Evaluation of fluid transport processes in dental enamel. Methods to assess the relevance of enamel permeability in caries prevention and etching treatments.

This thesis evaluated in vivo and in vitro enamel permeability in different physiological and clinical conditions by means of SEM inspection of replicas of enamel surface obtained from polyvinyl siloxane impressions subsequently later cast in polyether impression ma-terial. This technique, not invasive and risk-free, allows the evaluation of fluid outflow from enamel surface and is able to detect the presence of small quantities of fluid, visu-alized as droplets. Fluid outflow on enamel surface represents enamel permeability. This property has a paramount importance in enamel physiolgy and pathology although its ef-fective role in adhesion, caries pathogenesis and prevention today is still not fully under-stood. The aim of the studies proposed was to evaluate enamel permeability changes in differ-ent conditions and to correlate the findings with the actual knowledge about enamel physiology, caries pathogenesis, fluoride and etchinhg treatments. To obtain confirmed data the replica technique has been supported by others specific techniques such as Ra-man and IR spectroscopy and EDX analysis. The first study carried out visualized fluid movement through dental enamel in vivo con-firmed that enamel is a permeable substrate and demonstrated that age and enamel per-meability are closely related. Examined samples from subjects of different ages showed a decreasing number and size of droplets with increasing age: freshly erupted permanent teeth showed many droplets covering the entire enamel surface. Droplets in permanent teeth were prominent along enamel perikymata. These results obtained through SEM inspection of replicas allowed innovative remarks in enamel physiology. An analogous testing has been developed for evaluation of enamel permeability in primary enamel. The results of this second study showed that primary enamel revealed a substantive permeability with droplets covering the entire enamel sur-face without any specific localization accordingly with histological features, without changes during aging signs of post-eruptive maturation. These results confirmed clinical data that showed a higher caries susceptibility for primary enamel and suggested a strong relationship between this one and enamel permeability. Topical fluoride application represents the gold standard for caries prevention although the mechanism of cariostatic effect of fluoride still needs to be clarified. The effects of topical fluoride application on enamel permeability were evaluated. Particularly two dif-ferent treatments (NaF and APF), with different pH, were examined. The major product of topical fluoride application was the deposition of CaF2-like globules. Replicas inspec-tion before and after both treatments at different times intervals and after specific addi-tional clinical interventions showed that such globule formed in vivo could be removed by professional toothbrushing, sonically and chemically by KOH. The results obtained in relation to enamel permeability showed that fluoride treatments temporarily reduced enamel water permeability when CaF2-like globules were removed. The in vivo perma-nence of decreased enamel permeability after CaF2 globules removal has been demon-strated for 1 h for NaF treated teeth and for at least 7 days for APF treated teeth. Important clinical consideration moved from these results. In fact the caries-preventing action of fluoride application may be due, in part, to its ability to decrease enamel water permeability and CaF2 like-globules seem to be indirectly involved in enamel protection over time maintaining low permeability. Others results obtained by metallographic microscope and SEM/EDX analyses of or-thodontic resins fluoride releasing and not demonstrated the relevance of topical fluo-ride application in decreasing the demineralization marks and modifying the chemical composition of the enamel in the treated area. These data obtained in both the experiments confirmed the efficacy of fluoride in caries prevention and contribute to clarify its mechanism of action. Adhesive dentistry is the gold standard for caries treatment and tooth rehabilitation and is founded on important chemical and physical principles involving both enamel and dentine substrates. Particularly acid etching of dental enamel enamel has usually employed in bonding pro-cedures increasing microscopic roughness. Different acids have been tested in the litera-ture suggesting several etching procedures. The acid-induced structural transformations in enamel after different etching treatments by means of Raman and IR spectroscopy analysis were evaluated and these findings were correlated with enamel permeability. Conventional etching with 37% phosphoric acid gel (H3PO4) for 30 s and etching with 15 % HCl for 120 s were investigated. Raman and IR spectroscopy showed that the treatment with both hydrochloric and phosphoric acids induced a decrease in the carbonate content of the enamel apatite. At the same time, both acids induced the formation of HPO42- ions. After H3PO4 treatment the bands due to the organic component of enamel decreased in intensity, while in-creased after HCl treatment. Replicas of H3PO4 treated enamel showed a strongly reduced permeability while replicas of HCl 15% treated samples showed a maintained permeability. A decrease of the enamel organic component, as resulted after H3PO4 treatment, involves a decrease in enamel permeability, while the increase of the organic matter (achieved by HCl treat-ment) still maintains enamel permeability. These results suggested a correlation between the amount of the organic matter, enamel permeability and caries. The results of the different studies carried out in this thesis contributed to clarify and improve the knowledge about enamel properties with important rebounds in theoretical and clinical aspects of Dentistry.

