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Lemna L. sp. is a free-floating aquatic macrophyte that plays a key role as a standard test species in aquatic risk assessment for herbicides and other contaminants. Population modeling can be used to extrapolate from laboratory to field conditions. However, there are insufficient data on longer-term seasonal dynamics of this species to evaluate such models. Therefore, several long-term growth experiments were conducted in outdoor microcosms (surface area 0.174 m 2 ). Monitoring parameters included biomass, frond numbers, water parameters, and weather data. Three different datasets were generated: frond numbers and biomass from weekly to monthly destructively sampled microcosms; a year-round dataset of frond numbers from five continuously monitored microcosms; and seasonal growth rates without the effect of density dependence over 1-2 weeks in freshly inoculated microcosms. Lemna sp. reached a maximum of approximately 500 000 fronds m -2 and 190 g dry weight m -2 . During the first winter, the microcosms were covered by ice for approximately four weeks, and Lemna sp. populations collapsed. The second winter was warmer, without any ice cover, and Lemna sp. populations maintained high abundance throughout the winter. Dry weight per frond was not constant throughout the year but was highest in autumn and winter. Growth rates without density dependence under outdoor environmental conditions reached 0.29 day -1 for frond number, 0.43 day -1 for fresh weight, and 0.39 day -1 for dry weight. In linear regressions, these growth rates were best explained by water temperature. For the populations continuously monitored throughout a year, the nitrogen-to-phosphorus ratio best explained the growth rate of frond numbers. This study yielded a relevant dataset for testing and refining Lemna population models used in chemical risk assessment as well as for managing ecosystems and combating the effects of eutrophication. Integr Environ Assess Manag 2024;20:1625-1638. © 2024 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC)., (© 2024 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).)

Antibiotics are a contaminant class of worldwide concern as they are frequently detected in aquatic ecosystems. To better understand the impacts of antibiotics on aquatic ecosystems, we conducted an outdoor mesocosm experiment in which aquatic communities were exposed to different concentrations of the antibiotic sulfamethoxazole (0, 0.15, 1.5, 15 and 150 μg/L). These concentrations include mean (0.15 μg/L) and maximum detected concentrations (15 and 150 μg/L) in aquatic ecosystems worldwide. Sulfamethoxazole was applied once a week for eight consecutive weeks to 1530 L outdoor mesocosms in the Netherlands, followed by an eight-week recovery period. We evaluated phytoplankton-, bacterial- and invertebrate responses during and after sulfamethoxazole exposure and assessed impacts on organic matter decomposition. Contrary to our expectations, consistent treatment-related effects on algal and bacterial communities could not be demonstrated. In addition, sulfamethoxazole did not significantly affect zooplankton and macroinvertebrate communities. However, some effects on specific taxa were observed, with an increase in Mesostoma flatworm abundance (NOEC of <0.15 μg/L). In addition, eDNA analyses indicated negative impacts on the insects Odonata at a sulfamethoxazole concentration of 15 μg/L. Overall, environmentally relevant sulfamethoxazole concentration did not result in direct or indirect impairment of entire aquatic communities and ecological processes in our mesocosms. However, several specific macroinvertebrate taxa demonstrated significant (in)direct effects from sulfamethoxazole. Comparison of the results with the literature showed inconsistent results between studies using comparable, environmentally relevant, concentrations. Therefore, our study highlights the importance of testing the ecological impacts of pharmaceuticals (such as sulfamethoxazole) across multiple trophic levels spanning multiple aquatic communities, to fully understand its potential ecological threats., 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 © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The antidepressant fluoxetine is frequently detected in aquatic ecosystems, yet the effects on aquatic communities and ecosystems are still largely unknown. Therefore the aim of this study is to assess the effects of the long-term application of fluoxetine on key components of aquatic ecosystems including macroinvertebrate-, zooplankton-, phytoplankton- and microbial communities and organic matter decomposition by using traditional and non-traditional assessment methods. For this, we exposed 18 outdoor mesocosms (water volume of 1530 L and 10 cm of sediment) to five different concentrations of fluoxetine (0.2, 2, 20 and 200 μg/L) for eight weeks, followed by an eight-week recovery period. We quantified population and community effects by morphological identification, environmental DNA metabarcoding, in vitro and in vivo bioassays and measured organic matter decomposition as a measure of ecosystem functioning. We found effects of fluoxetine on bacterial, algal, zooplankton and macroinvertebrate communities and decomposition rates, mainly for the highest (200 μg/L) treatment. Treatment-related decreases in abundances were found for damselfly larvae (NOEC of 0.2 μg/L) and Sphaeriidae bivalves (NOEC of 20 μg/L), whereas Asellus aquaticus increased in abundance (NOEC <0.2 μg/L). Fluoxetine decreased photosynthetic activity and primary production of the suspended algae community. eDNA assessment provided additional insights by revealing that the algae belonging to the class Cryptophyceae and certain cyanobacteria taxa were the most negatively responding taxa to fluoxetine. Our results, together with results of others, suggest that fluoxetine can alter community structure and ecosystem functioning and that some impacts of fluoxetine on certain taxa can already be observed at environmentally realistic concentrations., 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 © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: The primary aim of this study was to investigate the effect of including the Dutch National Pharmacotherapy Assessment (DNPA) in the medical curriculum on the level and development of prescribing knowledge and skills of junior doctors. The secondary aim was to evaluate the relationship between the curriculum type and the prescribing competence of junior doctors., Methods: We re-analysed the data of a longitudinal study conducted in 2016 involving recently graduated junior doctors from 11 medical schools across the Netherlands and Belgium. Participants completed three assessments during the first year after graduation (around graduation (+ / - 4 weeks), and 6 months, and 1 year after graduation), each of which contained 35 multiple choice questions (MCQs) assessing knowledge and three clinical case scenarios assessing skills. Only one medical school used the DNPA in its medical curriculum; the other medical schools used conventional means to assess prescribing knowledge and skills. Five medical schools were classified as providing solely theoretical clinical pharmacology and therapeutics (CPT) education; the others provided both theoretical and practical CPT education (mixed curriculum)., Results: Of the 1584 invited junior doctors, 556 (35.1%) participated, 326 (58.6%) completed the MCQs and 325 (58.5%) the clinical case scenarios in all three assessments. Junior doctors whose medical curriculum included the DNPA had higher knowledge scores than other junior doctors (76.7% [SD 12.5] vs. 67.8% [SD 12.6], 81.8% [SD 11.1] vs. 76.1% [SD 11.1], 77.0% [12.1] vs. 70.6% [SD 14.0], p < 0.05 for all three assessments, respectively). There was no difference in skills scores at the moment of graduation (p = 0.110), but after 6 and 12 months junior doctors whose medical curriculum included the DNPA had higher skills scores (both p < 0.001). Junior doctors educated with a mixed curriculum had significantly higher scores for both knowledge and skills than did junior doctors educated with a theoretical curriculum (p < 0.05 in all assessments)., Conclusion: Our findings suggest that the inclusion of the knowledge focused DNPA in the medical curriculum improves the prescribing knowledge, but not the skills, of junior doctors at the moment of graduation. However, after 6 and 12 months, both the knowledge and skills were higher in the junior doctors whose medical curriculum included the DNPA. A curriculum that provides both theoretical and practical education seems to improve both prescribing knowledge and skills relative to a solely theoretical curriculum., (© 2023. The Author(s).)

