UV reflectance photography, while a powerful tool, is often misunderstood and misreported as to what is actually being imaged. By combining measurements on camera sensitivity, lens and filter transmission and light source spectra the researcher can more fully understand what is they are actually measuring, thereby enabling better communication with the consumer on what they are seeing and a more complete description for any claims support., (© 2019 Society of Cosmetic Scientists and Société Française de Cosmétologie.)

Apoptosis signal-regulating kinases (ASK1, ASK2, and ASK3) are activators of the p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways. ASK1-3 form oligomeric complexes known as ASK signalosomes that initiate signaling cascades in response to diverse stress stimuli. Here, we demonstrated that oligomerization of ASK proteins is driven by previously uncharacterized sterile-alpha motif (SAM) domains that reside at the carboxy-terminus of each ASK protein. SAM domains from ASK1-3 exhibited distinct behaviors, with the SAM domain of ASK1 forming unstable oligomers, that of ASK2 remaining predominantly monomeric, and that of ASK3 forming a stable oligomer even at a low concentration. In contrast to their behavior in isolation, the ASK1 and ASK2 SAM domains preferentially formed a stable heterocomplex. The crystal structure of the ASK3 SAM domain, small-angle x-ray scattering, and mutagenesis suggested that ASK3 oligomers and ASK1-ASK2 complexes formed discrete, quasi-helical rings through interactions between the mid-loop of one molecule and the end helix of another molecule. Preferential ASK1-ASK2 binding was consistent with mass spectrometry showing that full-length ASK1 formed hetero-oligomeric complexes incorporating large amounts of ASK2. Accordingly, disrupting the association between SAM domains impaired ASK activity in the context of electrophilic stress induced by 4-hydroxy-2-nonenal (HNE). These findings provide a structural template for how ASK proteins assemble foci that drive inflammatory signaling and reinforce the notion that strategies to target ASK proteins should consider the concerted actions of multiple ASK family members., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Calprotectin, the major neutrophil protein, is a critical alarmin that modulates inflammation and plays a role in host immunity by strongly binding trace metals essential for bacterial growth. It has two cysteine residues favourably positioned to act as a redox switch. Whether their oxidation occurs in vivo and affects the function of calprotectin has received little attention. Here we show that in saliva from healthy adults, and in lavage fluid from the lungs of patients with respiratory diseases, a substantial proportion of calprotectin was cross-linked via disulfide bonds between the cysteine residues on its S100A8 and S100A9 subunits. Stimulated human neutrophils released calprotectin and subsequently cross-linked it by myeloperoxidase-dependent production of hypochlorous acid. The myeloperoxidase-derived oxidants hypochlorous acid, taurine chloramine, hypobromous acid, and hypothiocyanous acid, all at 10 μM, cross-linked calprotectin (5 μM) via reversible disulfide bonds. Hypochlorous acid generated A9-A9 and A8-A9 cross links. Hydrogen peroxide (10 μM) did not cross-link the protein. Purified neutrophil calprotectin existed as a non-covalent heterodimer of A8/A9 which was converted to a heterotetramer - (A8/A9) 2 - with excess calcium ions. Low level oxidation of calprotectin with hypochlorous acid produced substantial proportions of high order oligomers, whether oxidation occurred before or after addition of calcium ions. At high levels of oxidation the heterodimer could not form tetramers with calcium ions, but prior addition of calcium ions afforded some protection for the heterotetramer. Oxidation and formation of the A8-A9 disulfide cross link enhanced calprotectin's susceptibility to proteolysis by neutrophil proteases. We propose that reversible disulfide cross-linking of calprotectin occurs during inflammation and affects its structure and function. Its increased susceptibility to proteolysis will ultimately result in a loss of function., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

In vitro permeation studies using nail clippings or nail plates are commonly used in the development of transungual formulations. However, there are ethical, safety and cost issues associated with sourcing such tissues. Herein, we describe a preliminary approach is described for the design and manufacture of a human nail model surrogate based on 3D printing. To evaluate these 3D printed constructs, nails were mounted in conventional glass Franz cells and a commercial antifungal lacquer formulation containing ciclopirox olamine was applied daily to the surrogate printed surfaces for a period of 14 days. On days 8 and 14, the surfaces of the 3D printed nails were washed with ethanol to remove excess formulation. Confocal Raman spectroscopy (CRS) was used to profile the drug in the 3D printed nail. At the end of the Franz cell studies, no drug was observed in the receptor phase. CRS studies confirmed penetration of the active into the model nails with reproducible depth profiles. Our ongoing work is focused on synthesising commercial and non-commercial printable resins that can replicate the physical and chemical characteristics of the human nail. This will allow further evaluation of actives for ungual therapy and advance the development of the surrogate nail tissue model., Competing Interests: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