β1-integrin/matrix interactions support blood-brain barrier integrity

Brain microvascular endothelium forms an active permeability barrier, the blood-brain barrier (BBB). In neurologic disorders, barrier properties of the BBB are often lost indicating their dependance on molecular cues of the brain microenvironment. In this issue, Osada et al demonstrate that the endothelial extracellular matrix (ECM) provides one of these cues. Their study shows that β1-integrin-mediated adhesion of brain endothelial cells to the surrounding ECM is critical for stabilizing claudin-5 in BBB tight junctions (TJs) and BBB integrity. These observations point to a novel intracellular signaling pathway from β1-integrin/ECM endothelial adhesions to BBB TJs contributing to BBB integrity.

The blood-brain and the blood-cerebrospinal fluid barriers: function and dysfunction

The central nervous system (CNS) is tightly sealed from the changeable milieu of blood by the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier (BCSFB). While the BBB is considered to be localized at the level of the endothelial cells within CNS microvessels, the BCSFB is established by choroid plexus epithelial cells. The BBB inhibits the free paracellular diffusion of water-soluble molecules by an elaborate network of complex tight junctions (TJs) that interconnects the endothelial cells. Combined with the absence of fenestrae and an extremely low pinocytotic activity, which inhibit transcellular passage of molecules across the barrier, these morphological peculiarities establish the physical permeability barrier of the BBB. In addition, a functional BBB is manifested by a number of permanently active transport mechanisms, specifically expressed by brain capillary endothelial cells that ensure the transport of nutrients into the CNS and exclusion of blood-borne molecules that could be detrimental to the milieu required for neural transmission. Finally, while the endothelial cells constitute the physical and metabolic barrier per se, interactions with adjacent cellular and acellular layers are prerequisites for barrier function. The fully differentiated BBB consists of a complex system comprising the highly specialized endothelial cells and their underlying basement membrane in which a large number of pericytes are embedded, perivascular antigen-presenting cells, and an ensheathment of astrocytic endfeet and associated parenchymal basement membrane. Endothelial cell morphology, biochemistry, and function thus make these brain microvascular endothelial cells unique and distinguishable from all other endothelial cells in the body. Similar to the endothelial barrier, the morphological correlate of the BCSFB is found at the level of unique apical tight junctions between the choroid plexus epithelial cells inhibiting paracellular diffusion of water-soluble molecules across this barrier. Besides its barrier function, choroid plexus epithelial cells have a secretory function and produce the CSF. The barrier and secretory function of the choroid plexus epithelial cells are maintained by the expression of numerous transport systems allowing the directed transport of ions and nutrients into the CSF and the removal of toxic agents out of the CSF. In the event of CNS pathology, barrier characteristics of the blood-CNS barriers are altered, leading to edema formation and recruitment of inflammatory cells into the CNS. In this review we will describe current knowledge on the cellular and molecular basis of the functional and dysfunctional blood-CNS barriers with focus on CNS autoimmune inflammation.

Modeling immune functions of the mouse blood-cerebrospinal fluid barrier in vitro: primary rather than immortalized mouse choroid plexus epithelial cells are suited to study immune cell migration across this brain barrier.

