Improving oral health of adolescent girls in Kenya through education - An observational study.

OBJECTIVES: Dental caries and periodontal diseases are significant health concerns in developing nations. This study assessed the impact of a comprehensive oral health education program on adolescent girls in rural Kenya. METHODS: Eighty-seven girls aged 13-18 years attending school in rural Nanyuki, Kenya, were enrolled in the study. The comprehensive program included personalized oral hygiene training, education and health advocacy coaching. Dental caries, gingival inflammation and dental plaque biofilm were assessed at baseline, 1- and 2.5-year post-implementation. RESULTS: The intervention was highly effective in arresting pre-existing carious lesions and preventing the formation of new ones in this population. The data revealed that there was a significant remineralization of incipient caries lesion, as shown by decreases in modified ICCMS™ scores from baseline to 1-year post-implementation. From baseline to the 2.5-year post-implementation assessment, only six new carious lesions developed. Dental plaque biofilm was reduced by 83.6%, and gingival inflammation was reduced by 81.6%. CONCLUSION: A comprehensive oral health program, which included behavioural awareness and educational approaches, resulted in significant positive oral health outcomes in caries, dental plaque biofilm and gingival inflammation.

Thermodynamic and structural study of DMPC-alkanol systems.

The thermodynamic and structural behaviors of lamellar dimyristoylphosphatidylcholine-alkanol (abbreviation DMPC-CnOH, n = 8-18 is the even number of carbons in the alkyl chain) systems were studied by using DSC and SAXD/WAXD methods at a 0-0.8 CnOH : DMPC molar ratio range. Up to n≤ 10 a significant biphasic effect depending on the main transition temperature tm on the CnOH concentration was observed. Two breakpoints were revealed: turning point (TP), corresponding to the minimum, and threshold concentration (cT), corresponding to the end of the biphasic tendency. These breakpoints were also observed in the alkanol concentration dependent change in the enthalpy of the main transition ΔHm. In the case of CnOHs with n > 10 we propose a marked shift of TP and cT to very low concentrations; consequently, only increase of tm is observed. A partial phase diagram was constructed for a pseudo-binary DMPC-C12OH system. We suggest a fluid-fluid immiscibility of the DMPC-C12OH system above cT with a consequent formation of domains with different C12OH contents. At a constant CnOH concentration, the effects of CnOHs on ΔHm and bilayer repeat distance were found to depend predominantly on the mismatch between CnOH and lipid chain lengths. Observed effects are suggested to be underlined by a counterbalancing effect of interchain van der Waals interactions and headgroup repulsion.

Metallo-responsive self-assembly of lipophilic guanines in hydrocarbon solvents: a systematic SAXS structural characterization.

Lipophilic guanines (LipoGs) in aprotic solvents undergo different self-assembly processes based on different H-bonded motifs. Cylindrical nanotubes made by π-π stacked guanine tetramers (G-quadruplexes) and flat, tape-like aggregates (G-ribbons) have been observed depending on the presence of alkali metal ions. To obtain information on the structural properties and stability of these LipoG aggregates, Small-Angle X-ray Scattering (SAXS) experiments have been performed in dodecane, both in the presence and in the absence of potassium ions. As a result, the occurrence of the two different metallo-responsive architectures (nanoribbons or columnar nanotubes) was confirmed and we reported here for the first time a systematic study on the dependence of the aggregate properties on composition, temperature and molecular unit structure. Even if dodecane was selected to favour LipoG solubility, a strong tendency to self-organize into ordered lyotropic phases was indeed detected.

Interactions of Cationic Lipids with DNA: A Structural Approach.

Colloidal nucleic acid carrier systems based on cationic lipids are a promising pharmaceutical tool in the implementation of gene therapeutic strategies. This study demonstrates the complex behavior of DNA at the lipid-solvent interface facilitating structural changes of the lyotropic liquid-crystalline phases. For this study, the structural properties of six malonic acid based cationic lipids were determined using small- and wide-angle X-ray scattering (SAXS and WAXS) as well as differential scanning calorimetry (DSC). Selected lipids (lipid 3 and lipid 6) with high nucleic acid transfer activity have been investigated in detail because of the strong influence of the zwitterionic helper lipid 1,2-di(9 Z-octadecenoyl)- sn-glycero-3-phosphoethanolamine (DOPE) on the structural properties as well as of the complex formation of lipid-DNA complexes (lipoplexes). In the case of lipid 3, DNA stabilizes a metastable cubic mesophase with Im3 m symmetry and an Im3 m Qαc lipoplex is formed, which is rarely described for DNA lipoplexes in literature. In the case of lipid 6, a cubic mesophase with Im3 m symmetry turns into a fluid lamellar phase while mixing with DOPE and complexing DNA.

