Phenotypic flexibility in cutaneous water loss and lipids of the stratum corneum in house sparrows (Passer domesticus) following acclimation to high and low humidity

Resistance to water-vapor diffusion through the skin is thought to be conferred by lipids in the stratum corneum (SC), the outer layer of the epidermis. We tested the effect of ambient humidity on cutaneous water loss (CWL) and lipid composition of the SC by acclimating house sparrows (Passer domesticus) to either a dry (6.5 g/m(3) absolute humidity) or a humid (31 g/m(3)) environment for 3 wk at a thermoneutral temperature (30 degrees C). Sparrows in the dry-acclimated group reduced CWL by 36% compared with those in the humid environment. Relative to initial values, both groups of sparrows decreased CWL, 45% in the dry-acclimated group and 23% in the humid group, suggesting that temperature is also an important stimulus for CWL apart from humidity. Both groups of acclimated sparrows decreased quantities of cholesterol, free fatty acids, and cerebrosides and increased the proportion of ceramides in their SC. Lipid amounts or proportions in the SC did not differ between dry- and humid-acclimated sparrows, but the free fatty acid : ceramide ratio was significantly lower in dry-acclimated birds. Also, lipid composition was only correlated with CWL in dry-acclimated sparrows, suggesting that structural changes to SC lipids are more tightly linked to CWL regulation in response to low humidity. Our results demonstrate phenotypic flexibility in CWL and lipid composition of the SC and provide support for a functional relationship between these traits.     

Cutaneous water loss and lipids of the stratum corneum in two syntopic species of bats

The lipid matrix of the stratum corneum (SC), the outer layer of the epidermis of mammals and birds, constitutes the barrier to diffusion of water vapor through the skin. The lipids of the SC are structured in the intercellular spaces of the mammalian epidermis in ordered layers, called lamellae, which have been postulated to prevent water loss. Lipids in the mammalian SC are mainly cholesterol, free fatty acids and ceramides, the latter forming the structural support for the lamellae. However, knowledge on how the lipid composition of the SC alters cutaneous water loss (CWL) in mammals is rudimentary, and is largely derived from studies on laboratory animals and humans. We measured CWL of individuals of two species of syntopic bats, Tadarida brasiliensis and Myotis velifer. In the first study of its kind on wild mammals, we correlated CWL with the lipid composition of the SC, measured using thin layer chromatography and high performance liquid chromatography coupled with atmospheric pressure photoionization mass spectrometry. Surface-specific CWL was 20.6% higher in M. velifer than in T. brasiliensis, although differences were not significant. Compared with individuals of M. velifer, individuals of T. brasiliensis had more classes, and a higher proportion, of polar ceramides in the SC, a feature associated with lower CWL. Individuals of T. brasiliensis also had a class of non-polar ceramides that presumably spans the lamellae and gives more cohesiveness to the lipid matrix of the SC. We conclude that qualitative and quantitative modifications of the lipid composition of the SC contribute to regulate CWL of these two species of bats.     

Cutaneous water loss and the development of the stratum corneum of nestling house sparrows (Passer domesticus) from desert and mesic environments

Evaporation through the skin contributes to more than half of the total water loss in birds. Therefore, we expect the regulation of cutaneous water loss (CWL) to be crucial for birds, especially those that live in deserts, to maintain a normal state of hydration. Previous studies in adult birds showed that modifications of the lipid composition of the stratum corneum (SC), the outer layer of the epidermis, were associated with changes in rates of CWL. However, few studies have examined the ontogeny of CWL and the lipids of the SC in nestling birds. In this study, we measured CWL and the lipid composition of the SC during development of nestlings from two populations of house sparrows, one from the deserts of Saudi Arabia and the other from mesic Ohio. We found that desert and mesic nestlings followed different developmental trajectories for CWL. Desert nestlings seemed to make a more frugal use of water than did mesic nestlings. To regulate CWL, nestlings appeared to modify the lipid composition of the SC during ontogeny. Our results also suggest a tighter regulation of CWL in desert nestlings, presumably as a result of the stronger selection pressures to which nestlings are exposed in deserts.     