Aims: Prescribing errors among junior doctors are common in clinical practice because many lack prescribing competence after graduation. This is in part due to inadequate education in clinical pharmacology and therapeutics (CP&T) in the undergraduate medical curriculum. To support CP&T education, it is important to determine which drugs medical undergraduates should be able to prescribe safely and effectively without direct supervision by the time they graduate. Currently, there is no such list with broad-based consensus. Therefore, the aim was to reach consensus on a list of essential drugs for undergraduate medical education in the Netherlands., Methods: A two-round modified Delphi study was conducted among pharmacists, medical specialists, junior doctors and pharmacotherapy teachers from all eight Dutch academic hospitals. Participants were asked to indicate whether it was essential that medical graduates could prescribe specific drugs included on a preliminary list. Drugs for which ≥80% of all respondents agreed or strongly agreed were included in the final list., Results: In all, 42 (65%) participants completed the two Delphi rounds. A total of 132 drugs (39%) from the preliminary list and two (3%) newly proposed drugs were included., Conclusions: This is the first Delphi consensus study to identify the drugs that Dutch junior doctors should be able to prescribe safely and effectively without direct supervision. This list can be used to harmonize and support the teaching and assessment of CP&T. Moreover, this study shows that a Delphi method is suitable to reach consensus on such a list, and could be used for a European list., (© 2022 The Authors. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

The relationship between race and biology is complex. In contemporary medical science, race is a social construct that is measured via self-identification of study participants. But even though race has no biological essence, it is often used as variable in medical guidelines (e.g., treatment recommendations specific for Black people with hypertension). Such recommendations are based on clinical trials in which there was a significant correlation between self-identified race and actual, but often unmeasured, health-related factors such as (pharmaco)genetics, diet, sun exposure, etc. Many teachers are insufficiently aware of this complexity. In their classes, they (unintentionally) portray self-reported race as having a biological essence. This may cause students to see people of shared race as biologically or genetically homogeneous, and believe that race-based recommendations are true for all individuals (rather than reflecting the average of a heterogeneous group). This medicalizes race and reinforces already existing healthcare disparities. Moreover, students may fail to learn that the relation between race and health is easily biased by factors such as socioeconomic status, racism, ancestry, and environment and that this limits the generalizability of race-based recommendations. We observed that the clinical case vignettes that we use in our teaching contain many stereotypes and biases, and do not generally reflect the diversity of actual patients. This guide, written by clinical pharmacology and therapeutics teachers, aims to help our colleagues and teachers in other health professions to reflect on and improve our teaching on race-based medical guidelines and to make our clinical case vignettes more inclusive and diverse., (© 2022 The Authors. Clinical Pharmacology & Therapeutics published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Aim: The aim of this study was to investigate how the prescribing knowledge and skills of junior doctors in the Netherlands and Belgium develop in the year after graduation. We also analysed differences in knowledge and skills between surgical and nonsurgical junior doctors., Methods: This international, multicentre (n = 11), longitudinal study analysed the learning curves of junior doctors working in various specialties via three validated assessments at about the time of graduation, and 6 months and 1 year after graduation. Each assessment contained 35 multiple choice questions (MCQs) on medication safety (passing grade ≥85%) and three clinical scenarios., Results: In total, 556 junior doctors participated, 326 (58.6%) of whom completed the MCQs and 325 (58.5%) the clinical case scenarios of all three assessments. Mean prescribing knowledge was stable in the year after graduation, with 69% (SD 13) correctly answering questions at assessment 1 and 71% (SD 14) at assessment 3, whereas prescribing skills decreased: 63% of treatment plans were considered adequate at assessment 1 but only 40% at assessment 3 (P < .001). While nonsurgical doctors had similar learning curves for knowledge and skills as surgical doctors (P = .53 and P = .56 respectively), their overall level was higher at all three assessments (all P < .05)., Conclusion: These results show that junior doctors' prescribing knowledge and skills did not improve while they were working in clinical practice. Moreover, their level was under the predefined passing grade. As this might adversely affect patient safety, educational interventions should be introduced to improve the prescribing competence of junior doctors., (© 2022 The Authors. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Purpose: In the wake of the coronavirus disease 2019 (COVID-19) pandemic, support in clinical trials by students of human medicine and related disciplines has become of even greater importance than in pre-pandemic times. Documentation in clinical trials adheres to the principles of Good Clinical Practice (GCP), and healthcare professionals involved in the conduct of clinical trials-including students-are obliged to perform documentation in accordance with GCP principles. Unprecedented challenges have arisen with regard to the appropriate training of students as training courses in presence had largely to be suspended due to social-distancing regulations during the heyday of the COVID-19 pandemic. Therefore, novel training formats and self-study training materials for students working in clinical trials are urgently warranted., Methods: To overcome this shortcoming and to define a common quality standard, an interdisciplinary, multiprofessional (physicians, study nurses, medical students), and binational (Germany, The Netherlands) expert panel convened and devised the Students' guide to documentation in clinical trials., Results: Following a brief description of the different roles in clinical trials (e.g., sponsor, (principal) investigator, monitor) and an introduction into the principles of GCP, the documentation of adverse events, concomitant medication, medical history, and quality control are comprehensively discussed. The Guide concludes with a trilingual medical dictionary (English, German, Dutch) and with recommendations of pertinent literature for further reading., Conclusion: Serving both as textbook for self-training and as (quick-) reference work for the daily routine, the Guide has specifically been designed to complement, but not to replace practical training courses for students. While primarily addressed at students of human medicine and related disciplines, the Guide can also be of high relevance and utility to other healthcare professionals involved in the conduct of clinical trials., (© 2022. The Author(s).)