meso -Diaminopimelate decarboxylase catalyzes the decarboxylation of meso -diaminopimelate, the final reaction in the diaminopimelate l-lysine biosynthetic pathway. It is the only known pyridoxal-5-phosphate-dependent decarboxylase that catalyzes the removal of a carboxyl group from a d-stereocenter. Currently, only prokaryotic orthologs have been kinetically and structurally characterized. Here, using complementation and kinetic analyses of enzymes recombinantly expressed in Escherichia coli , we have functionally tested two putative eukaryotic meso- diaminopimelate decarboxylase isoforms from the plant species Arabidopsis thaliana We confirm they are both functional meso- diaminopimelate decarboxylases, although with lower activities than those previously reported for bacterial orthologs. We also report in-depth X-ray crystallographic structural analyses of each isoform at 1.9 and 2.4 Å resolution. We have captured the enzyme structure of one isoform in an asymmetric configuration, with one ligand-bound monomer and the other in an apo-form. Analytical ultracentrifugation and small-angle X-ray scattering solution studies reveal that A. thaliana meso -diaminopimelate decarboxylase adopts a homodimeric assembly. On the basis of our structural analyses, we suggest a mechanism whereby molecular interactions within the active site transduce conformational changes to the active-site loop. These conformational differences are likely to influence catalytic activity in a way that could allow for d-stereocenter selectivity of the substrate meso -diaminopimelate to facilitate the synthesis of l-lysine. In summary, the A. thaliana gene loci At 3g14390 and At 5g11880 encode functional. meso -diaminopimelate decarboxylase enzymes whose structures provide clues to the stereochemical control of the decarboxylation reaction catalyzed by these eukaryotic proteins., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health., (© 2019 Crowther et al.)

Intrinsic and extrinsic factors that induce cellular oxidative stress damage tissue integrity and promote ageing, resulting in accumulative strand breaks to the mitochondrial DNA (mtDNA) genome. Limited repair mechanisms and close proximity to superoxide generation make mtDNA a prominent biomarker of oxidative damage. Using human DNA we describe an optimised long-range qPCR methodology that sensitively detects mtDNA strand breaks relative to a suite of short mitochondrial and nuclear DNA housekeeping amplicons, which control for any variation in mtDNA copy number. An application is demonstrated by detecting 16-36-fold mtDNA damage in human skin cells induced by hydrogen peroxide and solar simulated radiation., (Copyright © 2018 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Oligomeric proteins are abundant in nature and are useful for a range of nanotechnological applications; however, a key requirement in using these proteins is controlling when and how they form oligomeric assemblies. Often, protein oligomerisation is triggered by various cellular signals, allowing for controllable oligomerisation. An example of this is human peroxiredoxin 3 (Prx), a stable protein that natively forms dimers, dodecameric rings, stacks, and tubes in response to a range of environmental stimuli. Although we know the key environmental stimuli for switching between different oligomeric states of Prx, we still have limited molecular knowledge and control over the formation and size of the protein's stacks and tubes. Here, we have generated a range of Prx mutants with either a decreased or knocked out ability to stack, and used both imaging and solution studies to show that Prx stacks through electrostatic interactions that are stabilised by a hydrogen bonding network. Furthermore, we show that altering the length of the polyhistidine tag will alter the length of the Prx stacks, with longer polyhistidine tags giving longer stacks. Finally, we have analysed the effect a variety of heavy metals have on the oligomeric state of Prx, wherein small transition metals like nickel enhances Prx stacking, while larger positively charged metals like tungstate ions can prevent Prx stacking. This work provides further structural characterisation of Prx, to enhance its use as a platform from which to build protein nanostructures for a variety of applications., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: Franz cells are routinely used to measure in vitro skin permeation of actives and must be inert to the permeant under study. The aim of the present work was to develop and manufacture transparent Franz-type diffusion cells using 3D printing. Printouts were then tested using a range of model active compounds. The study also aims to identify the critical 3D-printing parameters necessary for the process, including object design, choice of printing resin, printout curing and post-curing settings and introduction of model coatings., Methods: Transparent Franz cells were constructed using an online computer aided design program and reproduced with different stereolithography 3D printers. The two acrylate-based resins used for the fabrication process were a commercially available product and a polymer synthesised in-house. Comparative studies between glass and 3D-printed Franz cells were conducted with selected model actives: terbinafine hydrochloride (TBF), niacinamide (NIA), diclofenac free acid (DFA) and n-methyl paraben (MPB). In preliminary studies, MPB showed the lowest recovery when exposed to the receptor compartment of 3D printed cells. Consequently, in vitro permeation studies were carried out using only MPB with polydimethylsiloxane (PDMS) membrane., Results: A decrease in the amounts of selected compounds was observed for transparent 3D-printed Franz cells compared to glass cells. MPB showed the lowest recovery (53.8 ± 13.1%) when compared with NIA (74.9 ± 4.0%), TBF (81.5 ± 12.0%) and DFA (90.2 ± 12.9%) after 72 h. Permeation studies conducted using 3D-printed transparent cells with PDMS membrane also showed a decrease in MPB recovery of 51.4 ± 3.7% for the commercial resin and 94.4 ± 3.5% for the polymer synthesised in-house, when compared to glass cells. Although hydrophobic coatings were subsequently applied to the 3D-printed cells, the same reduction in MPB concentration was observed in the receptor solution., Conclusion: Transparent Franz cells were successfully prepared using 3D printing and were observed to be robust and leak-proof. There are few resins currently available for preparation of transparent materials and incompatibilities between the actives investigated and the 3D-printed cells were evident. Hydrophobic coatings applied as barriers to the printed materials did not prevent these interactions., (© 2018 Society of Cosmetic Scientists and the Société Française de Cosmétologie.)