Cutaneous and respiratory water loss in larks from arid and mesic environments

Birds from deserts generally have lower total evaporative water loss (TEWL), the sum of cutaneous (CWL) and respiratory water loss (RWL), than species from mesic areas. We investigated the role of CWL and RWL as a function of air temperature (T(a)) in hoopoe larks (Alaemon alaudipes) and Dunn's larks (Eremalauda dunni) from the Arabian Desert and skylarks (Alauda arvensis) and woodlarks (Lullula arborea) from temperate mesic grasslands. The proportional contribution of CWL to TEWL in all larks at moderate T(a) ranged from 50% to 70%. At high T(a) (40 degrees -45 degrees C), larks enhanced CWL by only 45%-78% and relied on an increase in RWL by 676%-2,733% for evaporative cooling. Surface-specific CWL at 25 degrees C was 29% lower in the arid-zone species than in the mesic larks. When acclimated to constant T(a), 15 degrees C-acclimated hoopoe larks increased CWL by 22% compared with 35 degrees C-acclimated birds, but the other species did not change CWL. This study is consistent with the hypothesis that larks from deserts have a reduced CWL at moderate and low T(a) but provided no support for the hypothesis that at high T(a) larks from arid regions rely more on CWL than larks from mesic environments. Interspecific differences in CWL cannot be attributed to acclimation to environmental temperature and are possibly the result of genetic differences due to natural selection or of phenotypically plastic responses to divergent environments during ontogeny.     

beta-Glucocerebrosidase activity in the stratum corneum of house sparrows following acclimation to high and low humidity

Skin is an important avenue of water loss in terrestrial birds, so environmental conditions that necessitate water conservation should favor physiological mechanisms that reduce cutaneous water loss (CWL). Skin resistance to CWL is conferred by a barrier of lipid molecules located in the stratum corneum (SC), the outer layer of the epidermis. In mammals, SC barrier function depends on the conversion of cerebrosides to ceramides by the enzyme beta -glucocerebrosidase ( beta -GlcCer'ase). Avian SC contains both cerebrosides and ceramides, suggesting that observed plasticity in CWL may be mediated by changes in beta -GlcCer'ase activity and resultant SC lipid composition. We tested the hypothesis that changes in ambient humidity would alter beta -GlcCer'ase activity by acclimating house sparrows (Passer domesticus) to either dry (6.5 g H(2)O m(-3) absolute humidity) or humid (31 g H(2)O m(-3)) conditions for 5 and 21 d at 30 degrees C and then measuring beta -GlcCer'ase activity from SC homogenates. Our results provide the first characterization of beta -GlcCer'ase activity in any nonmammalian vertebrate. Relative to nonacclimated controls, both dry- and humid-acclimated sparrows had significantly elevated beta -GlcCer'ase activity at 21 d postacclimation. Across individuals, we observed negative correlations between beta -GlcCer'ase activity and both CWL and SC ceramide content. Although dry- and humid-acclimated sparrows did not differ in beta -GlcCer'ase activity, these results are consistent with our findings that both humidity treatments caused a reduction in CWL and similar changes in SC lipid composition. Our results demonstrate physiological plasticity in CWL and provide tentative support for a role of beta -GlcCer'ase in mediating this response.     

Cocoon and epidermis of Australian Cyclorana frogs differ in composition of lipid classes that affect water loss

For amphibians to survive in environments that experience annual droughts, they must minimize evaporative water loss. One genus of Australian hylid frogs, Cyclorana, prevents desiccation by burrowing in the soil and forming cocoons composed of alternating layers of shed epidermis and glandular secretions. Previous data are inconclusive about the role that lipids play in reducing evaporative water loss through skin (cutaneous water loss [CWL]) when Cyclorana spp. are within cocoons. In this study, we measured CWL and lipids in the epidermis and in cocoons of five species of Cyclorana. CWL was significantly lower in frogs within cocoons than in frogs without cocoons. Surface-area-specific CWL for the three small species was significantly higher than that of the two larger species of Cyclorana, but this difference was not apparent in frogs within cocoons. Although lipids were responsible for more of the dry mass of the epidermis (approximately 20%) than of the cocoons (approximately 7%) we found that cerebrosides and ceramides, two polar lipid classes, were almost exclusively found in cocoons. This suggests that these lipid classes are in the glandular secretions rather than in the epidermis. Because these polar lipids are the types that reduce water loss in birds (cerebrosides and ceramides) and mammals (ceramides), we conclude that they are important not only for holding together the shed layers of skin but also for contributing to the barrier against water loss.     

