RESUMO
The fate of sediment associated compounds is the combined result of chemical properties and biological activities. Yet, studies simultaneously addressing the effects of biota on the redistribution and bioaccumulation of contaminants are scarce. Our aim was therefore to assess the effect of benthic invertebrate activities on organic matter degradation and the redistribution of metals and Polycyclic Aromatic Hydrocarbons (PAHs) in contaminated sediment. To this end, we introduced egg ropes of the non-biting midge Chironomus riparius into wastewater treatment plant sludge and allowed these to either develop until fourth instar larvae or to fully complete their life cycle into terrestrial flying adults. Chironomid larvae enhanced sludge degradation, resulting in increased metal concentrations in the sludge and in a flux of metals into the overlying water. Moreover, they hampered PAH degradation in the sludge. Contaminant transport from aquatic to terrestrial ecosystems with emerging invertebrates as a vector is widely acknowledged, but here we showed that biomanipulation prevailed over bioaccumulation, since due to chironomid activity, the flux of metals from the sludge into the overlying water was larger than into chironomid biomass. It is therefore concluded that contaminant-macroinvertebrate interactions are bilateral relationships driven by the interplay between macroinvertebrate traits and contaminant properties.
Assuntos
Hidrocarbonetos Policíclicos Aromáticos , Poluentes Químicos da Água , Animais , Ecossistema , Sedimentos Geológicos/química , Invertebrados , Larva , Metais/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/análise , Esgotos , Água/metabolismo , Poluentes Químicos da Água/análiseRESUMO
Deep-sea sponges create hotspots of biodiversity and biological activity in the otherwise barren deep-sea. However, it remains elusive how sponge hosts and their microbial symbionts acquire and process food in these food-limited environments. Therefore, we traced the processing (i.e. assimilation and respiration) of 13C- and 15N-enriched dissolved organic matter (DOM) and bacteria by three dominant North Atlantic deep-sea sponges: the high microbial abundance (HMA) demosponge Geodia barretti, the low microbial abundance (LMA) demosponge Hymedesmia paupertas, and the LMA hexactinellid Vazella pourtalesii. We also assessed the assimilation of both food sources into sponge- and bacteria-specific phospholipid-derived fatty acid (PLFA) biomarkers. All sponges were capable of assimilating DOM as well as bacteria. However, processing of the two food sources differed considerably between the tested species: the DOM assimilation-to-respiration efficiency was highest for the HMA sponge, yet uptake rates were 4-5 times lower compared to LMA sponges. In contrast, bacteria were assimilated most efficiently and at the highest rate by the hexactinellid compared to the demosponges. Our results indicate that phylogeny and functional traits (e.g., abundance of microbial symbionts, morphology) influence food preferences and diet composition of sponges, which further helps to understand their role as key ecosystem engineers of deep-sea habitats.
Assuntos
Bactérias/metabolismo , Biodiversidade , Poríferos/metabolismo , Poríferos/microbiologia , Água do Mar/microbiologia , Simbiose , Animais , Bactérias/genética , Biomarcadores , Isótopos de Carbono , DNA Bacteriano , Ácidos Graxos/química , Microbiota , Isótopos de Nitrogênio , Material Particulado , Fosfolipídeos/química , Filogenia , Análise de Sequência de DNARESUMO
The skin of the ear and the back are frequently selected sites in skin research using mouse models. However, distinct responses to treatment have been described between these two sites in several studies. Despite the crucial role of the stratum corneum (SC) in the skin barrier function of both dorsal back and ear skin, it remains unclear whether differences in lipid composition might underlie altered responses. Here, we compared the skin morphology and the barrier lipid composition of the ear with the back skin of wild-type mice. The ear contained more corneocyte layers in the SC and its barrier lipid composition was enriched with sphingosine ceramide subclasses, especially the short ones with a total chain length of 33-34 carbons. The free fatty acid (FFA) profile in the ear skin shifted towards shorter chains, significantly reducing the mean chain length to 23.3 vs 24.7 carbons in the back skin. In line, FFA species in the ear displayed a twofold increase in unsaturation index (P < .001). Gene expression in the ear skin revealed low expression of genes involved in lipid synthesis and uptake, indicating a reduced metabolic activity. Finally, the effects of hypercholesterolaemia on SC FFA composition was compared in ear and back skin of apolipoprotein E knockout (APOE-/- ) mice. Interestingly, the FFA profile in APOE-/- ear skin was minimally affected, while the FFA composition in the back skin was markedly changed in response to hypercholesterolaemia. In conclusion, ear and back skin have distinct barrier lipids and respond differently to elevated plasma cholesterol.