BACKGROUND The blood-cerebrospinal fluid barrier (BCSFB) established by the choroid plexus (CP) epithelium has been recognized as a potential entry site of immune cells into the central nervous system during immunosurveillance and neuroinflammation. The location of the choroid plexus impedes in vivo analysis of immune cell trafficking across the BCSFB. Thus, research on cellular and molecular mechanisms of immune cell migration across the BCSFB is largely limited to in vitro models. In addition to forming contact-inhibited epithelial monolayers that express adhesion molecules, the optimal in vitro model must establish a tight permeability barrier as this influences immune cell diapedesis. METHODS We compared cell line models of the mouse BCSFB derived from the Immortomouse(®) and the ECPC4 line to primary mouse choroid plexus epithelial cell (pmCPEC) cultures for their ability to establish differentiated and tight in vitro models of the BCSFB. RESULTS We found that inducible cell line models established from the Immortomouse(®) or the ECPC4 tumor cell line did not express characteristic epithelial proteins such as cytokeratin and E-cadherin and failed to reproducibly establish contact-inhibited epithelial monolayers that formed a tight permeability barrier. In contrast, cultures of highly-purified pmCPECs expressed cytokeratin and displayed mature BCSFB characteristic junctional complexes as visualized by the junctional localization of E-cadherin, β-catenin and claudins-1, -2, -3 and -11. pmCPECs formed a tight barrier with low permeability and high electrical resistance. When grown in inverted filter cultures, pmCPECs were suitable to study T cell migration from the basolateral to the apical side of the BCSFB, thus correctly modelling in vivo migration of immune cells from the blood to the CSF. CONCLUSIONS Our study excludes inducible and tumor cell line mouse models as suitable to study immune functions of the BCSFB in vitro. Rather, we introduce here an in vitro inverted filter model of the primary mouse BCSFB suited to study the cellular and molecular mechanisms mediating immune cell migration across the BCSFB during immunosurveillance and neuroinflammation.

Percutaneous peptide immunization via corneum barrier-disrupted murine skin for experimental tumor immunoprophylaxis

H-2Kb-restricted tumor epitope peptides, including tyrosinase-related protein 2 residues 181–188 (TRP-2) and connexin 37 residues 52–59 (MUT1), were applied to permeability barrier-disrupted C57BL/6 (B6) mouse skin from which the stratum corneum of the epidermis had been removed by tape-stripping. This procedure primed tumor-specific cytotoxic T lymphocytes (CTLs) in the lymph nodes and spleen, protected mice against subsequent challenge with corresponding tumor cells, and suppressed the growth of established tumors. Preventive and therapeutic effectiveness was correlated with the frequency of tumor-specific CTL precursors. MHC class II Iab+ cells separated from tape-stripped skin, compared with those from intact skin, exhibited a strong antigen-presenting capacity for CTL, suggesting that CTL expansion after peptide application is primarily mediated by epidermal Langerhans cells. Thus, percutaneous peptide immunization via barrier-disrupted skin provides a simple and noninvasive means of inducing potent anti-tumor immunity which may be exploited for cancer immunotherapy.

Reconstitution and functional comparison of purified GlpF and AqpZ, the glycerol and water channels from Escherichia coli

A large family of membrane channel proteins selective for transport of water (aquaporins) or water plus glycerol (aquaglyceroporins) has been found in diverse life forms. Escherichia coli has two members of this family—a water channel, AqpZ, and a glycerol facilitator, GlpF. Despite having similar primary amino acid sequences and predicted structures, the oligomeric state and solute selectivity of AqpZ and GlpF are disputed. Here we report biochemical and functional characterizations of affinity-purified GlpF and compare it to AqpZ. Histidine-tagged (His-GlpF) and hemagglutinin-tagged (HA-GlpF) polypeptides encoded by a bicistronic construct were expressed in bacteria. HA-GlpF and His-GlpF appear to form oligomers during Ni-nitrilotriacetate affinity purification. Sucrose gradient sedimentation analyses showed that the oligomeric state of octyl glucoside-solubilized GlpF varies: low ionic strength favors subunit dissociation, whereas Mg2+ stabilizes tetrameric assembly. Reconstitution of affinity-purified GlpF into proteoliposomes increases glycerol permeability more than 100-fold and water permeability up to 10-fold compared with control liposomes. Glycerol and water permeability of GlpF both occur with low Arrhenius activation energies and are reversibly inhibited by HgCl2. Our studies demonstrate that, unlike AqpZ, a water-selective stable tetramer, purified GlpF exists in multiple oligomeric forms under nondenaturing conditions and is highly permeable to glycerol but less well permeated by water.

