A traditional Ugandan Ficus natalensis bark cloth exhibits antimicrobial activity against methicillin-resistant Staphylococcus aureus.

AIMS: Surgical site, soft tissue and wound infections are some of the most prominent causes of healthcare-associated infections (HCAIs). Developing novel antimicrobial textiles and wound dressings may help alleviate the risk of developing HCAIs. We aimed to determine the antimicrobial efficacy of natural Ugandan bark cloth derived exclusively from the Ficus natalensis tree. METHODS AND RESULTS: Antimicrobial contact and disc diffusion assays, coupled with time-kill kinetic assays, demonstrated that bark cloth inhibited the growth of a clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) strain and acted as a bactericidal agent causing a seven-log reduction in bacterial viability. Scanning electron microscopy was used to reveal morphological changes in the bacterial cell ultrastructure when exposed to bark cloth, which supported a proposed mechanism of antimicrobial activity. CONCLUSIONS: The observed antimicrobial properties, combined with the physical characteristics elicited by bark cloth, suggest this product is ideally suited for wound and other skin care applications. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report where a whole bark cloth product made by traditional methods has been employed as an antimicrobial fabric against MRSA. Bark cloth is a highly sustainable and renewable product and this study presents a major advance in the search for natural fabrics which could be deployed for healthcare applications.

Topical photodynamic therapy following excisional wounding of human skin increases production of transforming growth factor-ß3 and matrix metalloproteinases 1 and 9, with associated improvement in dermal matrix organization.

BACKGROUND: Animal studies report photodynamic therapy (PDT) to improve healing of excisional wounds; the mechanism is uncertain and equivalent human studies are lacking. OBJECTIVES: To explore the impact of methyl aminolaevulinate (MAL)-PDT on clinical and microscopic parameters of human cutaneous excisional wound healing, examining potential modulation through production of transforming growth factor (TGF)-ß isoforms. METHODS: In 27 healthy older men (60-77 years), a 4-mm punch biopsy wound was created in skin of the upper inner arm and treated with MAL-PDT three times over 5 days. An identical control wound to the contralateral arm was untreated and both wounds left to heal by secondary intention. Wounds were re-excised during the inflammatory phase (7 days, n = 10), matrix remodelling (3 weeks, n = 8) and cosmetic outcome/dermal structure (9 months, n = 9). Production of TGF-ß1, TGF-ß3 and matrix metalloproteinases (MMPs) was assessed by immunohistochemistry alongside microscopic measurement of wound size/area and clinical assessment of wound appearance. RESULTS: MAL-PDT delayed re-epithelialization at 7 days, associated with increased inflammation. However, 3 weeks postwounding, treated wounds were smaller with higher production of MMP-1 (P = 0·01), MMP-9 (P = 0·04) and TGF-ß3 (P = 0·03). TGF-ß1 was lower than control at 7 days and higher at 3 weeks (both P = 0·03). At 9 months, MAL-PDT-treated wounds showed greater, more ordered deposition of collagen I, collagen III and elastin (all P < 0·05). CONCLUSIONS: MAL-PDT increases MMP-1, MMP-9 and TGF-ß3 production during matrix remodelling, ultimately producing scars with improved dermal matrix architecture.

Clinically relevant doses of lidocaine and bupivacaine do not impair cutaneous wound healing in mice.

BACKGROUND: Lidocaine and bupivacaine are commonly infiltrated into surgical cutaneous wounds to provide local anaesthesia after surgical procedures. However, very little is known about their effects on cutaneous wound healing. If an inhibitory effect is demonstrated, then the balance between the benefits of postoperative local anaesthesia and the negatives of impaired cutaneous wound healing may affect the decision to use local anaesthesia or not. Furthermore, if a difference in the rate of healing of lidocaine- and bupivacaine-treated cutaneous wounds is revealed, or if an inhibitory effect is found to be dose-dependent, then this may well influence the choice of agent and its concentration for clinical use. METHODS: Immediately before incisional wounding, we administered lidocaine and bupivacaine intradermally to adult female mice, some of which had been ovariectomized to act as a model of post-menopausal women (like post-menopausal women, ovariectomized mice heal wounds poorly, with increased proteolysis and inflammation). Day 3 wound tissue was analysed histologically and tested for expression of inflammatory and proteolytic factors. RESULTS: On day 3 post-wounding, wound areas and extent of re-epithelialization were comparable between the control and local anaesthetic-treated animals, in both intact and ovariectomized groups. Both tested drugs significantly increased wound activity of the degradative enzyme matrix metalloproteinase-2 relative to controls, while lidocaine also increased wound neutrophil numbers. CONCLUSIONS: Although lidocaine and bupivacaine influenced local inflammatory and proteolytic factors, they did not impair the rate of healing in either of two well-established models (mimicking normal human wound healing and impaired age-related healing).