The European Open Platform for Prescribing Education (EurOP 2 E) seeks to improve and harmonize European clinical pharmacology and therapeutics (CPT) education by facilitating international collaboration and sharing problem-based, online, open educational resources. The COVID-19 pandemic forced teachers to switch to virtual modalities, highlighting the need for high-quality online teaching materials. The goal of this study was to establish the online problem-based teaching resources needed to sustain prescribing education during the pandemic and thereafter. A nominal group technique study was conducted with prescribing teachers from 15 European countries. Results were analyzed through thematic analysis. In four meetings, 20 teachers from 15 countries proposed and ranked 35 teaching materials. According to the participants, the most necessary problem-based-online teaching materials related to three overarching themes. Related to learning outcomes for CPT, participants proposed creating prescription scenarios, including materials focusing on background knowledge and resources on personalized medicine and topical/ethical issues such as the prescription's impact on planetary health. Second, related to teaching, they proposed online case discussions, gamification and decision support systems. Finally, in relation to faculty development, they recommend teacher courses, a repository of reusable exam questions and harmonized formularies. Future work will aim to collaboratively produce such materials., (© 2022. The Author(s).)

Myriophyllum spicatum is a sediment-rooted, aquatic macrophyte growing submerged, with a wide geographical distribution and high ecological relevance in freshwater ecosystems. It is used in testing and risk assessment for pesticides in water and sediment. Population models enable effects measured under laboratory conditions to be extrapolated to effects expected in the field with time-variable environmental factors including exposure. These models are a promising tool in higher-tier risk assessments. However, there is a lack of data on the seasonal dynamics of M. spicatum, which is needed to test model predictions of typical population dynamics in the field. To generate such data, a two-year study was set up in outdoor experimental systems from May 2017 to May 2019. The growth of M. spicatum was monitored in 0.2025 m 2 plant baskets installed in an experimental ditch. Parameters monitored included biomass (fresh weight [FW] and dry weight [DW]), shoot length, seasonal short-term growth rates of shoots, relevant environmental parameters, and weather data. The results showed a clear seasonal pattern of biomass and shoot length and their variability. M. spicatum reached a maximum total shoot length (TSL) of 279 m m -2 and a maximum standing crop above-ground DW of 262 g m -2 . Periodical growth rates reached up to 0.072, 0.095, and 0.085 day -1 for total length, FW, and DW, respectively. Multivariate regression revealed that pH (as a surrogate for the availability of carbon species) and water temperature could explain a significant proportion of the variability in M. spicatum growth rates (p < 0.05). This study has provided an ecologically relevant data set on seasonal population dynamics representative of shallow freshwater ecosystems, which can be used to test and refine population models for use in chemical risk assessment and ecosystem management. Integr Environ Assess Manag 2022;18:1375-1386. © 2021 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC)., (© 2021 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).)

The existence of glucosylated cholesterol (GlcChol) in tissue has recently been recognized. GlcChol is generated from glucosylceramide (GlcCer) and cholesterol through transglucosylation by two retaining β-glucosidases, GBA and GBA2. Given the abundance of GBA, GlcCer and cholesterol in the skin's stratum corneum (SC), we studied the occurrence of GlcChol. A significant amount of GlcChol was detected in SC (6 pmol/mg weight). The ratio GlcChol/GlcCer is higher in SC than epidermis, 0.083 and 0.011, respectively. Examination of GlcChol in patients with Netherton syndrome revealed comparable levels (11 pmol/mg). Concluding, GlcChol was identified as a novel component in SC and is likely locally metabolized by GBA. The physiological function of GlcChol in the SC warrants future investigation., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Patients with Atopic Dermatitis (AD) suffer from inflamed skin and skin barrier defects. Proper formation of the outermost part of the skin, the stratum corneum (SC), is crucial for the skin barrier function. In this study we analyzed the localization and activity of lipid enzymes β-glucocerebrosidase (GBA) and acid sphingomyelinase (ASM) in the skin of AD patients and controls. Localization of both the expression and activity of GBA and ASM in the epidermis of AD patients was altered, particularly at lesional skin sites. These changes aligned with the altered SC lipid composition. More specifically, abnormal localization of GBA and ASM related to an increase in specific ceramide subclasses [AS] and [NS]. Moreover we related the localization of the enzymes to the amounts of SC ceramide subclasses and free fatty acids (FFAs). We report a correlation between altered localization of active GBA and ASM and a disturbed SC lipid composition. Localization of antimicrobial peptide beta-defensin-3 (HBD-3) and AD biomarker Thymus and Activation Regulated Chemokine (TARC) also appeared to be diverging in AD skin compared to control. This research highlights the relation between correct localization of expressed and active lipid enzymes and a normal SC lipid composition for a proper skin barrier., 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 © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Individuals with Netherton syndrome (NTS) have increased serine protease activity, which strongly impacts the barrier function of the skin epidermis and leads to skin inflammation. Here, we investigated how serine protease activity in NTS correlates with changes in the stratum corneum (SC) ceramides, which are crucial components of the skin barrier. We examined two key enzymes involved in epidermal ceramide biosynthesis, β-glucocerebrosidase (GBA) and acid-sphingomyelinase (ASM). We compared in situ expression levels and activities of GBA and ASM between NTS patients and controls and correlated the expression and activities with i ) SC ceramide profiles, ii ) in situ serine protease activity, and iii ) clinical presentation of patients. Using activity-based probe labeling, we visualized and localized active epidermal GBA, and a newly developed in situ zymography method enabled us to visualize and localize active ASM. Reduction in active GBA in NTS patients coincided with increased ASM activity, particularly in areas with increased serine protease activity. NTS patients with scaly erythroderma exhibited more pronounced anomalies in GBA and ASM activities than patients with ichthyosis linearis circumflexa. They also displayed a stronger increase in SC ceramides processed via ASM. We conclude that changes in the localization of active GBA and ASM correlate with i ) altered SC ceramide composition in NTS patients, ii ) local serine protease activity, and iii ) the clinical manifestation of NTS., (Copyright © 2020 van Smeden et al.)