Milk components, such as proteins and lipids, have different physicochemical properties depending upon the mammalian species from which they come. Understanding the different responses of these milks to digestion, processing, and differences in their immunogenicity requires detailed knowledge of these physicochemical properties. Here we report on the oligomeric state of β-lactoglobulin from caprine milk, the most abundant protein present in the whey fraction. At pH 2.5 caprine β-lactoglobulin is predominantly monomeric, whereas bovine β-lactoglobulin exists in a monomer-dimer equilibrium at the same protein concentrations. This behaviour was also observed in molecular dynamics simulations and can be rationalised in terms of the amino acid substitutions present between caprine and bovine β-lactoglobulin that result in a greater positive charge on each subunit of caprine β-lactoglobulin at low pH. The denaturation of β-lactoglobulin when milk is heat-treated contributes to the fouling of heat-exchange surfaces, reducing yields and increasing cleaning costs. The bovine and caprine orthologues of β-lactoglobulin display different responses to thermal treatment, with caprine β-lactoglobulin precipitating at higher pH values than bovine β-lactoglobulin (pH 7.1 compared to pH 5.6) that are closer to the natural pH of these milks (pH 6.7). This property of caprine β-lactoglobulin likely contributes to the reduced heat stability of caprine milk compared to bovine milk at its natural pH.

Attachment of ubiquitin to lysine 119 of Histone 2A (H2AK119Ub) is an epigenetic mark characteristic of repressed developmental genes, which is removed by the Polycomb Repressive-Deubiquitinase (PR-DUB) complex. Here we report the crystal structure of the Drosophila PR-DUB, revealing that the deubiquitinase Calypso and its activating partner ASX form a 2:2 complex. The bidentate Calypso-ASX complex is generated by dimerisation of two activated Calypso proteins through their coiled-coil regions. Disrupting the Calypso dimer interface does not affect inherent catalytic activity, but inhibits removal of H2AK119Ub as a consequence of impaired recruitment to nucleosomes. Mutating the equivalent surface on the human counterpart, BAP1, also compromises activity on nucleosomes. Together, this suggests that high local concentrations drive assembly of bidentate PR-DUB complexes on chromatin-providing a mechanistic basis for enhanced PR-DUB activity at specific genomic foci, and the impact of distinct classes of PR-DUB mutations in tumorigenesis.

Objective: While established methods for the calibration of visible light photographs are well defined, the use of these approaches in UVA reflectance photography is less well understood. A systematic, low-cost and simple method for the production of well-defined grey calibration standard targets for UVA reflectance photography, with a particular emphasis on low reflectivity surfaces, along with a comparison with standard visible light photographic standards is presented here., Methods: Grey calibration standard targets suitable for use in the UVA region were produced, based on optimised methods from the literature. The standards were assessed using UV-Visible reflection spectroscopy, and visible and UVA light photography, and their behaviour compared with a commercially available visible light photographic calibration chart., Results: Calibration standards with a relatively flat reflection response in the UVA region with a variety of reflectances between 2% and 35% were prepared. Imaging of the standards in both UV and visible light demonstrated the differences between these standards and the ones specifically designed for visible light photography., Conclusions: A low cost and simple method for the production of low reflectance UVA calibration targets, suitable for UVA reflectance photography has been described and tested against commercially available visual light calibration standards. These new UV suitable standards have potential for use in a wide range of applications such as forensics, biology and cosmetic science.