Assuntos
Ceramidas/metabolismo , Hipercolesterolemia/metabolismo , Metabolismo dos Lipídeos , Lipídeos/sangue , Pele/anatomia & histologia , Pele/metabolismo , Animais , Dorso , Orelha , Ácidos Graxos Insaturados/metabolismo , Ácidos Graxos Voláteis/metabolismo , Feminino , Perfilação da Expressão Gênica , Metabolismo dos Lipídeos/genética , Lipídeos/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , RNA Mensageiro/metabolismo , Pele/fisiopatologia , Fenômenos Fisiológicos da PeleRESUMO
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, ß-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.
Assuntos
Ceramidas/metabolismo , Metabolismo dos Lipídeos , Síndrome de Netherton/metabolismo , Peptídeo Hidrolases/metabolismo , Pele/enzimologia , Humanos , Síndrome de Netherton/enzimologia , Pele/metabolismoRESUMO
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 ß-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.
Assuntos
Dermatite Atópica/imunologia , Epiderme/patologia , Glucosilceramidase/metabolismo , Metabolismo dos Lipídeos/imunologia , Esfingomielina Fosfodiesterase/metabolismo , Adolescente , Adulto , Biópsia , Estudos de Casos e Controles , Ceramidas/análise , Ceramidas/metabolismo , Quimiocina CCL17/metabolismo , Dermatite Atópica/patologia , Epiderme/química , Epiderme/enzimologia , Ácidos Graxos não Esterificados/análise , Ácidos Graxos não Esterificados/metabolismo , Feminino , Voluntários Saudáveis , Humanos , Masculino , Perda Insensível de Água/imunologia , Adulto Jovem , beta-Defensinas/metabolismoRESUMO
Scavenger receptor class B type I (SR-BI) mediates the selective uptake of cholesteryl esters (CE) from high-density lipoproteins (HDL). An impaired SR-BI function leads to hyperalphalipoproteinemia with elevated levels of cholesterol transported in the HDL fraction. Accumulation of cholesterol in apolipoprotein B (apoB)-containing lipoproteins has been shown to alter skin lipid composition and barrier function in mice. To investigate whether these hypercholesterolemic effects on the skin also occur in hyperalphalipoproteinemia, we compared skins of wild-type and SR-BI knockout (SR-BI-/-) mice. SR-BI deficiency did not affect the epidermal cholesterol content and induced only minor changes in the ceramide subclasses. The epidermal free fatty acid (FFA) pool was, however, enriched in short and unsaturated chains. Plasma CE levels strongly correlated with epidermal FFA C18:1 content. The increase in epidermal FFA coincided with downregulation of cholesterol and FFA synthesis genes, suggesting a compensatory response to increased flux of plasma cholesterol and FFAs into the skin. Importantly, the SR-BI-/- epidermal lipid barrier showed increased permeability to ethyl-paraminobenzoic acid, indicating an impairment of the barrier function. In conclusion, increased HDL-cholesterol levels in SR-BI-/- mice can alter the epidermal lipid composition and lipid barrier function similarly as observed in hypercholesterolemia due to elevated levels of apoB-containing lipoproteins.
Assuntos
Proteínas de Transferência de Ésteres de Colesterol/deficiência , Epiderme/metabolismo , Erros Inatos do Metabolismo Lipídico/metabolismo , Ácido 4-Aminobenzoico/farmacocinética , Animais , Apolipoproteínas B/metabolismo , Antígenos CD36/genética , Proteínas de Transferência de Ésteres de Colesterol/genética , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Ésteres do Colesterol/sangue , Ésteres do Colesterol/metabolismo , Epiderme/patologia , Ácidos Graxos Insaturados/metabolismo , Feminino , Lecitinas/genética , Lecitinas/metabolismo , Erros Inatos do Metabolismo Lipídico/genética , Erros Inatos do Metabolismo Lipídico/patologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Long-term exposure to hypercholesterolemia induces the development of skin xanthoma's characterized by the accumulation of lipid-laden foam cells in humans and in mice. Early skin changes in response to hypercholesterolemia are however unknown. In this study, we investigated the skin lipid composition and associated barrier function in young adult low-density lipoprotein receptor knockout (LDLR-/-) and apolipoprotein E knockout (APOE-/-) mice, two commonly used hypercholesterolemic mouse models characterized by the accumulation of apolipoprotein B containing lipoproteins. No differences were observed on cholesterol content in the epidermis in LDLR-/- mice nor in the more extremely hypercholesterolemic APOE-/- mice. Interestingly, the free fatty acid profile in the APOE-/- epidermis shifted towards shorter and unsaturated chains. Genes involved in the synthesis of cholesterol and fatty acids were downregulated in APOE-/- skin suggesting a compensation for the higher influx of plasma lipids, most probably as cholesteryl esters. Importantly, in vivo transepidermal water loss and permeability studies with murine lipid model membranes revealed that the lipid composition of the APOE-/- skin resulted in a reduced skin barrier function. In conclusion, severe hypercholesterolemia associated with increased apolipoprotein B containing lipoproteins affects the epidermal lipid composition and its protective barrier.