Local osmotic gradients drive the water flux associated with Na+/glucose cotransport

It recently was proposed [Loo, D. D. F., Zeuthen, T., Chandy, G. & Wright, E. M. (1996) Proc. Natl. Acad. Sci. USA 93, 13367–13370] that SGLT1, the high affinity intestinal and renal sodium/glucose cotransporter carries water molecules along with the cosubstrates with a strict stoichiometry of two Na+, one glucose, and ≈220 water molecules per transport cycle. Using electrophysiology together with sensitive volumetric measurements, we investigated the nature of the driving force behind the cotransporter-mediated water flux. The osmotic water permeability of oocytes expressing human SGLT1 (Lp ± SE) averaged 3.8 ± 0.3 × 10−4 cm⋅s−1 (n = 15) and addition of 100 μM phlorizin (a specific SGLT1 inhibitor) reduced the permeability to 2.2 ± 0.2 × 10−4 cm⋅s−1 (n = 15), confirming the presence of a significant water permeability closely associated with the cotransporter. Addition of 5 mM α-methyl-glucose (αMG) induced an average inward current of 800 ± 10 nA at −50 mV and a water influx reaching 120 ± 20 pL cm−2 ⋅s−1 within 5–8 min. After rapidly inhibiting the Na+/glucose cotransport with phlorizin, the water flux remained significantly elevated, clearly indicating the presence of a local osmotic gradient (Δπ) estimated at 16 ± 2 mOsm. In short-term experiments, a rapid depolarization from −100 to 0 mV in the presence of αMG decreased the cotransport current by 94% but failed to produce a comparable reduction in the swelling rate. A mathematical model depicting the intracellular accumulation of transported osmolytes can accurately account for these observations. It is concluded that, in SGLT1-expressing oocytes, αMG-dependent water influx is induced by a local osmotic gradient by using both endogenous and SGLT1-dependent water permeability.

Polarity and permeation profiles in lipid membranes

The isotropic 14N-hyperfine coupling constant, a\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{o}^{N}}}\end{equation*}\end{document}, of nitroxide spin labels is dependent on the local environmental polarity. The dependence of a\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{o}^{N}}}\end{equation*}\end{document} in fluid phospholipid bilayer membranes on the C-atom position, n, of the nitroxide in the sn-2 chain of a spin-labeled diacyl glycerophospholipid therefore determines the transmembrane polarity profile. The polarity variation in phospholipid membranes, with and without equimolar cholesterol, is characterized by a sigmoidal, trough-like profile of the form {1 + exp [(n − no)/λ]}−1, where n = no is the point of maximum gradient, or polarity midpoint, beyond which the free energy of permeation decreases linearly with n, on a characteristic length-scale, λ. Integration over this profile yields a corresponding expression for the permeability barrier to polar solutes. For fluid membranes without cholesterol, no ≈ 8 and λ ≈ 0.5–1 CH2 units, and the permeability barrier introduces an additional diffusive resistance that is equivalent to increasing the effective membrane thickness by 35–80%, depending on the lipid. For membranes containing equimolar cholesterol, no ≈ 9–10, and the total change in polarity is greater than for membranes without cholesterol, increasing the permeability barrier by a factor of 2, whereas the decay length remains similar. The permeation of oxygen into fluid lipid membranes (determined by spin-label relaxation enhancements) displays a profile similar to that of the transmembrane polarity but of opposite sense. For fluid membranes without cholesterol no ≈ 8 and λ ≈ 1 CH2 units, also for oxygen. The permeation profile for polar paramagnetic ion complexes is closer to a single exponential decay, i.e., no lies outside the acyl-chain region of the membrane. These results are relevant not only to the permeation of water and polar solutes into membranes and their permeabilities, but also to depth determinations of site-specifically spin-labeled protein residues by using paramagnetic relaxation agents.