The commercial farming of Atlantic salmon, Salmo salar, may require the periodic application of emamectin benzoate (EB) treatments to reduce the effects of biological pests, such as sea lice. As a result, EB is detected in sediments beneath these fish farms at considerable levels. Literature sediment toxicity data for EB for marine benthic species is only available for 10-day sediment toxicity tests, which might be too short to assess field effects. Here, we present a sediment toxicity test to determine 28-day mortality and growth effect concentrations for the non-target polychaete worm Arenicola marina, the crustacean Corophium volutator and the mollusk Cerastoderma edule using a marine microcosm setup. Results indicate that no concentration-dependent increase of mortality and growth rate was apparent to A. marina and C. edule. But for C. volutator, a concentration-dependent increase in mortality was observed, resulting in a calculated 28-d LC50 of 316 μg/kg dry sediment (95% confidence interval: 267-373 μg/kg dry sediment). There were significant effects on C. volutator growth rate at concentrations of 100 μg/kg dry sediment and above (NOEC = 30 μg/kg dry sediment). These observations show that C. volutator is more sensitive to EB than A. marina, which differs from results reported in previous studies. Comparison to the most sensitive NOEC (30 μg/kg dry sediment) found for C. volutator (organisms of 8-11 mm length), shows that the Environmental Quality Standard, derived by the Scottish Environment Protection Agency in 2017 which based on freshwater species data (NOEC = 1.175 μg/kg dry sediment), are relatively strict and is sufficiently protective for the marine species tested in this paper., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Glucocerebrosidase (GCase) is a retaining β-glucosidase with acid pH optimum metabolizing the glycosphingolipid glucosylceramide (GlcCer) to ceramide and glucose. Inherited deficiency of GCase causes the lysosomal storage disorder named Gaucher disease (GD). In GCase-deficient GD patients the accumulation of GlcCer in lysosomes of tissue macrophages is prominent. Based on the above, the key function of GCase as lysosomal hydrolase is well recognized, however it has become apparent that GCase fulfills in the human body at least one other key function beyond lysosomes. Crucially, GCase generates ceramides from GlcCer molecules in the outer part of the skin, a process essential for optimal skin barrier property and survival. This review covers the functions of GCase in and beyond lysosomes and also pays attention to the increasing insight in hitherto unexpected catalytic versatility of the enzyme.

The cornified lipid envelope (CLE) is a lipid monolayer covalently bound to the outside of corneocytes and is part of the stratum corneum (SC). The CLE is suggested to act as a scaffold for the unbound SC lipids. By profiling the bound CLE ceramides, a new subclass was discovered and identified as an omega-hydroxylated dihydrosphingosine (OdS) ceramide. Bound glucosylceramides were observed in superficial SC layers of healthy human skin. To investigate the relation between bound and unbound SC ceramides, the composition of both fractions was analyzed and compared. Selectivity in ceramide binding towards unsaturated ceramides and ceramides with a shorter chain length was observed. The selectivity in ceramide species bound to the cornified envelope is thought to have a physiological function in corneocyte flexibility. Next, it was examined if skin models exhibit an altered bound ceramide composition and if the composition was dependent on liver X-receptor (LXR) activation. The effects of an LXR agonist and antagonist on the bound ceramides composition of a full thickness model (FTM) were analyzed. In FTMs, a decreased amount of bound ceramides was observed compared to native human skin. Furthermore, FTMs had a bound ceramide fraction which consisted mostly of unsaturated and shorter ceramides. The LXR antagonist had a normalizing effect on the FTM bound ceramide composition. The agonist exhibited minimal effects. We show that ceramide binding is a selective process, yet, still is contingent on lipid synthesized., (Copyright © 2019. Published by Elsevier B.V.)

Restoring the lipid homeostasis of the stratum corneum (SC) is a common strategy to enhance skin barrier function. Here, we used a ceramide containing vernix caseosa (VC)-based formulation and were able to accelerate barrier recovery in healthy volunteers. The recovery was examined over 16 days by monitoring trans-epidermal water loss (TEWL) after barrier disruption by tape-stripping. Four skin sites were used to examine the effects of both treatment and barrier recovery. After 16 days, samples were harvested at these sites to examine the SC ceramide composition and lipid organization. Changes in ceramide profiles were identified using principal component analysis. After barrier recovery, the untreated sites showed increased levels of ceramide subclass AS and ceramides with a 34 total carbon-atom chain length, while the mean ceramide chain length was reduced. These changes were diminished by treatment with the studied formulation, which concurrently increased the formulated ceramides. Correlations were observed between SC lipid composition, lipid organization, and TEWL, and changes in the ceramide subclass composition suggest changes in the ceramide biosynthesis. These results suggest that VC-based formulations enhance skin barrier recovery and are attractive candidates to treat skin disorders with impaired barrier properties., (Copyright © 2018 by the American Society for Biochemistry and Molecular Biology, Inc.)

