Human axillary skin condition is improved following incorporation of glycerol into the stratum corneum from an antiperspirant formulation.

The study objectives were to demonstrate that glycerol, when topically applied from a roll-on antiperspirant formulation, can be delivered directly to human skin ex vivo and the axillary stratum corneum (SC) in vivo, and to assess whether it improves the quality of the axillary skin barrier. Ex vivo human skin absorption of glycerol was measured following application of a roll-on antiperspirant formulation containing 4% 13C3-glycerol. Skin distribution of 13C3-glycerol over 24 h was assessed using gas chromatography-mass spectrometry. In vivo axillary SC penetration was measured by confocal Raman spectroscopy and multivariate curve-resolution software 1 h after topical application of a roll-on antiperspirant formulation containing 8% deuterated glycerol (d5-glycerol). A clinical study was conducted to determine the efficacy of a roll-on antiperspirant formulation containing 4% glycerol in reducing shaving-induced visual irritation and in increasing axillary-skin hydration. Ex vivo skin absorption studies indicated that the formulation delivered 13C3-glycerol into the SC at all timepoints over the 24-h period. In vivo Raman measurements (1 h after application) demonstrated that d5-glycerol was detectable to a depth of at least 10 µm in the axillary SC. Application of 4% glycerol from a roll-on antiperspirant formulation to the axilla was associated with significantly less visible irritation and greater skin hydration than observed with the control (glycerol-free) product. These studies demonstrate that glycerol, incorporated in a roll-on antiperspirant formulation, is delivered directly and rapidly to all depths of the axillary SC, and results in improvements in visible irritation and hydration in the axilla.

Confocal Raman spectroscopy of whole hairs.

This paper describes the application of Raman spectroscopy to whole hair fibers. Previously this has proved difficult because the hairs are relatively opaque, and spatial resolution diminishes with depth because of the change in refractive index. A solution is to couple confocal Raman with multivariate curve resolution (MCR) data analysis, which separates spectral differences with depth despite this reduction in resolution. Initially, it is shown that the cuticle can be separated from the cortex, showing the differences in the proteins, which can then be plotted as a function of depth, with the cuticle factor being seen only at the surface as expected. Hairs that had been treated in different ways, e.g., by bleaching, treatment with the active molecule resorcinol followed by rinsing and treatment with a full hair care product, were also examined. In all cases, changes to the hair are identified and are associated with specific parts of the fiber. Since the hair fiber is kept intact, it can be repeatedly treated and measured, hence multistep treatment processes can be followed. This method expands the potential use of Raman spectroscopy in hair research.

A new in vivo Raman probe for enhanced applicability to the body.

This paper describes a new in vivo Raman probe that allows investigation of areas of the body that are otherwise difficult to access. It is coupled to a previously described commercially available in vivo Raman spectrometer that samples the skin through an optical flat. In the work presented here, the laser light emerges from a smaller pen-shaped probe. It thus works on the same principles as the original spectrometer, while its relative performance in terms of signal-to-noise ratio of the spectra and obtained spatial resolution is only slightly diminished. It allows the window to be placed against the subject in more curved and recessed areas of subject's body and also for them to be more comfortable while the measurements take place. Results from three areas of the body that have previously been very difficult to study are described, the mouth, axilla, and scalp. Results from the scalp and axilla strata cornea (SC) show significant differences from the "normal" SC of the volar forearm. For instance, the scalp is observed to have lower amounts of natural moisturizing factors (NMF) compared to the volar forearm within the same subjects. Also for both the axilla and scalp the lipids show a change in order as compared to the lipids in the volar forearm and also differences from each other. The potential significance of these observations is discussed. Further, we show how we can probe the mouth, in this case observing the presence of the astringent tea polyphenol epigallocatechin gallate within the oral mucosa.

Using the fluorescence decay of 2-aminopurine to investigate conformational change in the recognition sequence of the EcoRV DNA-(adenine-N6)-methyltransferase on enzyme binding.