Objectives: Objective methods for understanding sunscreen behaviour in vitro before they are applied to the skin have failed to keep pace with the ever-increasing demands for higher SPF scores where the products are absorbing more and more similar levels of UV. A novel method for visualizing the spreading and location of SPF ingredients based on cross-polarized UVA reflectance photography is described here which gives new insights into the formation of final film morphology and how it correlates with in vivo SPF efficacy for a set of test products., Methods: High-resolution UVA-based images of sunscreen films spread onto PMMA plates were captured using a modified commercial SLR camera in a custom imaging system. Visual grading and image analysis were used to describe the overall UVA absorbance and streakiness of the resultant films, and the data compared with both in vivo and calculated in vitro SPF scores for the products., Results: Differences were observed between the products in terms of how they spread during application. A strong correlation was observed between the evenness of the resultant film as determined from the photographs and final in vivo SPF scores., Conclusions: Cross-polarized UVA reflectance photography has been demonstrated to be a valuable new method for assessing sunscreen distribution after spreading and to differentiate product based on film morphology, as well as strongly correlating with final in vivo behaviour., (© 2017 Society of Cosmetic Scientists and the Société Française de Cosmétologie.)

Objective: Methods which assess skin moisturization based on changes in its electrical properties are widely used in both cosmetic and medical research industries. However, the devices themselves often give results which are significantly different to each other. Recently two-dimensional imaging moisturization systems have become commercially available, which have the capability to provide a more detailed assessment of what is contributing to measured skin moisturization. Presented here is a new in vitro method for preparing textured model test substrates for use with these devices, and results of their use to provide a clearer insight into the devices operation., Methods: A variety of different textured model test substrates were measured using a commercially available skin moisturization measurement device, the Epsilon. The response of the Epsilon was also tested against conventional skin moisturization devices., Results: Surface morphology of model test substrates was found to have a significant influence on the measurement of its electrical properties with both the conventional and two-dimensional skin moisturization measurement devices. Through modification of the areas of the image being assessed for the two-dimensional moisturization mapping device, the parts of the model test substrate in contact with the device were indentified and analysed separately to areas not in contact with the sensor. This provided a more robust assessment of the electrical properties of substrate itself, rather than being influenced by texture like the conventional skin moisturization measurement devices., Conclusions: While the two-dimensional moisturization mapping systems can be used like a conventional electrical skin measurement device giving a simple overall reading of skin moisturization for the test area, their true value over existing electrical measures comes from its ability to isolate the skin itself from areas which are not in contact with the sensor., (© 2017 Society of Cosmetic Scientists and the Société Française de Cosmétologie.)

Thin films of dispersions of light-absorbing solid particles or emulsions containing a light-absorbing solute all have a nonuniform distribution of light-absorbing species throughout the sample volume. This results in nonuniform light absorption over the illuminated area, which causes the optical absorbance, as measured using a conventional specular UV-vis spectrophotometer, to deviate from the Beer-Lambert relationship. We have developed a theoretical model to account for the absorbance properties of such films, which are shown to depend on the size and volume fraction of the light-absorbing particles plus other sample variables. We have compared model predictions with measured spectra for samples consisting of emulsions containing a dissolved light-absorbing solute. Using no adjustable parameters, the model successfully predicts the behavior of nonuniform, light-absorbing emulsion films with varying values of droplet size, volume fraction, and other parameters.