Epidermal β-glucocerebrosidase (GBA1), an acid β-glucosidase normally located in lysosomes, converts (glucosyl)ceramides into ceramides, which is crucial to generate an optimal barrier function of the outermost skin layer, the stratum corneum (SC). Here we report on two developed in situ methods to localize active GBA in human epidermis: i ) an optimized zymography method that is less labor intensive and visualizes enzymatic activity with higher resolution than currently reported methods using either substrate 4-methylumbelliferyl-β-D-glucopyranoside or resorufin-β-D-glucopyranoside; and ii ) a novel technique to visualize active GBA1 molecules by their specific labeling with a fluorescent activity-based probe (ABP), MDW941. The latter method pro-ved to be more robust and sensitive, provided higher resolution microscopic images, and was less prone to sample preparation effects. Moreover, in contrast to the zymography substrates that react with various β-glucosidases, MDW941 specifically labeled GBA1. We demonstrate that active GBA1 in the epidermis is primarily located in the extracellular lipid matrix at the interface of the viable epidermis and the lower layers of the SC. With ABP-labeling, we observed reduced GBA1 activity in 3D-cultured skin models when supplemented with the reversible inhibitor, isofagomine, irrespective of GBA expression. This inhibition affected the SC ceramide composition: MS analysis revealed an inhibitor-dependent increase in the glucosylceramide:ceramide ratio., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

Background: The barrier dysfunction in atopic dermatitis (AD) skin correlates with stratum corneum (SC) lipid abnormalities including reduction of global lipid content, shorter ceramide (CER) as well as free fatty acid (FFA) chain length and altered CER subclass levels. However, the underlying cause of these changes in lipid composition has not been fully investigated., Aim: We investigated whether the expression of CER and FFA biosynthesis enzymes are altered in AD skin compared with control skin and determine whether changes in enzyme expression can be related with changes in lipid composition., Methods: In AD patients and controls the expression of enzymes involved in the biosynthesis of FFAs and CERs was analyzed in relation to the SC lipid composition. These enzymes include stearoyl CoA desaturase (SCD), elongase 1 (ELOVL1) and ELOVL6 involved in FFA synthesis and β-glucocerebrosidase (GBA), acid-sphingomyelinase (aSmase), ceramide synthase 3 (CerS3) involved in CER synthesis. In TH2 treated human skin equivalents (AD HSEs) mimicking lesional AD skin, the mRNA expression of these enzymes was investigated., Results: The results reveal an altered expression of SCD and ELOVL1 in AD lesional skin. This was accompanied by functional changes displayed by increased unsaturated FFAs (SCD) and reduced FFA C22-C28 (ELOVL1) in AD lesional skin. The expression of GBA, aSmase and CerS3 were also altered in lesional skin. The CER composition in AD lesional skin showed corresponding changes such as increased CER AS and NS (aSmase) and decreased esterified ω-hydroxy CERs (CerS3). In support of the results from AD skin, the AD HSEs showed reduced mRNA ELOVL1, GBA and a Smase levels., Conclusion: This study shows that alterations in the expression of key enzymes involved in SC lipid synthesis contribute to changes in the lipid composition in AD skin and inflammation may influence expression of these enzymes., (Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Full thickness human skin models (FTMs) contain an epidermal and a dermal equivalent. The latter is composed of a collagen dermal matrix which harbours fibroblasts. Current epidermal barrier properties of FTMs do not fully resemble that of native human skin (NHS), which makes these human skin models less suitable for barrier related studies. To further enhance the resemblance of NHS for epidermal morphogenesis and barrier formation, we modulated the collagen dermal matrix with the biocompatible polymer chitosan. Herein, we report that these collagen-chitosan FTMs (CC-FTMs) possess a well-organized epidermis and maintain both the early and late differentiation programs as in FTMs. Distinctively, the epidermal cell activation is reduced in CC-FTMs to levels observed in NHS. Dermal-epidermal interactions are functional in both FTM types, based on the formation of the basement membrane. Evaluation of the barrier structure by the organization of the extracellular lipid matrix of the stratum corneum revealed an elongated repeat distance of the long periodicity phase. The ceramide composition exhibited a higher resemblance of the NHS, based on the carbon chain-length distribution and subclass profile. The inside-out barrier functionality indicated by the transepidermal water loss is significantly improved in the CC-FTMs. The expression of epidermal barrier lipid processing enzymes is marginally affected, although more restricted to a single granular layer. The novel CC-FTM resembles the NHS more closely, which makes them a promising tool for epidermal barrier related studies.

In several skin diseases, both the lipid composition and organization in the stratum corneum (SC) are altered which contributes to the impaired skin barrier function in patients. One of the approaches for skin barrier repair is treatment with topical formulations to normalize SC lipid composition and organization. Vernix caseosa (VC), a white cheesy cream on the skin during gestational delivery, has shown to enhance skin barrier repair. In this study, we examined how a fatty acid (FA) containing formulation mimicking the lipid composition of VC interacts with the lipid matrix in the SC. The formulation was applied on ex vivo human skin after SC removal. Subsequently, the ex vivo human skin generated SC during culture. The effect of FA containing formulations on the lipid organization and composition in the regenerated SC was analysed by Fourier transform infrared (FTIR) spectroscopy and liquid chromatography mass spectroscopy (LC/MS), respectively. FTIR results demonstrate that the FAs are intercalated in the lipid matrix of the regenerated SC and partition in the same lattice with the endogenous SC lipids, thereby enhancing the fraction of lipids forming an orthorhombic (very dense) packing in the SC. LC/MS data show that the topically applied FAs are elongated before intercalation in the lipid matrix and are thus involved in the lipid biosynthesis in the skin., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