Assuntos
Apolipoproteínas E/genética , Epiderme/química , Hipercolesterolemia/fisiopatologia , Lipídeos/química , Animais , Apolipoproteínas B/metabolismo , Apolipoproteínas E/deficiência , Ácidos Graxos/metabolismo , Hipercolesterolemia/metabolismo , Lipídeos/análise , Camundongos , Permeabilidade , Receptores de LDL/genéticaRESUMO
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.
Assuntos
Lipídeos/biossíntese , Pele/metabolismo , Verniz Caseoso/metabolismo , Adolescente , Adulto , Feminino , Humanos , Masculino , Verniz Caseoso/química , Adulto JovemRESUMO
Epidermal ß-glucocerebrosidase (GBA1), an acid ß-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-ß-D-glucopyranoside or resorufin-ß-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 ß-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.
Assuntos
Ensaios Enzimáticos , Corantes Fluorescentes/química , Glucosilceramidase/análise , Pele/enzimologia , Coloração e Rotulagem/métodos , Benzoxazinas/química , Compostos de Boro/química , Cicloexanóis/química , Compostos de Epóxi/química , Expressão Gênica , Glucosídeos/química , Glucosilceramidase/metabolismo , Humanos , Himecromona/análogos & derivados , Himecromona/química , Técnicas de Cultura de TecidosRESUMO
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 ß-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.
Assuntos
Ceramidas/metabolismo , Dermatite Atópica/patologia , Epiderme/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Lipogênese , Acetiltransferases/metabolismo , Adulto , Células Cultivadas , Ceramidas/análise , Ceramidas/biossíntese , Citocinas/imunologia , Dermatite Atópica/imunologia , Células Epidérmicas , Epiderme/patologia , Elongases de Ácidos Graxos , Ácidos Graxos não Esterificados/análise , Ácidos Graxos não Esterificados/biossíntese , Feminino , Glucosilceramidase/metabolismo , Humanos , Queratinócitos , Masculino , Esfingomielina Fosfodiesterase/metabolismo , Esfingosina N-Aciltransferase/metabolismo , Estearoil-CoA Dessaturase/metabolismo , Células Th2/metabolismo , Adulto JovemRESUMO
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.
Assuntos
Quitosana/metabolismo , Colágeno/metabolismo , Epiderme/metabolismo , Pele Artificial , Membrana Basal/citologia , Membrana Basal/metabolismo , Diferenciação Celular , Células Epidérmicas , Humanos , Queratinócitos/metabolismoRESUMO
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.
Assuntos
Epiderme/metabolismo , Ácidos Graxos/administração & dosagem , Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos , Fenômenos Fisiológicos da Pele , Administração Cutânea , Cromatografia Líquida , Epiderme/química , Ácidos Graxos/química , Humanos , Espectrometria de Massas , Espectroscopia de Infravermelho com Transformada de Fourier , Técnicas de Cultura de Tecidos , Verniz Caseoso/químicaRESUMO
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.
Assuntos
Ceramidas/análise , Metabolômica/métodos , Adulto , Calibragem , Ceramidas/química , Feminino , Humanos , Masculino , Padrões de Referência , Reprodutibilidade dos Testes , Pele/metabolismo , EstereoisomerismoRESUMO
Interdomain interactions between the CH3 domains of antibody heavy chains are the first step in antibody assembly and are of prime importance for maintaining the native structure of IgG. For human IgG4 it was shown that CH3-CH3 interactions are weak, resulting in the potential for half-molecule exchange ("Fab arm exchange"). Here we systematically investigated non-covalent interchain interactions for CH3 domains in the other human subclasses, including polymorphisms (allotypes), using real-time monitoring of Fab arm exchange with a FRET-based kinetic assay. We identified structural variation between human IgG subclasses and allotypes at three amino acid positions (Lys/Asn-392, Val/Met-397, Lys/Arg-409) to alter the strength of inter-domain interactions by >6 orders of magnitude. Each substitution affected the interactions independent from the other substitutions in terms of affinity, but the enthalpic and entropic contributions were non-additive, suggesting a complex interplay. Allotypic variation in IgG3 resulted in widely different CH3 interaction strengths that were even weaker for IgG3 than for IgG4 in the case of allotype G3m(c3c5*/6,24*), whereas G3m(s*/15*) was equally stable to IgG1. These interactions are sufficiently strong to maintain the structural integrity of IgG1 during its normal life span; for IgG2 and IgG3 the inter-heavy chain disulfide bonds are essential to prevent half-molecule dissociation, whereas the labile hinge disulfide bonds favor half-molecule exchange in vivo for IgG4.