DNA-DOPE-gemini surfactants complexes at low surface charge density: from structure to transfection efficiency.

DNA condensation, structure and transfection efficiency of complexes formed by gemini surfactants alkane-α,ω-diyl-bis(dodecyldimethylammonium bromide)s (CnGS12, n = 3, 6 and 12 is the number of alkane spacer carbons), dioleoylphosphatidylethanolamine (CnGS12/DOPE = 0.3 mol/mol) and DNA at low surface charge density were investigated through different techniques. Small angle X-ray diffraction showed a condensed lamellar phase with marked dependence of DNA-DNA distance on (+/-) charge ratio. High ionic strength of hydrating medium screens the interaction DNA - CnGS12/DOPE and complexed DNA represented maximally ~ 45-60% of total DNA in the solution as derived from fluorescence and UV-VIS spectroscopy. The in vitro transfection efficiency of CnGS12/DOPE liposomes on mammalian HEK 293 cell line was spacer length-dependent. C12GS12/DOPE/DNA complexes exhibited the best transfection efficiency (~ 18% GFP-expressing cells relative to all viable cells) accompanied by ~ 89% cell viability.

Unravelling a Mechanism of Action for a Cecropin A-Melittin Hybrid Antimicrobial Peptide: The Induced Formation of Multilamellar Lipid Stacks.

An understanding of the mechanism of action of antimicrobial peptides is fundamental to the development of new and more active antibiotics. In the present work, we use a wide range of techniques (SANS, SAXD, DSC, ITC, CD, and confocal and electron microscopy) in order to fully characterize the interaction of a cecropin A-melittin hybrid antimicrobial peptide, CA(1-7)M(2-9), of known antimicrobial activity, with a bacterial model membrane of POPE/POPG in an effort to unravel its mechanism of action. We found that CA(1-7)M(2-9) disrupts the vesicles, inducing membrane condensation and forming an onionlike structure of multilamellar stacks, held together by the intercalated peptides. SANS and SAXD revealed changes induced by the peptide in the lipid bilayer thickness and the bilayer stiffening in a tightly packed liquid-crystalline lamellar phase. The analysis of the observed abrupt changes in the repeat distance upon the phase transition to the gel state suggests the formation of an Lγ phase. To the extent of our knowledge, this is the first time that the Lγ phase is identified as part of the mechanism of action of antimicrobial peptides. The energetics of interaction depends on temperature, and ITC results indicate that CA(1-7)M(2-9) interacts with the outer leaflet. This further supports the idea of a surface interaction that leads to membrane condensation and not to pore formation. As a result, we propose that this peptide exerts its antimicrobial action against bacteria through extensive membrane disruption that leads to cell death.

Incorporation of mRNA in Lamellar Lipid Matrices for Parenteral Administration.

Insertion of high molecular weight messenger RNA (mRNA) into lyotropic lipid phases as model systems for controlled release formulations for the mRNA was investigated. Low fractions of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were used as an anchor to load the mRNA into a lamellar lipid matrix. Dispersions of zwitterionic lipid in the aqueous phase in the presence of increasing fractions of mRNA and cationic lipid were prepared, and the molecular organization was investigated as a function of mRNA and cationic lipid fraction. Insertion of both cationic lipid and mRNA was clearly proven from the physicochemical characteristics. The d-spacing of the lipid bilayers, as determined by small-angle X-ray scattering (SAXS) measurements, responded sensitively to the amount of inserted DOTAP and mRNA. A concise model of the insertion of the mRNA in the lipid matrices was derived, indicating that the mRNA was accommodated in the aqueous slab between lipid bilayers. Depending on the DOTAP and mRNA fraction, a different excess of water was present in this slab. Results from further physicochemical characterization, including determination of free and bound mRNA, zeta potential, and calorimetry data, were in line with this assumption. The structure of these concentrated lipid/mRNA preparations was maintained upon dilution. The functionality of the inserted mRNA was proven by cell culture experiments using C2C12 murine myoblast cells with the luciferase-encoding mRNA. The described lipid phases as carriers for the mRNA may be applicable for different routes of local administration, where control of the release kinetics and the form of the released mRNA (bound or free) is required.