In the outermost layer of the skin, the stratum corneum (SC), ceramides form a diverse and essential pool of lipids. Due to their diversity and the limited availability of synthetic standards it is challenging to quantitatively analyse all SC ceramides independently. We aim to perform a detailed analysis of ceramides on SC harvested from in vivo and ex vivo skin, therefore, a LC/MS method was developed in which all steps from sample acquisition until data analysis were examined and optimized. Improving extraction efficiency of ceramides resulted in an increase in efficiency from 71.5% to 99.3%. It was shown that sample harvesting by tape-stripping in vivo was accurate and precise. A full scan MS method was developed, compatible with all sample types, enabling simultaneously qualitative and quantitative data analysis. A novel three dimensional response model was constructed to quantify all detected ceramides from full scan data using a limited amount of synthetic ceramides. The application is demonstrated on various SC sample types. When ex vivo SC was regenerated during human skin culture, increases are observed in the amount of the ceramide sphingosine subclasses, in mono unsaturated ceramides (which have an cis-double bond in the acyl chain), and ceramides with a short C34 carbon chain (ceramides with a total carbon chain of 34 carbon atoms), compared with native human skin. These changes in ceramide levels are also often encountered in diseased skin., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Human skin acts as a primary barrier between the body and its environment. Crucial for this skin barrier function is the lipid matrix in the outermost layer of the skin, the stratum corneum (SC). Two of its functions are (1) to prevent excessive water loss through the epidermis and (2) to avoid that compounds from the environment permeate into the viable epidermal and dermal layers and thereby provoke an immune response. The composition of the SC lipid matrix is dominated by three lipid classes: cholesterol, free fatty acids and ceramides. These lipids adopt a highly ordered, 3-dimensional structure of stacked densely packed lipid layers (lipid lamellae): the lateral and lamellar lipid organization. The way in which these lipids are ordered depends on the composition of the lipids. One very common skin disease in which the SC lipid barrier is affected is atopic dermatitis (AD). This review addresses the SC lipid composition and organization in healthy skin, and elaborates on how these parameters are changed in lesional and nonlesional skin of AD patients. Concerning the lipid composition, the changes in the three main lipid classes and the importance of the carbon chain lengths of the lipids are discussed. In addition, this review addresses how these changes in lipid composition induce changes in lipid organization and subsequently correlate with an impaired skin barrier function in both lesional and nonlesional skin of these patients. Furthermore, the effect of filaggrin and mutations in the filaggrin gene on the SC lipid composition is critically discussed. Also, the breakdown products of filaggrin, the natural moisturizing factor molecules and its relation to SC-pH is described. Finally, the paper discusses some major changes in epidermal lipid biosynthesis in patients with AD and other related skin diseases, and how inflammation has a deteriorating effect on the SC lipids and SC biosynthesis. The review ends with perspectives on future studies in relation to other skin diseases., (© 2016 S. Karger AG, Basel.)

Our in-house human skin equivalents contain all stratum corneum (SC) barrier lipid classes, but have a reduced level of free fatty acids (FAs), of which a part is mono-unsaturated. These differences lead to an altered SC lipid organization and thereby a reduced barrier function compared to human skin. In this study, we aimed to improve the SC FA composition and, consequently, the SC lipid organization of the Leiden epidermal model (LEM) by specific medium supplements. The standard FA mixture (consisting of palmitic, linoleic and arachidonic acids) supplemented to the medium was modified, by replacing protonated palmitic acid with deuterated palmitic acid or by the addition of deuterated arachidic acid to the mixture, to determine whether FAs are taken up from the medium and are incorporated into SC of LEM. Furthermore, supplementation of the total FA mixture or that of palmitic acid alone was increased four times to examine whether this improves the SC FA composition and lipid organization of LEM. The results demonstrate that the deuterated FAs are taken up into LEMs and are subsequently elongated and incorporated in their SC. However, a fourfold increase in palmitic acid supplementation does not change the SC FA composition or lipid organization of LEM. Increasing the concentration of the total FA mixture in the medium resulted in a decreased level of very long chain FAs and an increased level of mono-unsaturated FAs, which lead to deteriorated SC lipid properties. These results indicate that SC lipid properties can be modulated by specific medium supplements., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Explant human skin equivalents (Ex-HSEs) can be generated by placing a 4mm skin biopsy onto a dermal equivalent. The keratinocytes migrate from the biopsy onto the dermal equivalent, differentiate and form the epidermis of 1(st) generation Ex-HSEs. This is especially suitable for the expansion of skin material from which only small fragments of skin can be harvested e.g. diseased skin., Objective: We evaluated whether 2(nd) and 3(rd) generation Ex-HSEs can also be generated from a single skin biopsy whilst maintaining the epidermal properties of 1(st) generation Ex-HSEs and native human skin., Methods: 2(nd) generation Ex-HSEs were produced by placing a biopsy from the 1(st) generation Ex-HSE onto a new dermal equivalent. Likewise, the 3(rd) generation Ex-HSEs were generated from a 2(nd) generation Ex-HSE biopsy., Results: We show for the first time that Ex-HSEs can be passaged to the 2(nd) and 3(rd) generation and display similar epidermal morphology and expression of differentiation markers as in native human skin and 1(st) generation Ex-HSEs except for involucrin. The 2(nd) and 3(rd) generation Ex-HSEs also show many similarities with 1(st) generation Ex-HSEs in lipid properties e.g. presence of all lipid classes, similar fatty acid chain length distribution and lamellar lipid organization. However, some differences arise in increased level of hexagonal lateral packing and a change in ceramide profiling. The changes in specific lipid classes were also accompanied by changes in the expression of the enzymes responsible for their synthesis., Conclusion: The expansion of skin biopsies to the 2(nd) and 3(rd) generation Ex-HSEs could be a promising method to expand valuable epidermal tissue to analyze morphological and differentiation parameters in the native epidermis., (Copyright © 2015 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Human skin equivalents (HSEs) can be considered a valuable tool to study aspects of human skin, including the skin barrier, or to perform chemical or toxicological screenings. HSEs are three-dimensional skin models that are usually established using primary keratinocytes and closely mimic human skin. The use of primary keratinocytes has several drawbacks, including a limited in vitro life span and large donor-donor variation. This makes them less favorable for in vitro toxicity screenings. Usage of an established keratinocyte cell line circumvents these drawbacks and enables the generation of easy-to-generate and reproducible HSEs, which can be used for pharmacological and/or toxicological screenings. For such screenings, a proper barrier function is required. In this study, we investigated the barrier properties of HSEs established with the keratinocyte cell line N/TERT (N-HSEs). N-HSEs showed comparable tissue morphology and expression of several epidermal proteins compared with HSEs established with primary keratinocytes. Our results clearly demonstrate that N-HSEs not only contain several stratum corneum (SC) barrier properties similar to HSEs, including the presence of the long periodicity phase and a comparable SC permeability, but also show some differences in lipid composition. Nonetheless, the similarities in barrier properties makes N/TERT cells a promising alternative for primary keratinocytes to generate HSEs.