Novel lipid mixtures based on synthetic ceramides reproduce the unique stratum corneum lipid organization

Lipid lamellae present in the outermost layer of the skin protect the body from uncontrolled water loss. In human stratum corneum (SC), two crystalline lamellar phases are present, which contain mostly cholesterol, free fatty acids, and nine types of free ceramides. Previous studies have demonstrated that the SC lipid organization can be mimicked with model mixtures based on isolated SC lipids. However, those studies are hampered by low availability and high interindividual variability of the native tissue. To elucidate the role of each lipid class in the formation of a competent skin barrier, the use of synthetic lipids would offer an alternative. The small- and wide-angle X-ray diffraction results of the present study show for the first time that synthetic lipid mixtures, containing only three synthetic ceramides, reflect to a high extent the SC lipid organization. Both an appropriately chosen preparation method and lipid composition promote the formation of two characteristic lamellar phases with repeat distances similar to those found in native SC. From all synthetic lipid mixtures examined, equimolar mixtures of cholesterol, ceramides, and free fatty acids equilibrated at 80 degrees C resemble to the highest extent the lamellar and lateral SC lipid organization, both at room and increased temperatures.     

Phase behaviour of skin barrier model membranes at pH 7.4.

The main function of the skin is to protect the body against exogenous substances. The skin barrier is located in the outermost layer of the skin, the stratum corneum (SC). This layer consists of keratin enriched cells embedded in lipid lamellae that form the main barrier for diffusion of substances through the skin. The main lipid classes in this barrier are ceramides, cholesterol and free fatty acids. Cholesterol sulfate and calcium are also present in SC. Furthermore it has been suggested that a pH gradient exists. In a previous paper the effect of cholesterol sulfate and calcium on the lipid phase behaviour of mixtures prepared from cholesterol, ceramides and free fatty acids at pH 5 was reported (approximate pH at the skin surface). In the present study the phase behaviour of mixtures prepared from cholesterol, ceramides and free fatty acids prepared at pH 7.4 (the pH of viable cells) has been examined between 25 and 95 degrees C. Our studies reveal that a reversed hexagonal phase has been formed at elevated temperatures. Addition of calcium inhibits the formation of the reversed hexagonal phase, while cholesterol sulfate promotes the presence of the reversed hexagonal phase at increased temperatures. From our results we can conclude that the lipid mixtures prepared at pH 5 resemble more closely the lipid phase behaviour in intact SC than the lipid mixtures prepared at pH 7.4.     

Preparation and characterization of a stratum corneum substitute for in vitro percutaneous penetration studies

The intercellular stratum corneum (SC) lipids form the main barrier for diffusion of substances through the skin. A porous substrate covered with synthetic SC lipids would be an attractive model to study percutaneous penetration, hereby replacing native human SC. Prerequisite is that this stratum corneum substitute (SCS) is prepared with a uniform lipid composition and layer thickness. Furthermore, the lipid organization and orientation should resemble that in SC. The objective of this study was to investigate the utility of an airbrush spraying device to prepare a SCS composed of cholesterol, ceramides and free fatty acids on a polycarbonate filter. The results demonstrate that a proper choice of solvent mixture and lipid concentration is crucial to achieve a uniform distribution of the applied lipids over the filter surface. A smooth and tightly packed lipid layer is only obtained when the equilibration conditions are appropriately chosen. The SCS possesses two crystalline lamellar phases with periodicities similar to those present in native SC. The orientation of these lamellae is mainly parallel to the surface of the polycarbonate filter, which resembles the orientation of the intercellular SC lipids. In conclusion, the airbrush technique enables generation of a homogeneous SCS, which ultimately may function as a predictive in vitro percutaneous penetration model.     