5alpha-dihydrotestosterone (DHT) retards wound closure by inhibiting re-epithelialization.

The ongoing search for explanations as to why elderly males heal acute skin wounds more slowly than do their female counterparts (and are more strongly disposed to conditions of chronic ulceration) has identified endogenous oestrogens and androgens as being respectively enhancers and inhibitors of repair. We previously demonstrated that blocking the conversion of testosterone to 5alpha-dihydrotestosterone (DHT) limits its ability to impair healing, suggesting that DHT is a more potent inhibitor of repair than is testosterone. The present study aimed to delineate the central mechanisms by which androgens delay repair. Whilst the contractile properties of neither rat wounds in vivo nor fibroblast-impregnated collagenous discs in vitro appeared to be influenced by androgen manipulations, the global blockade of DHT biosynthesis markedly accelerated re-epithelialization of incisional and excisional wounds and reduced local expression of beta-catenin, a key inhibitor of repair. Moreover, DHT retarded the in vitro migration of epidermal keratinocytes following scratch wounding. By contrast, it failed to influence the migratory and proliferative properties of dermal fibroblasts, suggesting that its primary inhibitory effect is upon re-epithelialization. These novel findings may be of particular significance in the context of chronic ulceration, for which being male is a key risk factor.

A cluster randomised controlled trial to evaluate the effectiveness of fluoride varnish as a public health measure to reduce caries in children.

This cluster randomised controlled study assessed the effectiveness of twice-yearly applications of fluoride varnish as a public health measure to reduce dental caries in children living in relatively deprived communities. The test (n = 334) and control (n = 330) children in 2 school years (unit of randomisation) attended 24 state primary schools and were 6-8 years of age at the start. Good baseline balance was found. Duraphat varnish was applied at school on 5 occasions over 26 months, by dental therapists. A combined visual and fibre-optic transillumination examination included all surfaces of primary and first permanent molars at baseline and after 26 months for small and large enamel and dentine lesions. At the final examination the only statistically significant difference was in the caries increment for small enamel lesions in the primary dentition, with the test children having fewer lesions. This study failed to demonstrate that the twice-yearly application of fluoride varnish provided at school reduced dental caries in children living in this community. The low level of response and a lower than expected caries increment had a major impact on the effectiveness of the intervention, since the children who participated were least likely to have benefited from the programme, whereas those who might have benefited did not consent.

Differentially expressed late constituents of the epidermal cornified envelope.

Barrier activity of skin and internal barrier-forming epithelial linings are conferred by a lipid-corneocyte structure (stratum corneum in skin). The integrity of the corneocytes depends on the outer cornified envelope and is essential for maintenance of barrier function. During epidermal development and differentiation, proteins are sequentially incorporated into the envelope via action of epidermal transglutaminases in a well documented process. However, recent knockouts of major cornified envelope constituents have failed to disrupt barrier function significantly, suggesting that additional unidentified components are involved. We report a new gene cluster in the epidermal differentiation complex at human 1q21 encoding a family of 18 proteins that are substrates for epidermal transglutaminases. These proteins incorporate into the cornified envelope late in development and late in the process of envelope maturation during epidermal differentiation. The genes cluster within the epidermal differentiation complex according to expression pattern, i.e., epidermally expressed proteins cluster together while proteins from internal barrier-forming epithelia also cluster. We propose that these proteins modulate barrier activity over the surface of the animal, in a manner analogous to that proposed for the well characterized cornified envelope precursors, the small proline-rich proteins. To emphasize the incorporation of these proteins late in envelope assembly, we call the human proteins late envelope proteins.

Crystal structure of a truncated mutant of glucose-fructose oxidoreductase shows that an N-terminal arm controls tetramer formation.

