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Supplementary Figures 1-4 from Complex N-Glycan and Metabolic Control in Tumor Cells

Golgi β1,6N-acetylglucosaminyltransferase V (Mgat5) produces β1,6GlcNAc-branched complex N-glycans on cell surface glycoproteins that bind to galectins and promote surface residency of glycoproteins, including cytokine receptors. Carcinoma cells from polyomavirus middle T (PyMT) transgenic mice on a Mgat5−/− background have reduced surface levels of epidermal growth factor (EGF) and transforming growth factor-β (TGF-β) receptors and are less sensitive to acute stimulation by cytokines in vitro compared with PyMT Mgat5+/+ tumor cells but are nonetheless tumorigenic when injected into mice. Here, we report that PyMT Mgat5−/− cells are reduced in size, checkpoint impaired, and following serum withdrawal, fail to down-regulate glucose transport, protein synthesis, reactive oxygen species (ROS), and activation of Akt and extracellular signal-regulated kinase. To further characterize Mgat5+/+ and Mgat5−/− tumor cells, a screen of pharmacologically active compounds was done. Mgat5−/− tumor cells were comparatively hypersensitive to the ROS inducer 2,3-dimethoxy-1,4-naphthoquinone, hyposensitive to tyrosine kinase inhibitors, to Golgi disruption by brefeldin A, and to mitotic arrest by colcemid, hydroxyurea, and camptothecin. Finally, regulation of ROS, glucose uptake, and sensitivities to EGF and TGF-β were rescued by Mgat5 expression or by hexosamine supplementation to complex N-glycan biosynthesis in Mgat5−/− cells. Our results suggest that complex N-glycans sensitize tumor cells to growth factors, and Mgat5 is required to balance responsiveness to growth and arrest cues downstream of metabolic flux. [Cancer Res 2007;67(20):9771–80]

Folate receptor (FR) is a typical tumor-associated antigen that has been widely used as a biomarker for the determination of cancer. A biosensing platform functionalized with folic acid (FA) is essential to FR detection because FA specifically binds FR. We demonstrate an MgZnO dual-gate thin-film transistor (MZO DGTFT) based biosensor for the sensitive determination of FA derivative, hexadecyl alkynated folic acid (HAFA). The receptor is an MZO nanostructure (MZO $_{\mathrm {nano}}$ ) modified sensing pad which electrically connects to the top gate of DGTFT. The bottom gate of DGTFT is used to bias the device to its optimum operation point, enabling high sensitivity. First, 11-azidoundecanoic acid (AA) was bond onto the MZO $_{\mathrm {nano}}$ surface as the linker layer, resulting in negative charges to MZO $_{\mathrm {nano}}$ due to carboxylic acid binding chemistry. HAFA was then immobilized on MZO $_{\mathrm {nano}}$ via click reaction with AA. HAFA is a polar molecule with the positive end adjacent to AA/MZO $_{\mathrm {nano}}$ , and therefore, the electrostatic condition of MZO $_{\mathrm {nano}}$ is impacted again. Such changes were detected by DGTFT that displayed significant drain current variations. Fourier-transform infrared spectroscopy (FTIR) imaging confirms such successful chemical processes. This MZO DGTFT biosensor with HAFA is promising for potential applications in the detection of FR overexpressed cancer cells.

IPr (IPr = 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene) represents the most important NHC (NHC = N-heterocyclic carbene) ligand throughout the field of homogeneous catalysis. Herein, we report the synthesis, catalytic activity, and full structural and electronic characterization of novel, sterically-bulky, easily-accessible NHC ligands based on the hash peralkylation concept, including IPr#, Np# and BIAN-IPr#. The new ligands have been commercialized in collaboration with Millipore Sigma: IPr#HCl, 915653; Np#HCl; 915912; BIAN-IPr#HCl, 916420, enabling broad access of the academic and industrial researchers to new ligands for reaction optimization and screening. In particular, the synthesis of IPr# hinges upon cost-effective, modular alkylation of aniline, an industrial chemical that is available in bulk. The generality of this approach in ligand design is demonstrated through facile synthesis of BIAN-IPr# and Np#, two ligands that differ in steric properties and N-wingtip arrangement. The broad activity in various cross-coupling reactions in an array of N–C, O–C, C–Cl, C–Br, C–S and C–H bond cross-couplings is demonstrated. The evaluation of steric, electron-donating and π-accepting properties as well as coordination chemistry to Au(i), Rh(i) and Pd(ii) is presented. Given the tremendous importance of NHC ligands in homogenous catalysis, we expect that this new class of NHCs will find rapid and widespread application., We report novel, sterically-bulky, easily-accessible NHC ligands based on the hash peralkylation concept. The new ligands have been commercialized in collaboration with Millipore Sigma: IPr#HCl, 915653; Np#HCl; 915912; BIAN-IPr#HCl, 916420.