Preparation and characterization of a stratum corneum substitute for in vitro percutaneous penetration studies

The intercellular stratum corneum (SC) lipids form the main barrier for diffusion of substances through the skin. A porous substrate covered with synthetic SC lipids would be an attractive model to study percutaneous penetration, hereby replacing native human SC. Prerequisite is that this stratum corneum substitute (SCS) is prepared with a uniform lipid composition and layer thickness. Furthermore, the lipid organization and orientation should resemble that in SC. The objective of this study was to investigate the utility of an airbrush spraying device to prepare a SCS composed of cholesterol, ceramides and free fatty acids on a polycarbonate filter. The results demonstrate that a proper choice of solvent mixture and lipid concentration is crucial to achieve a uniform distribution of the applied lipids over the filter surface. A smooth and tightly packed lipid layer is only obtained when the equilibration conditions are appropriately chosen. The SCS possesses two crystalline lamellar phases with periodicities similar to those present in native SC. The orientation of these lamellae is mainly parallel to the surface of the polycarbonate filter, which resembles the orientation of the intercellular SC lipids. In conclusion, the airbrush technique enables generation of a homogeneous SCS, which ultimately may function as a predictive in vitro percutaneous penetration model.     

Skin lipids from Saudi Arabian birds

Skin lipids play an important role in the regulation of cutaneous water loss (CWL). Earlier studies have shown that Saudi desert birds exhibit a tendency of reduced CWL than birds from temperate environment due to adaptive changes in composition of their skin lipids. In this study, we used thin-layer chromatography (TLC) for separation and detection of non-polar and polar lipids from the skin of six bird species including sooty gull, brown booby, house sparrow, Arabian waxbill, sand partridge, and laughing dove. The lipids were separated and detected on Silica gel G coated TLC plates and quantified by using densitometric image analysis. Rf values of the non-polar lipids were as follows: cholesterol (0.29), free fatty acids (0.58), triacylglycerol (0.69), fatty acids methyl esters (0.84) and cholesterol ester (0.97). Rf values for the polar lipids were: cerebroside (0.42), ceramide (0.55) and cholesterol (0.73). The results showed the abundance of fatty acids methyl esters (47.75–60.46%) followed by triacylglycerol (12.69–24.14%). The remaining lipid compositions were as follows: cholesterol (4.09–13.18%), ceramide (2.18–13.27%), and cerebroside (2.53–12.81%). In conclusion, our findings showed that TLC is a simple and sensitive method for the separation and quantification of skin lipids. We also reported a new protocol for lipid extraction using the zirconia beads for efficient disruption of skin tissues. This study will help us better understand the role of skin lipids in adaptive physiology towards adverse climatic conditions.     

Physiological adjustments to arid and mesic environments in larks (Alaudidae)

Because deserts are characterized by low food availability, high ambient temperature extremes, and absence of drinking water, one might expect that birds that live in these conditions exhibit a lower basal metabolic rate (BMR), reduced total evaporative water loss (TEWL), and greater ability to cope with high air temperatures than their mesic counterparts. To minimize confounding effects of phylogeny, we compared the physiological performance of four species of larks at ambient temperatures (T(a)'s) ranging from 0 degrees to 50 degrees C: hoopoe larks (Alaemon alaudipes) and Dunn's larks (Eremalauda dunni) live in hot and dry deserts, whereas skylarks (Alauda arvensis) and woodlarks (Lullula arborea) occur in temperate mesic areas. Mass-adjusted BMR and TEWL were indistinguishable between hoopoe lark and Dunn's lark and between skylark and woodlark. When grouping the data of the two desert larks in one set and the data of the two mesic larks in another, desert larks are shown to have 43% lower BMR levels and 27% lower TEWL values than the mesic species. Their body temperatures (T(b)'s) were 1.1 degrees C lower, and the minimal dry heat transfer coefficients (h) were 26% below values for the mesic larks. When T(a) exceeded T(b), the h of hoopoe larks and Dunn's larks was high and indistinguishable from h at 40 degrees C, in contrast to the prediction that h should be decreased to minimize heat gain through conductance, convection, or radiation from the environment when T(a) exceeds T(b).     

The Lipid Organisation of the Skin Barrier: Liquid and Crystalline Domains Coexist in Lamellar Phases

The superficial layer of the skin, the stratum corneum, is the main barrier for diffusion of substances across the skin. The stratum corneum is composed of corneocytes embedded in lipid lamellae. In previous studies two lamellar phases have been identified with periodicities of 6.4 and 13.4 nm of which the 13.4 nm phase (long periodicity phase = LPP) is considered to be very important for the skin banier function. The main lipid classes in stratumcorneum are ceramides, free fatty acids and cholesterol. Until now 8 subclassesof ceramides are identified in human stratum corneum referred to as ceramide 1 to 8. Studies with mixtures prepared with isolated human ceramides revealed that cholesterol and ceramides are very important for the formation of the lamellar phases. After addition of free fatty acids the lipids are organised in an orthorhombic packing with a small proportion of lipids in a liquid phase. Our most recent results show that the presence of ceramide 1 and the formation of a liquid phase are crucial elements for the formation of the LPP. These observations and the broad-narrowbroad sequence of lipid layers in the LPP led us to propose a molecular model for this phase. This consists of one narrow central lipid layer with fluid domains with on both sides a broad layer with a crystalline structure. This model is referred to as `the sandwich model'.     