Niacinamide (NIA) is an amide form of vitamin B3 which is used in cosmetic formulations to improve various skin conditions and it has also been shown to increase stratum corneum thickness following repeated application. In this study, three doses (5, 20 and 50μL per cm 2 ) of two NIA containing oil-in-water skin barrier-mimetic formulations were evaluated in silicone membrane and porcine ear skin and compared with a commercial control formulation. Permeation studies were conducted over 24h in Franz cells and at the end of the experiment membranes were washed and niacinamide was extracted. For the three doses, retention or deposition of NIA was generally higher in porcine skin compared with silicone membrane, consistent with the hydrophilic nature of the active. Despite the control containing a higher amount of active, comparable amounts of NIA were deposited in skin for all formulations for all doses; total skin absorption values (permeation and retention) of NIA were also comparable across all formulations. For infinite (50μL) and finite (5μL) doses the absolute permeation of NIA from the control formulation was significantly higher in porcine skin compared with both test formulations. This likely reflects differences in formulation components and/or presence of skin penetration enhancers in the formulations. Higher permeation for the 50 and 20μL dose was also evident in porcine skin compared with silicone membrane but the opposite is the case for the finite dose. The findings point to the critical importance of dose and occlusion when evaluating topical formulations in vitro and also the likelihood of exaggerated effects of excipients on permeation at infinite and pseudo-finite dose applications., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective: Methods that assess skin hydration based on changes in its electrical properties are widely used in both cosmetic and medical research. However, the devices themselves often give results which are significantly different to each other. Although some work has previously been carried out to try and understand what these devices are actually reading, it was based on a technique for measuring the devices' responses to filter discs impregnated with different liquids, which could in itself be influencing the measurements. Presented here is a new method for measuring the devices' direct responses to different materials and solutions which removes any other confounding effects, thereby providing a clearer insight into their operation., Methods: The responses of a variety of different liquids and solutions were measured using the Corneometer ® and Skicon ® . A new method is presented, based on the use of a custom-designed PTFE block to hold the liquids, allowing their measurement without using a filter paper. This method was developed and tested against the existing filter paper-based approach., Results: Differences were observed in results between filter paper- and PTFE block-based approach, indicating that the filter paper itself is capable of influencing the measurements and as such is not to be recommended for assessing how different liquids impact on results from the devices. A positive correlation was observed between Corneometer ® and Skicon ® readings for certain solutions and under certain conditions. A large influence of salt concentration was noted for the Skicon ® device with no or minimal impact from the actual water itself, humectants and emollients. Salts, emollients, water and humectants were observed to have an effect on Corneometer ® readings., Conclusions: Both the Corneometer ® and Skicon ® were influenced to different extents by chemicals other than water and therefore cannot be seen purely as measures of skin 'hydration'. Although there is strong evidence that the devices do correlate with expert assessment of skin dryness, the level of water in the skin is only part of the story when it comes to understanding the benefits of topical moisturizing products applied to the skin. An alternative approach would be to consider skin 'moisturization' as a property which is influenced by water, salts and other materials such as humectants and emollients, which is more consistent with how the stratum corneum itself helps to maintain its plasticity and flexibility. In the work presented here, the Corneometer ® was more suited to providing a measurement which reflects the impact of multiple different components., (© 2016 Society of Cosmetic Scientists and the Société Française de Cosmétologie.)

We recently showed (Binks et al., ACS Appl. Mater. Interfaces, 2016, DOI: 10.1021/acsami.6b02696) how evaporation of sunscreen films consisting of solutions of molecular UV filters leads to loss of UV light absorption and derived sun protection factor (SPF). In the present work, we investigate evaporation-induced effects for sunscreen films consisting of particle-stabilized emulsions containing a dissolved UV filter. The emulsions contained either droplets of propylene glycol (PG) in squalane (SQ), droplets of SQ in PG or droplets of decane in PG. In these different emulsion types, the SQ is involatile and shows no evaporation, the PG is volatile and evaporates relatively slowly, whereas the decane is relatively very volatile and evaporates quickly. We have measured the film mass and area, optical micrographs of the film structure, and the UV absorbance spectra during evaporation. For emulsion films containing the involatile SQ, evaporation of the PG causes collapse of the emulsion structure with some loss of specular UV absorbance due to light scattering. However, for these emulsions with droplets much larger than the wavelength of light, the light is scattered only at small forward angles so does not contribute to the diffuse absorbance and the film SPF. The UV filter remains soluble throughout the evaporation and thus the UV absorption by the filter and the SPF remain approximately constant. Both PG-in-SQ and SQ-in-PG films behave similarly and do not show area shrinkage by dewetting. In contrast, the decane-in-PG film shows rapid evaporative loss of the decane, followed by slower loss of the PG resulting in precipitation of the UV filter and film area shrinkage by dewetting which cause the UV absorbance and derived SPF to decrease. Measured UV spectra during evaporation are in reasonable agreement with spectra calculated using models discussed here.