This work reports our discoveries from the first exploration in microwave pyrolysis of a metal–organic framework. A time- and energy-efficient approach was developed for direct fabrication of electrochemical single-atom catalysts (E-SACs) without the requirement of post-treatment. The most unique structure of the fabricated E-SAC is that the Cu catalytic sites were not in the amorphous carbon matrix as those achieved via traditional pyrolysis but in the basal planes of pristine holey graphene nanoplatelets. The as-prepared Cu-E-SAC exhibits excellent catalytic activity and selectivity in reducing oxygen to water in both acidic and alkaline media. The desired direct 4e– pathway is more favorable in acidic versus alkaline media, which is different from all the Cu-E-SACs reported so far and most transition-metal-based E-SACs. The superior performance is attributed to the unique structure of the catalytic sites. The large graphene domains in the holey graphene materials provide higher delocalized electron-rich π band and increase the d-orbital energy level of the Cu centers. Consequently, their binding strength for molecular oxygen is largely enhanced, improving the oxygen reduction reaction and likely promoting a direct 4e– pathway with minimized generation of a peroxide byproduct. Considering the high conductivity and excellent stability against oxidation of the holey graphene material, this work, for the first time, suggests that the contradictory structural requirement of a carbon matrix for high catalytic activity and long-term durability can be unified and simultaneously satisfied. Combined with the merits of simplicity and rapidness for fabricating both holey graphene and E-SACs, this work provides a possible strategy to address the critical challenges of precious metal-free single-atom catalysts.

Purpose This work investigated whether topical pain relief diclofenac gels can form a diclofenac reservoir in the epidermal and dermal layers of human skin. Methods Excised human skin samples were treated with three topical diclofenac gels ex vivo and examined using Raman microscopy of transversally microtomed sections. The relative diclofenac concentration in the skin layers was calculated as the ratio of the integrated areas of bands characteristic of diclofenac (~445 cm-1) and skin (Amide I). A customized masking algorithm ensured that only diclofenac-specific signal was mapped in the resulting Raman images. Results A heterogenous spatial distribution of diclofenac was clearly visible in both the epidermis and the dermis in all samples, with a markedly higher diclofenac relative content and number of pixels above the detection limit in the epidermis compared to the dermis. Conclusion The Raman images evidenced that the studied topical gels deliver diclofenac through the stratum corneum skin barrier and form a drug depot localized in the epidermis. The data are in line with earlier clinical findings that this depot acts like a true reservoir and enables sustained drug release.

BACKGROUND Skin hydration is essential for maintaining stratum corneum (SC) flexibility and facilitating maturation events. Moisturizers contain multiple ingredients to maintain and improve skin hydration although a complete understanding of hydration mechanisms is lacking. The ability to differentiate the source of the hydration (water from the environment or deeper skin regions) upon application of product will aid in designing more efficacious formulations. MATERIALS AND METHODS Novel confocal Raman microscopy (CRM) experiments allow us to investigate mechanisms and levels of hydration in the SC. Using deuterium oxide (D2 O) as a probe permits the differentiation of endogenous water (H2 O) from exogenous D2 O. Following topical application of D2 O, we first compare in vivo skin depth profiles with those obtained using ex vivo skin. Additional ex vivo experiments are conducted to quantify the kinetics of D2 O diffusion in the epidermis by introducing D2 O under the dermis. RESULTS Relative D2 O depth profiles from in vivo and ex vivo measurements compare well considering procedural and instrumental differences. Additional in vivo experiments where D2 O was applied following topical glycerin application increased the longevity of D2 O in the SC. Reproducible rates of D2 O diffusion as a function of depth have been established for experiments where D2 O is introduced under ex vivo skin. CONCLUSION Unique information regarding hydration mechanisms are obtained from CRM experiments using D2 O as a probe. The source and relative rates of hydration can be delineated using ex vivo skin with D2 O underneath. One can envision comparing these depth-dependent rates in the presence and absence of topically applied hydrating agents to obtain mechanistic information.