Correction: Cooling induces phase separation in membranes derived from isolated CNS myelin.

[This corrects the article DOI: 10.1371/journal.pone.0184881.].

Cooling induces phase separation in membranes derived from isolated CNS myelin.

Purified myelin membranes (PMMs) are the starting material for biochemical analyses such as the isolation of detergent-insoluble glycosphingolipid-rich domains (DIGs), which are believed to be representatives of functional lipid rafts. The normal DIGs isolation protocol involves the extraction of lipids under moderate cooling. Here, we thus address the influence of cooling on the structure of PMMs and its sub-fractions. Thermodynamic and structural aspects of periodic, multilamellar PMMs are examined between 4°C and 45°C and in various biologically relevant aqueous solutions. The phase behavior is investigated by small-angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC). Complementary neutron diffraction (ND) experiments with solid-supported myelin multilayers confirm that the phase behavior is unaffected by planar confinement. SAXS and ND consistently show that multilamellar PMMs in pure water become heterogeneous when cooled by more than 10-15°C below physiological temperature, as during the DIGs isolation procedure. The heterogeneous state of PMMs is stabilized in physiological solution, where phase coexistence persists up to near the physiological temperature. This result supports the general view that membranes under physiological conditions are close to critical points for phase separation. In presence of elevated Ca2+ concentrations (> 10 mM), phase coexistence is found even far above physiological temperatures. The relative fractions of the two phases, and thus presumably also their compositions, are found to vary with temperature. Depending on the conditions, an "expanded" phase with larger lamellar period or a "compacted" phase with smaller lamellar period coexists with the native phase. Both expanded and compacted periods are also observed in DIGs under the respective conditions. The observed subtle temperature-dependence of the phase behavior of PMMs suggests that the composition of DIGs is sensitive to the details of the isolation protocol.

Structural Characterization of Lecithin-Stabilized Tetracosane Lipid Nanoparticles. Part I: Emulsions.

The structure of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)-stabilized colloidal tetracosane emulsions was investigated by photon correlation spectroscopy and small-angle X-ray and neutron scattering, using emulsions with different neutron scattering contrasts. Special emphasis was placed on the structure of the DMPC stabilizer layer covering the emulsion droplets. A monolayer, structurally similar to a half DMPC bilayer, with a thickness of 16 Å is found. Thereby, the phosphocholine headgroups arrange flat at the oil-water interface. A deep penetration of the tetracosane oil into the stabilizer layer can be ruled out.

Structural Characterization of Lecithin-Stabilized Tetracosane Lipid Nanoparticles. Part II: Suspensions.

Using photon correlation spectroscopy, transmission electron microscopy, microcalorimetry, wide-angle X-ray scattering (WAXS), and small-angle X-ray and neutron scattering (SAXS, SANS), the structure of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)-stabilized colloidal tetracosane suspensions was studied from the molecular level to the microscopic scale as a function of the temperature. The platelike nanocrystals exhibit for tetracosane an unusual orthorhombic low-temperature crystal structure. The corresponding WAXS pattern can be reproduced with a predicted orthorhombic unit cell (space group Pca21), which usually occurs only for much longer even-numbered n-alkanes. Special emphasis was placed on the structure of the DMPC stabilizer layer covering the nanocrystals. Their structure was investigated by SAXS and SANS, using suspensions with different neutron scattering contrasts. As for the emulsions in Part I , the crystallized nanoparticles are covered by a DMPC monolayer. Their significant smaller thickness of 10.5 Å (for the emulsions in Part I : 16 Å) could be related to a more tilted orientation of the DMPC molecules to cover the expanded surface of the crystallized nanoparticles.