Constitutive and regulated membrane expression of aquaporin 1 and aquaporin 2 water channels in stably transfected LLC-PK1 epithelial cells.

The aquaporins (AQPs) are a family of homologous water-channel proteins that can be inserted into epithelial cell plasma membranes either constitutively (AQP1) or by regulated exocytosis following vasopressin stimulation (AQP2). LLC-PK1 porcine renal epithelial cells were stably transfected with cDNA encoding AQP2 (tagged with a C-terminal c-Myc epitope) or rat kidney AQP1 cDNA in an expression vector containing a cytomegalovirus promoter. Immunofluorescence staining revealed that AQP1 was mainly localized to the plasma membrane, whereas AQP2 was predominantly located on intracellular vesicles. After treatment with vasopressin or forskolin for 10 min, AQP2 was relocated to the plasma membrane, indicating that this relocation was induced by cAMP. The location of AQP1 did not change. The basal water permeability of AQP1-transfected cells was 2-fold greater than that of nontransfected cells, whereas the permeability of AQP2-transfected cells increased significantly only after vasopressin treatment. Endocytotic uptake of fluorescein isothiocyanate-coupled dextran was stimulated 6-fold by vasopressin in AQP2-transfected cells but was only slightly increased in wild-type or AQP1-transfected cells. This vasopressin-induced endocytosis was inhibited in low-K+ medium, which selectively affects clathrin-mediated endocytosis. These water channel-transfected cells represent an in vitro system that will allow the detailed dissection of mechanisms involved in the processing, targeting, and trafficking of proteins via constitutive versus regulated intracellular transport pathways.

Nano-biocomposite films with modified cellulose nanocrystals and synthesized silver nanoparticles

Ternary nano-biocomposite films based on poly(lactic acid) (PLA) with modified cellulose nanocrystals (s-CNC) and synthesized silver nanoparticles (Ag) have been prepared and characterized. The functionalization of the CNC surface with an acid phosphate ester of ethoxylated nonylphenol favoured its dispersion in the PLA matrix. The positive effects of the addition of cellulose and silver on the PLA barrier properties were confirmed by reductions in the water permeability (WVP) and oxygen transmission rate (OTR) of the films tested. The migration level of all nano-biocomposites in contact with food simulants were below the permitted limits in both non-polar and polar simulants. PLA nano-biocomposites showed a significant antibacterial activity influenced by the Ag content, while composting tests showed that the materials were visibly disintegrated after 15 days with the ternary systems showing the highest rate of disintegration under composting conditions.

Human aquaporins:regulators of transcellular water flow

Background - Emerging evidence supports the view that (AQP) aquaporin water channels are regulators of transcellular water flow. Consistent with their expression in most tissues, AQPs are associated with diverse physiological and pathophysiological processes. Scope of review - AQP knockout studies suggest that the regulatory role of AQPs, rather than their action as passive channels, is their critical function. Transport through all AQPs occurs by a common passive mechanism, but their regulation and cellular distribution varies significantly depending on cell and tissue type; the role of AQPs in cell volume regulation (CVR) is particularly notable. This review examines the regulatory role of AQPs in transcellular water flow, especially in CVR. We focus on key systems of the human body, encompassing processes as diverse as urine concentration in the kidney to clearance of brain oedema. Major conclusions - AQPs are crucial for the regulation of water homeostasis, providing selective pores for the rapid movement of water across diverse cell membranes and playing regulatory roles in CVR. Gating mechanisms have been proposed for human AQPs, but have only been reported for plant and microbial AQPs. Consequently, it is likely that the distribution and abundance of AQPs in a particular membrane is the determinant of membrane water permeability and a regulator of transcellular water flow. General significance - Elucidating the mechanisms that regulate transcellular water flow will improve our understanding of the human body in health and disease. The central role of specific AQPs in regulating water homeostasis will provide routes to a range of novel therapies. This article is part of a Special Issue entitled Aquaporins.

Rapid tonicity induced re-localisation of endogenous aquaporin 4 in primary rat astrocytes - a therapeutic target for cytotoxic brain oedema?