Long periodicity phase in extracted lipids of vernix caseosa obtained with equilibration at physiological temperature

The outermost layer of the skin, the stratum corneum (SC), comprises the main barrier function between body and environment. The SC features a highly structured lipid organization: a short periodicity phase and a long periodicity phase (LPP) with a repeat distance of 6 and 13 nm, respectively. Like SC, vernix caseosa (VC), the creamy white skin-surface biofilm of the newborn, also contains barrier lipids, i.e. ceramides, cholesterol and free fatty acids. Aim of this study was to investigate whether isolated VC lipids also form the characteristic LPP. Several preparation methods were examined and only when the solution of the lipid mixture, isolated either from VC or SC, was dried under nitrogen at 37 °C and subsequently spread onto a support, the LPP was formed. When VC barrier lipids were first exposed to elevated temperatures and subsequently cooled down, the LPP was formed at around 34 °C, which is at a much lower temperature than observed with the lipids in SC. In conclusion, we showed for the first time that depending on the preparation method, (i) VC lipids also form the LPP and (ii) the LPP in VC lipids and SC lipids was obtained at a low equilibration temperature, mimicking the physiological condition.     

Interactions of dipalmitoylphosphatidylcholine with ceramide-based mixtures

The outermost layer of the skin, the stratum corneum (SC), acts as the natural physical barrier. The SC consists of corneocytes embedded in a crystalline lipid matrix consisting of ceramides, free fatty acids and cholesterol.Although phospholipids are frequently present in topical formulations, no detailed information is reported on the interactions between phospholipids and SC lipids. The aim of this study was to examine the interactions between a model phospholipid, dipalmitoylphosphatidylcholine (DPPC) and synthetic ceramide-based mixtures (referred to as SC lipids).(Perdeuterated) DPPC was mixed with SC lipids and the lipid organization and mixing properties were examined. The studies revealed that DPPC participates in the same lattice as SC lipids thereby enhancing a hexagonal packing. Even at a high DPPC level, no phase separated pure DPPC was observed.When a DPPC containing formulation is applied to the skin surface it must partition into the SC lipid matrix prior to any mixing with the SC lipids. To mimic this, DPPC was applied on top of a SC lipid membrane. DPPC applied in a liquid crystalline state was able to mix with the SC lipids and participated in the same lattice as the SC lipids. However, when DPPC was applied in a rippled gel-state very limited partitioning of DPPC into the SC lipid matrix occurred. Thus, when applied to the skin, liquid crystalline DPPC will have very different interactions with SC lipids than DPPC in a (rippled-)gel phase.     

A survey of polar and nonpolar lipids from epidermis and epidermal appendages of the chicken (Gallus domesticus)

Total lipid was extracted from chicken (Gallus domesticus) epidermis, leg scale, claws, feathers and preen glands and analyzed by quantitative thin-layer chromatography. All of the tissue lipids contained large proportions of wax diesters, triglycerides, and free sterols and variable proportions of phospholipids, steryl esters and free fatty acids. All of the keratinized tissues, but not the preen gland, contained ceramides, acylceramides and cholesteryl sulfate. Acylglucosylceramides were found only in full thickness epidermis. Glucosylsterols and acylglucosylsterols were found in the keratinized tissues, and may be of significance in the evolutionary history of the epidermal water barrier.     