Cytochrome-c Affects the Monoolein Polymorphism: Consequences for Stability and Loading Efficiency of Drug Delivery Systems.

Structural properties and polymorphism of monoolein (MO) in aqueous solutions have been studied for a long time, and the final picture can be considered definite. The presence of bicontinuous phases and the ability to encapsulate hydrophilic, hydrophobic, and amphiphilic compounds, together with the capability to protect and slowly release the entrapped molecules, designated MO phases as good matrices for the sustained release of drugs. Because phase stability, loading efficiency, and bioavailability are strongly correlated, the interplay between MO phases and entrapped compounds is worthy of investigation. In this paper, low angle X-ray diffraction has been used to describe the effects of a model protein (the cytochrome-c) on the monoolein cubic phases as a function of both incubation time and protein concentration in the soaking solutions. Results show that the MO polymorphism is strongly modified by the protein, underlying the very large affinity of the cytochrome-c toward monoolein. However, the different phases have a different sensibility to cytochrome-c, as phase transitions occur when the protein amount exceeds some different critical values, probably related to the structure characteristics (2 cytochrome-c per unit cell at the Pn3m to Im3m cubic phase transition and 10-20 cytochrome-c per unit cell at the Im3m to P4332 cubic phase transition). Moreover, although equilibration times resulted to be quite long (more than 10 days), the fraction of cytochrome-c incorporated into the MO phases is very high (up to 20% v/v inside the P4332 cubic phase). Such results are intriguing: even if they may be specific to the cytochrome-c/MO case, the need of assessing the structural characteristics of lipid matrices before their use as drug delivery systems is evident.

The location of coenzyme Q10 in phospholipid membranes made of POPE: a small-angle synchrotron X-ray diffraction study.

The location of coenzyme Q10 (Q10) inside the inner mitochondrial membrane is a topic of research aiming at a deeper understanding of the function of the mitochondrial respiratory chain. We investigated the location of Q10 inside model membranes made of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine by means of small-angle synchrotron X-ray diffraction. Q10, which stands for ubiquinone-10 (UQ) or ubihydroquinone-10 (UH), did not remarkably influence the main phase transition temperature, but significantly decreased the lamellar-inverse hexagonal phase transition temperature (T(h)). The effect of UH on T(h) was stronger than the effect of UQ and the effect of liquid Q10 on T(h) was stronger than the effect of crystalline Q10. In the presence of Q10, the lattice parameters of the lamellar phases remained unchanged, whereas the H II lattice parameter was clearly influenced: While UQ had an increasing effect, UH had a decreasing effect. Furthermore, Q10 prevented the formation of cubic phases. The results give new evidence that the headgroup of Q10 is distant from the center of the membrane, which might be important for the function of the mitochondrial respiratory chain.

Analysis of the structure of nanocomposites of triglyceride platelets and DNA.

DNA-complexes with platelet-like, cationically modified lipid nanoparticles (cLNPs) are studied with regard to the formation of nanocomposite structures with a sandwich-like arrangement of the DNA and platelets. For this purpose suspensions of platelet-like triglyceride nanocrystals, stabilized by a mixture of two nonionic (lecithin plus polysorbate 80 or poloxamer 188) and one cationic stabilizer dimethyldioctadecylammonium (DODAB), are used. The structure of the platelets in the native suspensions and their DNA-complexes, ranging from the sub-nano to the micron scale, is investigated with small- and wide-angle scattering (SAXS, SANS, WAXS), calorimetry, photon correlation spectroscopy, transmission electron microscopy and computer simulations. The appearance of strong, lamellarly ordered peaks in the SAXS patterns of the DNA-complexes suggests a stacked arrangement of the nanocrystals, with the DNA being partially condensed between the platelets. This finding is supported with computer simulated small-angle scattering patterns of nanocrystal stacks, which can reproduce the measured small-angle scattering patterns on an absolute scale. The influence of the choice of the nonionic stabilizers and the amount of the cationic stabilizer DODAB on the structure of the native suspensions and the inner structure of their DNA-complexes is studied, too. Using high amounts of DODAB, lecithins with saturated acyl chains and polysorbate 80 instead of poloxamer 188 produces thinner nanocrystals, and thus decreases their repeat distances in the nanocomposites. Such nanocomposites could be of interest as DNA carriers, where the triglyceride platelets protect the sandwiched DNA from degradation.