Atopic dermatitis (AD) is a chronic inflammatory skin disease in which the skin barrier function is disrupted. In this inflammatory AD environment, cytokines are upregulated, but the cytokine effect on the AD skin barrier is not fully understood. We aimed to investigate the influence of Th2 (IL-4, IL-13, IL-31) and pro-inflammatory (tumor necrosis factor alpha (TNF-α)) cytokines on epidermal morphogenesis, proliferation, differentiation, and stratum corneum lipid properties. For this purpose, we used the Leiden epidermal model (LEM) in which the medium was supplemented with these cytokines. Our results show that IL-4, IL-13, IL-31, and TNF-α induce spongiosis, augment TSLP secretion by keratinocytes, and alter early and terminal differentiation-protein expression in LEMs. TNF-α alone or in combination with Th2 cytokines decreases the level of long chain free fatty acids (FFAs) and ester linked ω-hydroxy (EO) ceramides, consequently affecting the lipid organization. IL-31 increases long chain FFAs in LEMs but decreases relative abundance of EO ceramides. These findings clearly show that supplementation with TNF-α and Th2 cytokines influence epidermal morphogenesis and barrier function. As a result, these LEMs show similar characteristics as found in AD skin and can be used as an excellent tool for screening formulations and drugs for the treatment of AD.

Netherton syndrome (NTS) is a rare genetic skin disease caused by mutations in the serine protease inhibitor Kazal-type 5 gene, which encodes the lympho-epithelial Kazal-type-related inhibitor. NTS patients have profoundly impaired skin barrier function. As stratum corneum (SC) lipids have a crucial role in the skin barrier function, we investigated the SC lipid composition and organization in NTS patients. We studied the SC lipid composition by means of mass spectrometry, and the lipid organization was examined by infrared spectroscopy and X-ray diffraction. Decreased free fatty acid (FFA) chain length and increased levels of monounsaturated FFAs were observed in the SC of NTS patients compared with controls. Furthermore, the level of short-chain ceramides (CERs) was enhanced in NTS patients and a strong reduction in long-chain CER levels was seen in several patients. The changes in lipid composition modified the lipid organization leading to an increased disordering of the lipids compared with the controls. In addition, in a subgroup of patients the organization of the lipid layers changed dramatically. The altered FFA and CER profiles in NTS patients corresponded to changes in the expression of enzymes involved in SC lipid processing. The observed changes in lipid composition, lipid organization, and enzyme expression are likely to contribute to the barrier dysfunction in NTS.

An important feature of atopic eczema (AE) is a decreased skin barrier function. The stratum corneum (SC) lipids - comprised of ceramides (CERs), free fatty acids (FFAs) and cholesterol - fulfil a predominant role in the skin barrier function. In this clinical study, the carbon chain length distribution of SC lipids (FFAs and CERs) and their importance for the lipid organization and skin barrier function were examined in AE patients and compared with control subjects. A reduction in FFA chain length and an increase in unsaturated FFAs are observed in non-lesional and lesional SC of AE patients. The reduction in FFA chain length associates with a reduced CER chain length, suggesting a common synthetic pathway. The lipid chain length reduction correlates with a less dense lipid organization and a decreased skin barrier function. All changes are more pronounced in lesional SC compared with non-lesional skin. No association was observed between lipid properties and filaggrin mutations, an important predisposing factor for developing AE. The results of this study demonstrate an altered SC lipid composition and signify the importance of these changes (specifically regarding the CER and FFA chain lengths) for the impaired skin barrier function in AE. This provides insights into epidermal lipid metabolism as well as new opportunities for skin barrier repair., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Ceramides (CERs), cholesterol, and free fatty acids (FFAs) are the main lipid classes in human stratum corneum (SC, outermost skin layer), but no studies report on the detailed analysis of these classes in a single platform. The primary aims of this study were to 1) develop an LC/MS method for (semi-)quantitative analysis of all main lipid classes present in human SC; and 2) use this method to study in detail the lipid profiles of human skin substitutes and compare them to human SC lipids. By applying two injections of 10μl, the developed method detects all major SC lipids using RPLC and negative ion mode APCI-MS for detection of FFAs, and NPLC using positive ion mode APCI-MS to analyze CERs and cholesterol. Validation showed this lipid platform to be robust, reproducible, sensitive, and fast. The method was successfully applied on ex vivo human SC, human SC obtained from tape strips and human skin substitutes (porcine SC and human skin equivalents). In conjunction with FFA profiles, clear differences in CER profiles were observed between these different SC sources. Human skin equivalents more closely mimic the lipid composition of human stratum corneum than porcine skin does, although noticeable differences are still present. These differences gave biologically relevant information on some of the enzymes that are probably involved in SC lipid processing. For future research, this provides an excellent method for (semi-)quantitative, 'high-throughput' profiling of SC lipids and can be used to advance the understanding of skin lipids and the biological processes involved., (© 2013.)