N-terminal or C-terminal arms that extend from folded protein domains can play a critical role in quaternary structure and other intermolecular associations and/or in controlling biological activity. We have tested the role of an extended N-terminal arm in the structure and function of a periplasmic enzyme glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis. We have determined the crystal structure of the NAD(+) complex of a truncated form of the enzyme, GFORDelta, in which the first 22 residues of the N-terminal arm of the mature protein have been deleted. The structure, refined at 2.7 A resolution (R(cryst)=24.1%, R(free)=28.4%), shows that the truncated form of the enzyme forms a dimer and implies that the N-terminal arm is essential for tetramer formation by wild-type GFOR. Truncation of the N-terminal arm also greatly increases the solvent exposure of the cofactor; since GFOR activity is dependent on retention of the cofactor during the catalytic cycle we conclude that the absence of GFOR activity in this mutant results from dissociation of the cofactor. The N-terminal arm thus determines the quaternary structure and the retention of the cofactor for GFOR activity and during translocation into the periplasm. The structure of GFORDelta also shows how an additional mutation, Ser64Asp, converts the strict NADP(+) specificity of wild-type GFOR to a dual NADP(+)/NAD(+) specificity.

SPRR1 gene induction and barrier formation occur as coordinated moving fronts in terminally differentiating epithelia.

Stratified, terminally differentiated epithelia, such as epidermis and oral epithelia, provide protective barriers against the environment. We recently developed wholemount assays that demonstrate epidermal barrier function during late gestation and showed that epidermal barrier forms at specific sites (epidermal initiation sites), and then spreads around the body as apparent moving fronts. We now ask if this is a fundamental and widespread mode of epithelial developmental change. If so, then the pattern should be apparent when assaying for developmental change other than barrier institution (e.g., gene induction) and similar types of patterned change should be apparent in other types of epithelia. In this study we demonstrate patterned barrier function in a range of additional stratified epithelia from the oral cavity and show that the gene induction pattern of a stratum corneum precursor small proline-rich region protein 1 (SPRR1) precedes barrier function and occurs in the barrier pattern, i.e., gene induction occurs first at initiation sites and propagates across epithelia as apparent moving fronts. These results demonstrate that late gestational developmental change in multiple terminally differentiating epithelia occurs via initiation sites and moving fronts. The pattern precedes barrier formation and results in a developmental gradient that influences gene induction.

Effect of heat treatment on the circular dichroism spectra of bovine beta-lactoglobulin A, B, and C.

Dilute solutions of beta-lactoglobulin (beta-Lg) A, B, and C were heated in phosphate buffer at temperatures between 40 and 94 degrees C for 10 min, cooled, and analyzed using near-UV and far-UV circular dichroism (CD). The decrease in near-UV CD intensity at 293 nm (Deltaepsilon(293)) could be analyzed in terms of a two-state model, and the stability was beta-Lg C > beta-Lg A > beta-Lg B on the basis of the midpoint temperatures for samples heated at pH 6.7 and 7.4. However, the slopes of the curves at the midpoint temperature for variant A were generally less than those for beta-Lg B and beta-Lg C, indicating that the substitution of Val (beta-Lg A) for Ala (beta-Lg B or beta-Lg C) at position 118 had altered the entropic contribution to unfolding of the protein. The changes in CD at 270 nm (Deltaepsilon(270)), an index of significant alteration to disulfide bond dihedral angles, occurred at higher temperatures than those for the Deltaepsilon(293) results. The far-UV CD showed some small changes as a consequence of heat treatment, and the shifts at 205 nm ([theta](205)) fitted a two-state model. Plotting the changes in both Deltaepsilon(293) and [theta](205) against the loss of nativelike and sodium dodecyl sulfate-monomeric protein (assessed by polyacrylamide gel electrophoresis) showed a strong 1:1 relationship between Deltaepsilon(293) or [theta](205) and the loss of nativelike beta-Lg. These results indicated that the initial irreversible stage in the heat-induced aggregation of beta-Lg (nativelike monomer to unfolded monomer) altered the chirality of the environment of Trp(19) and modified the secondary structure of beta-Lg slightly. The differences in the behavior of variants A-C were explicable on the basis of generalized electrostatic and hydrophobicity effects as well as specific amino acid effects.

Barrier formation in the human fetus is patterned.

We recently demonstrated patterned stratum corneum maturation and skin barrier formation during fetal development in rodents and rabbit. The presence of skin patterning in these mammals led us to predict patterned barrier formation during human infant development. Here we extend our mammalian study and demonstrate patterned stratum corneum development and skin barrier formation in the pre-term human infant. Surprisingly, we show initiation of human barrier regionally as early as 20-24 wk gestational age (22-26 wk menstrual age), bringing barrier formation close to the time of periderm disaggregation. We use the mouse model to show that patterns of periderm disaggregation mirrors barrier formation. Periderm disaggregation follows and recapitulates barrier pattern, suggesting a relationship between the processes. This work reveals regional patterning in skin maturation and barrier formation in the human infant and demonstrates that initiation of human skin barrier formation in utero coincides with the current lower limit of viability of the pre-term infant.