Stimuli responsive polymorphism of C12NO/DOPE/DNA complexes: Effect of pH, temperature and composition.

N,N-dimethyldodecylamine-N-oxide (C12NO) is a surfactant that may exist either in a neutral or cationic protonated form depending on the pH of aqueous solutions. Using small angle X-ray diffraction (SAXD) we observe the rich structural polymorphism of pH responsive complexes prepared due to DNA interaction with C12NO/dioleoylphosphatidylethanolamine (DOPE) vesicles and discuss it in view of utilizing the surfactant for the gene delivery vector of a pH sensitive system. In neutral solutions, the DNA uptake is low, and a lamellar Lα phase formed by C12NO/DOPE is prevailing in the complexes at 0.2≤C12NO/DOPE<0.6 mol/mol. A maximum of ~30% of the total DNA volume in the sample is bound in a condensed lamellar phase LαC at C12NO/DOPE=1 mol/mol and pH7.2. In acidic conditions, a condensed inverted hexagonal phase HIIC was observed at C12NO/DOPE=0.2 mol/mol. Commensurate lattice parameters, aHC≈dLC, were detected at 0.3≤C12NO/DOPE≤0.4 mol/mol and pH=4.9-6.4 suggesting that LαC and HIIC phases were epitaxially related. While at the same composition but pH~7, the mixture forms a cubic phase (Pn3m) when the complexes were heated to 80°C and cooled down to 20°C. Finally, a large portion of the surfactant (C12NO/DOPE>0.5) stabilizes the LαC phase in C12NO/DOPE/DNA complexes and the distance between DNA strands (dDNA) is modulated by the pH value. Both the composition and pH affect the DNA binding in the complexes reaching up to ~95% of the DNA total amount at acidic conditions.

Phase behavior of the DOPE + DOPC + alkanol system.

Small- and wide-angle X-ray diffraction was used to study the effect of 1-alkanols, as simple models of general anesthetics, (abbreviation CnOH, n = 8-18 is the even number of carbons in the aliphatic chain) on the lamellar to hexagonal Lα→ H(II) phase transition in the dioleoylphosphatidylethanolamine-dioleoylphosphatidylcholine = 3 : 1 mol/mol (DOPE + DOPC) system. All studied CnOHs were found to decrease the phase transition temperature of the DOPE + DOPC system in a CnOH chain length and concentration dependent manner and thus promote the formation of the HII phase. Anesthetically active C8OH and C10OH were found to decrease the lattice parameter d of the Lα phase, however longer non-anesthetic CnOHs increased the parameter d; this effect being more pronounced with increasing CnOH concentration. The lattice parameter of the HII phase was decreased in the presence of all CnOHs, even at the lowest concentrations studied. In the scope of the indirect mechanism of general anesthesia observed changes in the lattice parameter d (reflecting changes in the bilayer thickness) due to the intercalation of C8OH and C10OH might induce changes in the activity of integral membrane proteins engaged in neuronal pathways.

Size limit on the formation of periodic mesoporous organosilicas (PMOs).

The decrease of the lattice size of periodic mesoporous organosilicas (PMOs) is one important goal in obtaining a microporous material for storage or adsorption of small molecules. To determine the influence of different synthesis parameters in the lattice size, here we performed in situ small-angle X-ray diffraction studies and show that a variation of the surfactant's headgroup size is not directly followed by the lattice parameter of the resulting structure. We show that in the surfactant series of penta-, hexa-, hepta-, octa-, nona-, and decaethylene glycol monododecyl ether (C12(EO)n, n = 5, 6, 7, 8, 9, 10) the lattice size decreases between n = 5 and n = 8 and then increases, while the ordering of the materials is always cubic (space group Fd3m). This size effect is due to the ethylene oxide (EO) chain conformation that changes as the number of EO groups increases. Short ethylene oxide chains tend to have a so-called "zigzag" conformation while an increase of the chain length leads to a "Mäander" (coiling) conformation. Although this phenomenon is most commonly observed for chains consisting of more than 10 ethylene oxide units, we found a minimum PMO lattice size for 8 EO units and intermediate values for 6 and 7 EO units. The increase of the lattice parameter for more than 9 EO units is attributed to the increasing number of "Mäander" configurated EO units.