The objective of the present study was to evaluate the fate of three chemical sunscreens, isoamyl p-methoxycinnamate (IPMC), diethylamino hydroxybenzoyl hexyl benzoate (DHHB), and bis-ethylhexylphenol methoxyphenyl triazine (BEMT), topically applied to mammalian skin from a skin barrier mimetic oil-in-water formulation. High Performance Liquid Chromatography (HPLC) methods were developed for the analysis of each molecule and validated. Franz cell permeation studies were conducted following application of finite doses of the formulations to excised porcine skin. A vehicle formulation containing no sunscreens was evaluated as a control. Permeation studies were conducted for 12h after which full mass balance studies were carried out. Analysis of individual UV sunscreens was achieved with HPLC following application of the formulation to the skin with no interference from the vehicle components. No skin permeation of any of the chemical sunscreens was evident after 12h. While sunscreens were detected in up to 12 tape strips taken from the SC, 87% or more of the applied doses recovered in the first 5 tape strips. When corrected for the amount of protein removed per tape strip this corresponded to a penetration depth in porcine stratum corneum of ∼1.7μm. Mass balance studies indicated total recovery values were within accepted guidelines for cosmetic formulations. Overall, only superficial penetration into the SC was observed for each compound. These findings are consistent with the physicochemical properties of the selected UV absorbing molecules and their formulation into an ordered biomimetic barrier formulation thus support their intended use in topical consumer formulations designed to protect from UV exposure. To our knowledge this is the first report of depth profiling of chemical sunscreens in the SC that combines tape stripping and protein determination following in vitro Franz cell studies., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We have investigated the evaporation of thin sunscreen films and how the light absorption and the derived sun protection factor (SPF) change. For films consisting of solutions of common UV filters in propylene glycol (PG) as solvent, we show how evaporation generally causes three effects. First, the film area can decrease by dewetting leading to a transient increase in the average film thickness. Second, the film thins by evaporative loss of the solvent. Third, precipitation of the UV filter occurs when solvent loss causes the solubility limit to be reached. These evaporation-induced changes cause the UV absorbance of the film to decrease with resultant loss of SPF over the time scale of the evaporation. We derive an approximate model which accounts semiquantitatively for the variation of SPF with evaporation. Experimental results for solutions of different UV filters on quartz, different skin mimicking substrates, films with added nanoparticles, films with an added polymer and films with fast-evaporating decane as solvent (instead of slow evaporating PG) are discussed and compared with model calculations. Addition of either nanoparticles or polymer suppress film dewetting. Overall, it is hoped that the understanding gained about the mechanisms whereby film evaporation affects the SPF will provide useful guidance for the formulation of more effective sunscreens.

Objective: The Sebumeter(®) is widely used in both cosmetic and medical research, for measuring changes in sebum levels on skin. It is commonly reported that the units correlated to a mass of sebum on the skin in μg cm(-2) ; however, validation for this has not been published. Also, its use for assessing the presence of other oily materials which are widely utilized in topical skincare products on skin has not been widely discussed. Determining a calibration scale and whether the response of the device is linear with the level of oils present enables quantification of the output of the device, and would validate the device for claims substantiation., Methods: Different doses of a variety of oily materials (paraffin oil, white soft paraffin, capric-caprylic triglyceride, 350cSt silicone fluid and synthetic sebum) were applied to skin, and the Sebumeter(®) used to collect and quantify them. The mass per square centimetre of the oily material delivered to the skin was then compared to the Sebumeter(®) output to develop calibration curves for the different materials. Measurements were carried out on a single volunteer as this work was to verify the concept of quantitative oil assessment using the device., Results: A linear correlation between the mass of the oily material and the Sebumeter(®) output was seen for all the materials tested. However, the absolute response of the device was different for each material, and the output values did not directly give the mass of material on the skin in μg cm(-2) . As part of the calibration, it was also demonstrated that to remove all the oily material from a given area of the skin required multiple 30-s applications of the Sebumeter(®) cartridge., Conclusions: The Sebumeter(®) is a precise analytical instrument capable of quantitative measurement of deposition of oily materials onto skin from topical products (down to the μg cm(-2) level), as well as its traditional use of measuring sebum levels. However, the output values do not directly correlate with the mass of oil present, and generation of a calibration curve is necessary for any ingredient of interest to produce quantitative data for claim support and formulation development., (© 2015 Society of Cosmetic Scientists and the Société Française de Cosmétologie.)