The EcoRV DNA methyltransferase methylates the first adenine in the GATATC recognition sequence. It is presumed that methylation proceeds by a nucleotide flipping mechanism but no crystal structure is available to confirm this. A popular solution-phase assay for nucleotide flipping employs the fluorescent adenine analogue, 2-aminopurine (2AP), substituted at the methylation target site; a substantial increase in fluorescence intensity on enzyme binding indicates flipping. However, this appeared to fail for M.EcoRV, since 2AP substituted for the non-target adenine in the recognition sequence showed a much greater intensity increase than 2AP at the target site. This anomaly is resolved by recording the fluorescence decay of 2AP which shows that the target 2AP is indeed flipped by the enzyme, but its fluorescence is quenched by interaction with aromatic residues in the catalytic site, whereas bending of the duplex at the non-target site alleviates inter-base quenching and exposes the 2AP to solvent.

Dynamic in vivo mapping of model moisturiser ingress into human skin by GARfield MRI.

We describe the development of in vivo one-dimensional MRI (profiling) using a GARField (Gradient At Right angles to Field) magnet for the characterisation of side-of-hand human skin. For the first time and in vivo, we report measurements of the NMR longitudinal and transverse relaxation parameters and self-diffusivity of the upper layers of human skin with a nominal spatial resolution better than 10 µm. The results are correlated with in vivo confocal Raman spectroscopy measurements of water concentration and natural moisturiser factors, and discussed in terms of known skin biology and microstructure of the stratum corneum and viable epidermis. The application of model moisturiser solutions to the skin is followed and their dynamics of ingress are characterised using the MRI methodology developed. Selected hydrophilic and lipophilic formulations are studied. The results are corroborated by standard in vivo measurements of transepidermal water loss and hydration status. A further insight into moisturisation mechanisms is gained. The effect of two different penetration enhancers on a commonly used skin care oil is also discussed, and different timescales of oil penetration into the skin are reported depending on the type of enhancer.

Long-wavelength fluorescence from 2-aminopurine-nucleobase dimers in DNA.

When 2-aminopurine (2AP) is substituted for adenine in DNA, it is widely accepted that its fluorescence spectrum is essentially unchanged from that of the free fluorophore. We show that 2AP in DNA exhibits long-wavelength emission and excitation bands, in addition to the familiar short-wavelength spectra, as a result of formation of a ground-state heterodimer with an adjacent, pi-stacked, natural base. The observation of dual emission from 2AP in a variety of oligodeoxynucleotide duplexes and single strands demonstrates the generality of this phenomenon. The photophysical and conformational properties of the long-wavelength-emitting 2AP-nucleobase dimer are examined. Analogous long-wavelength fluorescence is seen when 2AP pi-stacks with aromatic amino acid sidechains in the active sites of methyltransferase enzymes during DNA nucleotide flipping.

2-Aminopurine flipped into the active site of the adenine-specific DNA methyltransferase M.TaqI: crystal structures and time-resolved fluorescence.

We report the crystal structure of the DNA adenine-N6 methyltransferase, M.TaqI, complexed with DNA, showing the fluorescent adenine analog, 2-aminopurine, flipped out of the DNA helix and occupying virtually the same position in the active site as the natural target adenine. Time-resolved fluorescence spectroscopy of the crystalline complex faithfully reports this state: base flipping is accompanied by the loss of the very short ( approximately 50 ps) lifetime component associated with fully base-stacked 2-aminopurine in DNA, and 2-aminopurine is subject to considerable quenching by pi-stacking interactions with Tyr108 in the catalytic motif IV (NPPY). This proves 2-aminopurine to be an excellent probe for studying base flipping by M.TaqI and suggests similar quenching in the active sites of DNA and RNA adenine-N6 as well as DNA cytosine-N4 methyltransferases sharing the conserved motif IV. In solution, the same distinctive fluorescence response confirms complete destacking from DNA and is also observed when the proposed key residue for base flipping by M.TaqI, the target base partner thymine, is substituted by an abasic site analog. The corresponding cocrystal structure shows 2-aminopurine in the active site of M.TaqI, demonstrating that the partner thymine is not essential for base flipping. However, in this structure, a shift of the 3' neighbor of the target base into the vacancy left after base flipping is observed, apparently replicating a stabilizing role of the missing partner thymine. Time-resolved fluorescence and acrylamide quenching measurements of M.TaqI complexes in solution provide evidence for an alternative binding site for the extra-helical target base within M.TaqI and suggest that the partner thymine assists in delivering the target base into the active site.