Effect of heat treatment on bovine beta-lactoglobulin A, B, and C explored using thiol availability and fluorescence.

Dilute solutions of beta-lactoglobulin (beta-Lg) A, B, and C were heated at temperatures between about 40 and 94 degrees C for 10 min, cooled, and analyzed using Trp fluorescence and extrinsic fluorescence spectra of the probe 1,8-anilinonaphthalene sulfonate (ANS). Thiol availabilities using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) were determined using a separate set of samples. The normalized ANS fluorescence emission intensity and the thiol availability results showed a 1:1 relationship with the loss of nativelike but not SDS-monomeric protein, as determined by PAGE analysis. The normalized Trp emission intensity results did not show a comparable 1:1 relationship with the loss of nativelike protein, indicating that the Trp intensity arose from consequential disulfide bond reorganization and not the initial unfolding reaction. The results were also analyzed in terms of two-state models, and the midpoint temperatures (T(mid)) for the proteins were generally beta-Lg C > beta-Lg A > beta-Lg B, and the slopes at the midpoint temperatures for the A variant were generally less than those for the B and C variants indicating that beta-Lg A may denature by a different mechanism from that of beta-Lg B or beta-Lg C. The T(mid) parameters derived from the ANS fluorescence intensity results were similar to those for thiol availability and both were lower than the T(mid) values for Trp emission intensity showing that creation of an ANS binding site on a beta-Lg molecule was linked to the irreversible exposure of a thiol group and the loss of native beta-Lg but preceded the decrease in Trp(61) fluorescence quenching. These results for the differences between the behavior of the A and B or the C variants involved the creation of a destabilizing cavity by the Val(118)Ala (A --> B) substitution and the changed charge distribution within the CD loop caused by the Asp(64)Gly (A --> B) substitution.

Patterned acquisition of skin barrier function during development.

Skin barrier function is conferred by the outer layer of epidermis, the stratum corneum, and is essential for terrestrial life. Quantitative trans-epidermal water loss assays show that barrier forms late in embryogenesis, permitting the foetus to survive a terrestrial environment at birth. Using qualitative in situ assays for skin permeability, we show that barrier forms in a patterned manner late in mouse gestation. Barrier forms at specific epidermal sites, then spreads around the embryo as a moving front. The moving front of permeability change is accompanied by multiple changes in the outer, stratum corneum-precursor cells. We use the permeability assays to show that final stages of cornified envelope assembly are coordinated with initial stages of barrier formation. Hence the whole-mount permeability assays record developmental acquisition of a known, essential component of the adult barrier. We demonstrate the authenticity of the whole-mount assays after maternal glucocorticoid therapy (known to accelerate barrier formation) and in additional species including the rat where barrier formation is well characterized by TEWL assay (Aszterbaum, M., Menon, G. K., Feingold, K. R. and Williams, M. L. Pediatr. Res. 31, 308-317). The demonstration of patterned barrier formation in other species suggests patterned change as the universal mode of embryonic barrier acquisition. These results highlight the importance of patterning as a mode of epidermal maturation during development.

Occurrence of arginine deiminase pathway enzymes in arginine catabolism by wine lactic Acid bacteria.

l-Arginine, an amino acid found in significant quantities in grape juice and wine, is known to be catabolized by some wine lactic acid bacteria. The correlation between the occurrence of arginine deiminase pathway enzymes and the ability to catabolize arginine was examined in this study. The activities of the three arginine deiminase pathway enzymes, arginine deiminase, ornithine transcarbamylase, and carbamate kinase, were measured in cell extracts of 35 strains of wine lactic acid bacteria. These enzymes were present in all heterofermentative lactobacilli and most leuconostocs but were absent in all the homofermentative lactobacilli and pediococci examined. There was a good correlation among arginine degradation, formation of ammonia and citrulline, and the occurrence of arginine deiminase pathway enzymes. Urea was not detected during arginine degradation, suggesting that the catabolism of arginine did not proceed via the arginase-catalyzed reaction, as has been suggested in some earlier studies. Detection of ammonia with Nessler's reagent was shown to be a simple, rapid test to assess the ability of wine lactic acid bacteria to degrade arginine, although in media containing relatively high concentrations (>0.5%) of fructose, ammonia formation is inhibited.