It is estimated that 69-75 million people worldwide will suffer a traumatic brain injury (TBI) or stroke each year. Brain oedema caused by TBI or following a stroke, together with other disorders of the brain cost Europe €770 billion in 2014. Aquaporins (AQP) are transmembrane water channels involved in many physiologies and are responsible for the maintenance of water homeostasis. They react rapidly to changes in osmolarity by transporting water through their highly selective central pore to maintain tonicity and aid in cell volume regulation. We have previously shown that recombinant AQP1-GFP trafficking occurs in a proteinkinase C-microtubule dependant manner in HEK-293 cells in response to hypotonicity. This trafficking mechanism is also reliant on the presence of calcium and its messenger-binding protein calmodulin and results in increased cell surface expression of AQP1 in a time-scale of ~30 seconds. There is currently very little research into the trafficking mechanisms of endogenous AQPs in primary cells. AQP4 is the most abundantly expressed AQP within the brain, it is localised to the astrocytic end-feet, in contact with the blood vessels at the blood-brain-barrier. In situations where the exquisitely-tuned osmotic balance is disturbed, high water permeability can become detrimental. AQP4-mediated water influx causes rapid brain swelling, resulting in death or long term brain damage. Previous research has shown that AQP4 knock-out mice were protected from the formation of cytotoxic brain oedema in a stroke model, highlighting AQP4 as a key drug target for this pathology. As there are currently no treatments available to restrict the flow of water through AQP4 as all known inhibitors are either cytotoxic or non-specific, controlling the mechanisms involved in the regulation of AQP4 in the brain could provide a therapeutic solution to such diseases. Using cell surface biontinylation of endogenous AQP4 in primary rat astrocytes followed by neutraavidin based ELISA we have shown that AQP4 cell surface localisation increases by 2.7 fold after 5 minutes hypotonic treatment at around 85 mOsm/kg H2O. We have also shown that this rapid relocalisation of AQP4 is regulated by PKA, calmodulin, extra-cellular calcium and actin. In summary we have shown that rapid translocation of endogenous AQP4 occurs in primary rat astrocytes in response to hypotonic stimuli; this mechanism is PKA, calcium, actin and calmodulin dependant. AQP4 has the potential to provide a treatment for the development of brain oedema.

Characterization of a biodegradable starch based film. Application on the preservation of fresh spinach

Mestrado em Engenharia Alimentar - Instituto Superior de Agronomia - UL

Impact and energy absorption of road safety barriers by coupled SPH/FEM

Road safety barriers are used to minimise the severity of road accidents and protect lives and property. There are several types of barrier in use today. This paper reports the initial phase of research carried out to study the impact response of portable water-filled barrier (PWFB) which has the potential to absorb impact energy and hence provide crash mitigation under low to moderate speeds. Current research on the impact and energy absorption capacity of water-filled road safety barriers is limited due to the complexity of fluid-structure interaction under dynamic impact. In this paper, a novel fluid-structure interaction method is developed based on the combination of Smooth Particle Hydrodynamics (SPH) and Finite Element Method (FEM). The sloshing phenomenon of water inside a PWFB is investigated to explore the energy absorption capacity of water under dynamic impact. It was found that water plays an important role in energy absorption. The coupling analysis developed in this paper will provide a platform to further the research in optimising the behaviour of the PWFB. The effect of the amount of water on its energy absorption capacity is investigated and the results have practical applications in the design of PWFBs.

High-strength high-performance lightweight concrete : a review (Invited)

In the last two decades, there are developments that lead to greater understanding on how and why lightweight concretes (LWC) may achieve similar or higher performance than their normal weight counterparts. The present paper reviews some of these aspects beginning with basic properties such as unit weight, compressive strength and specific strength (strength/ unit weight). Stability and workability of LWC is discussed from rheological perspective. The volumetric stability of LWC in terms of shrinkage and creep are presented with some recent published data. Transport properties of the LWC in terms of sorptivity, water permeability and resistance to chloride-ion penetration are reviewed in comparison with normal weight concrete. Fire resistance of LWC and some current measures used to improve the resistance are discussed. With continual research and development, the performance of LWC is being enhanced to provide new opportunities for practical applications.