Differential effect of 2-hydroxyoleic acid enantiomers on protein (sphingomyelin synthase) and lipid (membrane) targets.

The complex dual mechanism of action of 2-hydroxyoleic acid (2OHOA), a potent anti-tumor compound used in membrane lipid therapy (MLT), has yet to be fully elucidated. It has been demonstrated that 2OHOA increases the sphingomyelin (SM) cell content via SM synthase (SGMS) activation. Its presence in membranes provokes changes in the membrane lipid structure that induce the translocation of PKC to the membrane and the subsequent overexpression of CDK inhibitor proteins (e.g., p21(Cip1)). In addition, 2OHOA also induces the translocation of Ras to the cytoplasm, provoking the silencing of MAPK and its related pathways. These two differential modes of action are triggered by the interactions of 2OHOA with either lipids or proteins. To investigate the molecular basis of the different interactions of 2OHOA with membrane lipids and proteins, we synthesized the R and S enantiomers of this compound. A molecular dynamics study indicated that both enantiomers interact similarly with lipid bilayers, which was further confirmed by X-ray diffraction studies. By contrast, only the S enantiomer was able to activate SMS in human glioma U118 cells. Moreover, the anti-tumor efficacy of the S enantiomer was greater than that of the R enantiomer, as the former can act through both MLT mechanisms. The present study provides additional information on this novel therapeutic approach and on the magnitude of the therapeutic effects of type-1 and type-2 MLT approaches. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.

Comparative SAXS and DSC study on stratum corneum structural organization in an epidermal cell culture model (ROC): impact of cultivation time.

Cell cultured skin equivalents present an alternative for dermatological in vitro evaluations of drugs and excipients as they provide the advantage of availability, lower variability and higher assay robustness compared to native skin. For penetration/permeation studies, an adequate stratum corneum barrier similar to that of human stratum corneum is, however, a prerequisite. In this study, the stratum corneum lipid organization in an epidermal cell culture model based on rat epidermal keratinocytes (REK organotypic culture, ROC) was investigated by small-angle X-ray scattering (SAXS) in dependence on ROC cultivation time and in comparison to native human and rat stratum cornea. In addition, the thermal phase behavior was studied by differential scanning calorimetry (DSC) and barrier properties were checked by measurements of the permeability of tritiated water. The development of the barrier of ROC SC obtained at different cultivation times (7, 14 and 21 days at the air-liquid interface) was connected with an increase in structural order of the SC lipids in SAXS measurements: Already cultivation for 14 days at the air-liquid interface resulted overall in a competent SC permeability barrier and SC lipid organization. Cultivation for 21 days resulted in further minor changes in the structural organization of ROC SC. The SAXS patterns of ROC SC had overall large similarities with that of human SC and point to the presence of a long periodicity phase with a repeat distance of about 122Å, e.g. slightly smaller than that determined for human SC in the present study (127Å). Moreover, SAXS results also indicate the presence of covalently bound ceramides, which are crucial for a proper SC barrier, although the corresponding thermal transitions were not clearly detectable by DSC. Due to the competent SC barrier properties and high structural and organizational similarity to that of native human SC, ROC presents a promising alternative for in vitro studies, particularly as it can be obtained under overall rather straightforward cell culture conditions and thus low assay costs.

Modification of phospholipid bilayers induced by sulfurated naphthoquinones.

New thionaphthoquinones and their hydroxyl derivatives, bearing alkyl side chains that match the phospholipids POPC and POPE, were synthesized in order to investigate their interactions with lipids. It was observed that, in general, these additives destabilize the lipid bilayer and induce less organized structures with higher curvature, in particular the induction of an hexagonal phase on aqueous POPC mixtures. Moreover, cubic phases, not normally observed in the pure lipids when fully hydrated, were detected. Coexistence of lamellar phases was interpreted as a consequence of microsegregation of the components in the mixtures. These results are in line with previous observations on the effect of structurally similar (hydro)quinones in phase behavior of these lipids.