Human skin mainly functions as an effective barrier against unwanted environmental influences. The barrier function strongly relies on the outermost layer of the skin, the stratum corneum (SC), which is composed of corneocytes embedded in an extracellular lipid matrix. The importance of a proper barrier function is shown in various skin disorders such as atopic dermatitis (AD), a complex human skin disorder strongly associated with filaggrin (FLG) null mutations, but their role in barrier function is yet unclear. To study the role of FLG in SC barrier properties in terms of SC lipid organization and lipid composition, we generated an N/TERT-based 3D-skin equivalent (NSE) after knock-down of FLG with shRNA. In these NSEs, we examined epidermal morphogenesis by evaluating the expression of differentiation markers keratin 10, FLG, loricrin and the proliferation marker ki67. Furthermore, the SC was extensively analysed for lipid organization, lipid composition and SC permeability. Our results demonstrate that FLG knock-down (FLG-KD) did not affect epidermal morphogenesis, SC lipid organization, lipid composition and SC permeability for a lipophilic compound in NSEs. Therefore, our findings indicate that FLG-KD alone does not necessarily affect the functionality of a proper barrier function., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Previous results showed that our in-house human skin equivalents (HSEs) differ in their stratum corneum (SC) lipid organization compared with human SC. To elucidate the cause of the altered SC lipid organization in the HSEs, a recently developed liquid chromatography/mass spectrometry method was used to study the free fatty acid (FFA) and ceramide composition in detail. In addition, the SC lipid composition of the HSEs and human skin was examined quantitatively with high-performance thin-layer chromatography. Our results reveal that all our HSEs have an increased presence of monounsaturated FFAs compared with human SC. Moreover, the HSEs display the presence of ceramide species with a monounsaturated acyl chain, which are not detected in human SC. All HSEs also exhibit an altered expression of stearoyl-CoA desaturase, the enzyme that converts saturated FFAs to monounsaturated FFAs. Furthermore, the HSEs show the presence of 12 ceramide subclasses, similar to native human SC. However, the HSEs have increased levels of ceramides EOS and EOH and ceramide species with short total carbon chains and a reduced FFA level compared with human SC. The presence of unsaturated lipid chains in HSE offers new opportunities to mimic the lipid properties of human SC more closely.

A hallmark of atopic eczema (AE) is skin barrier dysfunction. Lipids in the stratum corneum (SC), primarily ceramides, fatty acids, and cholesterol, are crucial for the barrier function, but their role in relation to AE is indistinct. Filaggrin is an epithelial barrier protein with a central role in the pathogenesis of AE. Nevertheless, the precise causes of AE-associated barrier dysfunction are largely unknown. In this study, a comprehensive analysis of ceramide composition and lipid organization in nonlesional SC of AE patients and control subjects was performed by means of mass spectrometry, infrared spectroscopy, and X-ray diffraction. In addition, the skin barrier and clinical state of the disease were examined. The level of ceramides with an extreme short chain length is drastically increased in SC of AE patients, which leads to an aberrant lipid organization and a decreased skin barrier function. Changes in SC lipid properties correlate with disease severity but are independent of filaggrin mutations. We demonstrate for the first time that changes in ceramide chain length and lipid organization are directly correlated with the skin barrier defects in nonlesional skin of AE patients. We envisage that these insights will provide a new therapeutic entry in therapy and prevention of AE.

Ceramides (CERs) in the upper layer of the skin, the stratum corneum (SC), play a key role in the skin barrier function. In human SC, the literature currently reports 11 CER subclasses that have been identified. In this paper, a novel quick and robust LC/MS method is presented that allows the separation and analysis of all known human SC CER subclasses using only limited sample preparation. Besides all 11 known and identified subclasses, a 3D multi-mass chromatogram shows the presence of other lipid subclasses. Using LC/MS/MS with an ion trap (IT) system, a Fourier transform-ion cyclotron resonance system, and a triple quadrupole system, we were able to identify one of these lipid subclasses as a new CER subclass: the ester-linked ω-hydroxy fatty acid with a dihydrosphingosine base (CER [EOdS]). Besides the identification of a new CER subclass, this paper also describes the applicability and robustness of the developed LC/MS method by analyzing three (biological) SC samples: SC from human dermatomed skin, human SC obtained by tape stripping, and SC from full-thickness skin explants. All three biological samples showed all known CER subclasses and slight differences were observed in CER profile.

A characterization and optimization of the in vitro transdermal iontophoretic transport of 5-hydroxy-2-(N,N,-di-n-propylamino)tetralin (5-OH-DPAT) is presented. The utility of acetaminophen as a marker of electroosmotic flow was studied as well. The following parameters of iontophoretic transport of 5-OH-DPAT were examined: drug donor concentration, electroosmotic contribution, influence of co-ions, current density, and composition of the acceptor phase. The steady-state flux (Flux(ss)) of acetaminophen was linearly correlated with the donor concentration and co-iontophoresis of acetaminophen did not influence the iontophoretic flux of 5-OH-DPAT, indicating that acetaminophen is an excellent marker of electroosmotic flow. Lowering the Na(+) concentration from 78 to 10 mM in the donor phase, resulted in a 2.5-fold enhancement of the Flux(ss). The Flux(ss) showed a nonlinear relation with the drug donor concentration and an excellent linear correlation with the current density. Reducing the pH of the acceptor phase from 7.4 to 6.2 resulted in a dramatic decrease of the Flux(ss) of 5-OH-DPAT, explained by a reduced electroosmotic flow and an increased counter-ion flow. Optimization of the conditions resulted in a maximum Flux(ss) of 5-OH-DPAT of 1.0 micromol x cm(-2) h(-1) demonstrating the potential of the iontophoretic delivery of this dopamine agonist for the symptomatic treatment of Parkinson's disease.