β-Lactoglobulin (βlg) is the most abundant whey protein in the milks of ruminant animals. While bovine βlg has been subjected to a vast array of studies, little is known about the caprine ortholog. We present an ultra-high resolution crystal structure of caprine βlg complemented by analytical ultracentrifugation and small-angle X-ray scattering data. In both solution and crystalline states caprine βlg is dimeric (K(D)<5 μM); however, our data suggest a flexible quaternary arrangement of subunits within the dimer. These structural findings will provide insight into relationships among structural, processing, nutritional and immunological characteristics that distinguish cow's and goat's milk., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Diaminopimelate decarboxylase catalyses the last step in the diaminopimelate-biosynthetic pathway leading to S-lysine: the decarboxylation of meso-diaminopimelate to form S-lysine. Lysine biosynthesis occurs only in microorganisms and plants, and lysine is essential for the growth and development of animals. Thus, the diaminopimelate pathway represents an attractive target for antimicrobial and herbicide treatments and has received considerable attention from both a mechanistic and a structural viewpoint. Diaminopimelate decarboxylase has only been characterized in prokaryotic species. This communication describes the first structural studies of two diaminopimelate decarboxylase isoforms from a plant. The Arabidopsis thaliana diaminopimelate decarboxylase cDNAs At3g14390 (encoding DapDc1) and At5g11880 (encoding DapDc2) were cloned from genomic DNA and the recombinant proteins were expressed and purified from Escherichia coli Rosetta (DE3) cells. The crystals of DapDc1 and DapDc2 diffracted to beyond 2.00 and 2.27 Å resolution, respectively. Understanding the structural biology of diaminopimelate decarboxylase from a eukaryotic species will provide insights for the development of future herbicide treatments, in particular.

L,L-Diaminopimelate aminotransferase (DapL) is an enzyme required for the biosynthesis of meso-diaminopimelate (m-DAP) and L-lysine (Lys) in some bacteria and photosynthetic organisms. m-DAP and Lys are both involved in the synthesis of peptidoglycan (PG) and protein synthesis. DapL is found in specific eubacterial and archaeal lineages, in particular in several groups of pathogenic bacteria such as Leptospira interrogans (LiDapL), the soil/water bacterium Verrucomicrobium spinosum (VsDapL) and the alga Chlamydomonas reinhardtii (CrDapL). Here we present the first comprehensive inhibition study comparing the kinetic activity of DapL orthologs using previously active small molecule inhibitors formerly identified in a screen with the DapL of Arabidopsis thaliana (AtDapL), a flowering plant. Each inhibitor is derived from one of four classes with different central structural moieties: a hydrazide, a rhodanine, a barbiturate, or a thiobarbituate functionality. The results show that all five compounds tested were effective at inhibiting the DapL orthologs. LiDapL and AtDapL showed similar patterns of inhibition across the inhibitor series, whereas the VsDapL and CrDapL inhibition patterns were different from that of LiDapL and AtDapL. CrDapL was found to be insensitive to the hydrazide (IC₅₀ >200 μM). VsDapL was found to be the most sensitive to the barbiturate and thiobarbiturate containing inhibitors (IC₅₀ ∼5 μM). Taken together, the data shows that the homologs have differing sensitivities to the inhibitors with IC₅₀ values ranging from 4.7 to 250 μM. In an attempt to understand the basis for these differences the four enzymes were modeled based on the known structure of AtDapL. Overall, it was found that the enzyme active sites were conserved, although the second shell of residues close to the active site were not. We conclude from this that the altered binding patterns seen in the inhibition studies may be a consequence of the inhibitors forming additional interactions with residues proximal to the active site, or that the inhibitors may not act by binding to the active site. Compounds that are specific for DapL could be potential biocides (antibiotic, herbicide or algaecide) that are nontoxic to animals since animals do not contain the enzymes necessary for PG or Lys synthesis. This study provides important information to expand our current understanding of the structure/activity relationship of DapL and putative inhibitors that are potentially useful for the design and or discovery of novel biocides., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Niacinamide-containing moisturisers are known be efficacious in alleviating dry skin conditions and improving stratum corneum (SC) barrier function. However, the mechanisms of action of niacinamide at the molecular level in the SC are still not well understood. Previously, we have reported the development of novel methods to probe SC barrier properties in vivo. The aim of the present study was to characterise changes in Trans Epidermal Water Loss (TEWL), corneocyte surface area and maturity, selected protease activities and SC thickness after repeated application of a simple vehicle containing niacinamide. A commercial formulation was also included as a reference. The left and right mid-volar forearms of 20 healthy volunteers were used as study sites, to which topical formulations were applied twice daily for 28 days. After successive tape-stripping, corneocyte maturity and surface area were assessed. In addition, activity of the desquamatory kallikrein (KLK) protease enzymes KLK5 and KLK7, and tryptase and plasmin (implicated in inflammatory process) were measured using a fluorogenic probe assay. The amount of protein removed and TEWL were also recorded. SC thickness before and after treatment was determined using Confocal Raman Spectroscopy (CRS). Overall (i) corneocyte maturity and surface area decreased with increasing number of tape strips, (ii) activity of both