The phase behaviour of skin lipid mixtures based on synthetic ceramides

The lipid lamellae present in the outermost layer of the skin, the stratum corneum (SC), form the main barrier for diffusion of molecules across the skin. The main lipid classes in SC are cholesterol (CHOL), free fatty acids (FFA) and at least nine classes of ceramides (CER), referred to as CER1 to CER9. In the present study the phase behaviour of four synthetic CER, either single or mixed with CHOL or CHOL and FFA, has been studied using small and wide angle X-ray diffraction. The lipid mixtures showed complex phase behaviour with coexistence of several phases. The results further revealed that the presence of synthetic CER1 as well as a proper composition of the other CER in the mixture were crucial for the formation of a phase with a long periodicity, characteristic for SC lipid phase behaviour. Only a mixture containing synthetic CER1 and CER3, CHOL and FFA showed similar phase behaviour to that of SC.     

Influence of different ceramides on the structure of in vitro model lipid systems of the stratum corneum lipid matrix

Human stratum corneum (SC) consists of several layers of keratinized corneocytes embedded in a lipid matrix of ordered lamellar structure which is considered to constitute the major barrier to percutaneous penetration. Artificial mixtures of SC lipids are often used as model systems to mimic the skin barrier or to investigate the effects of substances on the phase behaviour of the models. In the present study a SC lipid model composed of cholesterol, fatty acids and ceramides was used to investigate the effect of three different commercially available ceramide types on the microstructure and the physicochemical behaviour of the lipids. Polarized light microscopy, transmission electron microscopy, small-angle X-ray diffraction, wide-angle X-ray diffraction and differential scanning calorimetry (DSC) were used for physicochemical characterization. The results revealed a lamellar structure for all models but showed differences with regard to the thermal and optical behaviour depending obviously on the composition of the ceramide mixtures. A model containing a mixture of Cer[AS] was comparable to human SC lipids.     

Effect of sphingosine and phytosphingosine ceramide ratio on lipid arrangement and barrier function in skin lipid models

The lipids in the uppermost layer of the skin, the stratum corneum (SC), play an important role in the skin barrier function. The three main subclasses in the SC lipid matrix are ceramides (CER), cholesterol, and free fatty acids. In inflammatory skin diseases, such as atopic dermatitis and psoriasis, the SC lipid composition is modulated compared to the composition in healthy SC. One of the main alterations is the molar ratio between the concentration of CER N-(tetracosanoyl)-sphingosine (CER NS) and CER N-(tetracosanoyl)-phytosphingosine (CER NP), which correlated with an impaired skin barrier function. In the present study, we investigated the impact of varying the CER NS:CER NP ratios on the lipid organization, lipid arrangement, and barrier functionality in SC lipid model systems. The results indicate that a higher CER NS:CER NP ratio as observed in diseased skin did not alter the lipid organization or lipid arrangement in the long periodicity phase encountered in SC. The trans-epidermal water loss, an indication of the barrier functionality, was significantly higher for the CER NS:CER NP 2:1 model (mimicking the ratio in inflammatory skin diseases) compared to the CER NS:CER NP 1:2 ratio (in healthy skin). These findings provide a more detailed insight into the lipid organization in both healthy and diseased skin and suggest that in vivo the molar ratio between CER NS:CER NP contributes to barrier impairment as well but might not be the main factor.     

The role of ceramide chain length distribution on the barrier properties of the skin lipid membranes

The skin barrier function is provided by the stratum corneum (SC). The lipids in the SC are composed of three lipid classes: ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs) which form two crystalline lamellar structures. In the present study, we investigate the effect of CER chain length distribution on the barrier properties of model lipid membranes mimicking the lipid composition and organization of SC. The membranes were prepared with either isolated pig CERs (PCERs) or synthetic CERs. While PCERs have a wide chain length distribution, the synthetic CERs are quite uniform in chain length. The barrier properties were examined by means of permeation studies using hydrocortisone as a model drug. Our studies revealed a reduced barrier in lipid membranes prepared with PCERs compared to synthetic CERs. Additional studies revealed that a wider chain length distribution of PCERs results in an enhanced hexagonal packing and increased conformational disordering of the lipid tails compared to synthetic CERs, while the lamellar phases did not change. This demonstrates that the chain length distribution affects the lipid barrier by reducing the lipid ordering and density within the lipid lamellae. In subsequent studies, the effect of increased levels of FFAs or CERs with a long acyl chain in the PCERs membranes was also studied. These changes in lipid composition enhanced the level of orthorhombic packing, reduced the conformational disordering and increased the barrier of the lipid membranes. In conclusion, the CER chain length distribution is an important key factor for maintaining a proper barrier.