Introduction: As skin ages, it loses its ability to retain moisture and becomes rough and dry. This results in a clinically dull appearance with a loss of radiance, firmness, and suppleness. Symptoms can be improved with use of a moisturizer that builds and maintains skin hydration over time; however, most moisturizers that occlude the skin surface are perceived as heavy and greasy and are not consumer preferred. Methods: A unique, consumer-preferred gel matrix formula was developed by combining liquid crystal structures, which mimic skin barrier lipid assembly, with specific emulsifiers that deliver water deep into skin. Ex vivo studies were conducted to investigate the superior hydrating effects of the gel matrix formula. Confocal Raman microscopy studies assessed the spatial distribution of water in ex vivo skin after application of the gel matrix formula. To determine the effects of the gel matrix formula on dry facial skin, a 12-week clinical study was conducted with subjects with self-perceived skin dryness and dullness. Results: The formulation significantly increased the relative water content throughout epidermal regions, which was not observed with the application of a competitive gel formula. Instrumental measurements assessed improvements in skin surface moisturization and barrier function. Clinical grading showed significant improvements in hydration-related endpoints including radiance, clarity, and texture. Subject self-agree assessment demonstrated that subjects observed improvements in the appearance of their facial skin. Conclusion: These studies demonstrated that the gel matrix formula increased skin water content in deeper layers, and resulted in significant clinical improvements in hydration, barrier function, and clinical appearance of radiance.

Direct α-alkylation of carbonyl compounds represents a fundamental bond forming transformation in organic synthesis. We report the first ketone-alkylation using olefins and alcohols as simple alkylating agents catalyzed by graphene oxide. Extensive studies of the graphene surface suggest a pathway involving dual activation of both coupling partners. Notably, we show that polar functional groups have a stabilizing effect on the GO surface, which results in a net enhancement of the catalytic activity. The method represents the first alkylation of carbonyl compounds using graphenes, which opens the door for the development of an array of protocols for ketone functionalization employing common carbonyl building blocks and readily available graphenes.

Purpose This work investigated whether topical pain relief diclofenac gels can form a diclofenac reservoir in the epidermal and dermal layers of human skin. Methods Excised human skin samples were treated with three topical diclofenac gels ex vivo and examined using Raman microscopy of transversally microtomed sections. The relative diclofenac concentration in the skin layers was calculated as the ratio of the integrated areas of bands characteristic of diclofenac (~445 cm−1) and skin (Amide I). A customized masking algorithm ensured that only diclofenac-specific signal was mapped in the resulting Raman images. Results A heterogenous spatial distribution of diclofenac was clearly visible in both the epidermis and the dermis in all samples, with a markedly higher diclofenac relative content and number of pixels above the detection limit in the epidermis compared to the dermis. Conclusion The Raman images evidenced that the studied topical gels deliver diclofenac through the stratum corneum skin barrier and form a drug depot localized in the epidermis. The data are in line with earlier clinical findings that this depot acts like a true reservoir and enables sustained drug release.

Metal organic chemical vapor deposition grown films consisting of MgxZn1-xO (4%

Little is known about how stony corals build their calcareous skeletons. There are two prevailing hypotheses: that it is a physicochemically dominated process and that it is a biologically mediated one. Using a combination of ultrahigh-resolution three-dimensional imaging and two-dimensional solid-state nuclear magnetic resonance (NMR) spectroscopy, we show that mineral deposition is biologically driven. Randomly arranged, amorphous nanoparticles are initially deposited in microenvironments enriched in organic material; they then aggregate and form ordered aragonitic structures through crystal growth by particle attachment. Our NMR results are consistent with heterogeneous nucleation of the solid mineral phase driven by coral acid-rich proteins. Such a mechanism suggests that stony corals may be able to sustain calcification even under lower pH conditions that do not favor the inorganic precipitation of aragonite.