the desquamatory and inflammatory enzymes was highest in the outer layers of the SC and decreased with depth (iii) TEWL increased as more SC layers were removed. Furthermore, areas treated with formulations containing niacinamide were significantly different to pre-treatment baseline and untreated/vehicle-control treated sites, with larger and more mature corneocytes, decreased inflammatory activity, decreased TEWL and increased SC thickness. These data (a) confirm the utility of measures and metrics developed previously for the non-invasive assay of SC barrier function, (b) present an holistic picture of a SC compartment managing barrier function through dynamic optimisation of pathlength and quality of building materials used, and (c) shed new light on niacinamide as a topical formulation adjunct with unique SC barrier-augmentation properties., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Moisturizers are the most commonly used topically applied product for the treatment of dry skin conditions. They affect many properties and functions of the stratum corneum but some moisturizers have been reported to be detrimental to barrier function. Stratum corneum barrier function is a composite of its total structure and thickness but few studies have taken this into account. As a biosensor, the stratum corneum (SC) will change its structure in response to treatment and a swelling effect has been clearly demonstrated by skin hydration. Recently several moisturizing agents have been shown to have an effect on SC swelling behaviour with conflicting results. However, there is a paucity of data reported for measuring the effects of long-term usage of moisturizers on SC thickness in vivo as, until recently, traditional techniques did not have the resolution to measure the effects of moisturizers on nonpalmoplantar body sites. The development of confocal Raman spectroscopy for use in human subjects provides noninvasive, real-time, in vivo measurement of SC water concentration profiles and we have also used this state of the art equipment to measure the effect of the long-term use of moisturizers on SC thickness for the first time., Objectives: To validate the use of confocal Raman spectroscopy (CRS) to measure SC thickness and then use it to investigate the short- and long-term effects of moisturizers (one of which is known to improve SC barrier function) on SC thickness, water gradients and hydration., Methods: Two studies were conducted: (i) to validate the use of CRS for measuring SC thickness through comparison with optical coherence tomography (OCT); and (ii) once validated to use CRS to measure the long-term effects of three commercially available moisturizers (A, B, C) on SC thickness and water gradients, together with total hydration, over a 3-week period (2 weeks of treatment and 1 week regression) and compare the spectroscopy-derived hydration value with instrumentally derived capacitance hydration values., Results: (i) A strong, positive correlation in SC thickness was obtained between CRS and OCT (OCT-derived thickness = 0.96 x CRS-derived thickness, r(2) = 0.93; P <0.0001). OCT was shown, however, to have a lower resolution than CRS in distinguishing SC thickness on thinner nonpalmoplantar body sites. Using the CRS method, differences in SC thickness were readily apparent on different body sites (cheek 12.8 +/- 0.9 microm, volar forearm 18.0 +/- 3.9 microm, leg 22.0 +/- 6.9 microm). (ii) Examining the effects of moisturizers in a blinded, randomized 3-week study in human volunteers (n = 14) demonstrated that only one commercially available formulation (A) changed SC water gradients, thickness and hydration as measured by CRS. These hydration data did not directly correlate with capacitance hydration values., Conclusions: (i) In vivo CRS was validated as a technique to measure SC thickness on both palmoplantar and, particularly, on nonpalmoplantar skin sites. (ii) Moisturizers improve skin moisturization but in this study only formulation A improved SC thickness, water gradients and hydration as measured by CRS. We hypothesize that this was due to compositional differences between the products. We believe that niacinamide (nicotinamide, vitamin B(3)) is probably contributing significantly to this effect, as it has been proven to increase epidermal lipogenesis and SC barrier function in other studies. These results show that by using CRS, we were able for the first time to determine the effect of moisturizer on multiple SC barrier endpoints including SC thickness, and water content as a function of depth and total SC water content.

Variations in precipitation chemistry between and within rain events have been examined in order to identify possible relationships with synoptic, mesoscale and micrometeorological processes. A microprocessor-based acid rain monitor was used to provide high resolution meteorological and rain chemistry data from which two case study events have been selected to illustrate event and sub-event rainfall chemistry characteristics. Event rainfall chemistry is strongly influenced by the history of the prevailing air mass and the synoptic situation. From back trajectories calculated at the 950 mbar level it is clear that air mass history can change markedly within a few hours. These observations emphasise the value of high resolution rainfall chemistry measurements. Pollutant concentrations in rainwater have been shown to fluctuate markedly within the course of individual events as a result of both advective and scavenging processes. Advective effects may result from: (a) air mass discontinuities at frontal zones; and/or (b) variable rainfall interception of the air mass prior to arrival at the site. A simple mathematical model has been developed to describe the scavenging mechanisms and it shows good agreement with field observations. Theoretical considerations suggest that in-cloud processes give rise to most of the observed decline in concentrations.