A highly porous graphitic carbon material, dually-doped with P and S, was studied as a metal free catalyst for aerobic oxidation reactions. Catalytic mechanism studies suggest that the active centers, originated from P-and S-doping, additively/synergistically catalyze the aerobic oxidation of benzylic alcohols but with different pathways. For the first time, catalytic centers stemming from S-doping were experimentally identified to be exocyclic S species (C-S-C, sulfur out of the carbon ring), which are different from those proposed for electrochemical oxygen reduction reactions (ORR) with a 4e− pathway and oxygen evaluation reactions (OER). Notably, all the catalytic sites from both P and S doping share a similar “protruding out” pyramid structure, which is in contrast to the planar structure of the catalytic sites in N- or B-doped graphitic materials. The unique geometric structure of the catalytic sites can minimize substrate steric hindrance effects, endowing the P, S co-doped catalysts with a wide substrate scope and functional group tolerance. Furthermore, the unambiguous distinguishment of the catalytic sites from those in OER and ORR provides valuable guidance for designing and developing carbon materials with controlled active sites to satisfy different catalytic applications.

During lactation, mammals resorb mineral from the maternal skeleton to provide calcium to milk. Rodents lose 25% to 35% of skeletal ash weight, ash calcium content, and bone mineral content as measured by dual-energy X-ray absorptiometry (DXA), and have compromised material properties of bone as assessed by crushing vertebrae and 3-point bend tests of femora or tibias. The strength, stiffness, and toughness of vertebrae, femora, and tibias are reduced by as much as 60%. The effects of lactation are not uniform throughout the skeleton, but instead resorption is much more marked in the trabecular-rich spine than in the appendicular skeleton or whole body. Women who breastfeed exclusively lose an average of 210 mg calcium in milk each day, whereas nursing of twins or triplets can double and triple the output of calcium. Clinical data are also consistent with skeletal calcium being released during lactation to provide much of the calcium needed for milk production. Lumbar spine bone mineral density (BMD), as assessed by DXA, declines by a mean of 5% to 10% among numerous studies during 3 to 6 months of exclusive lactation, whereas largely cortical sites (hip, forearm, whole body) show half that loss or no significant changes. Micro-CT of rodents and high-resolution peripheral quantitative computed tomography (HR-pQCT) imaging of women confirm that lactation causes microarchitectural deterioration of bone. These skeletal losses occur through two pathways: upregulated osteoclast-mediated bone resorption and osteocytic osteolysis, in which osteocytes remove mineral from their lacunae and pericanalicular spaces. After weaning, the skeleton is fully restored to its prior mineral content and strength in both animal models and humans, despite persistent microarchitectural changes observed in high-resolution imaging. Osteoblasts upregulate to lay down new osteoid, while osteocytes remineralize their surroundings. The factors that stimulate this post-weaning skeletal recovery remain unclear. In most studies, a history of lactation does not increase the risk, but may protect against, low BMD and fragility fractures. © 2017 American Society for Bone and Mineral Research.

Transition-metal-catalyzed alkylation reactions of arenes have become a central transformation in organic synthesis. Herein, we report the first general strategy for alkylation of arenes with styrenes and alcohols catalyzed by carbon-based materials, exploiting the unique property of graphenes to produce valuable diarylalkane products in high yields and excellent regioselectivity. The protocol is characterized by a wide substrate scope and excellent functional group tolerance. Notably, this process constitutes the first general application of graphenes to promote direct C-C bond formation utilizing polar functional groups anchored on the GO surface, thus opening the door for an array of functional group alkylations using benign and readily available graphene materials. Mechanistic studies suggest that the reaction proceeds via a tandem catalysis mechanism in which both of the coupling partners are activated by interaction with the GO surface.

Terbinafine (Tbf) is a well-established anti-fungal agent used for management of a variety of dermal conditions including ringworm and athlete's foot. Both the biochemical mechanism of Tbf fungicidal action (based on squalene epoxidase inhibition) and the target region for Tbf in vivo (the stratum corneum (SC)) are well determined. However, the biochemical and pharmacokinetic approaches used to evaluate Tbf biochemistry provide no biophysical information about molecular level physical changes in the SC upon Tbf binding. Such information is necessary for improved drug and formulation design. IR spectroscopic methods were used to evaluate the effects of Tbf on keratin structure in environments commonly used in pharmaceutics to mimic those in vivo. The Amide I and II spectral regions (1500–1700 cm−1) provided an effective means to monitor keratin secondary structure changes, while a Tbf spectral feature near 775 cm−1 provides a measure of relative Tbf levels in skin. Interaction of Tbf with the SC induced substantial β-sheet formation in the keratin, an effect which was partially reversed both by ethanol washing and by exposure to high relative humidity. The irreversibility suggests the presence of a Tbf reservoir (consistent with kinetic studies), permitting the drug to be released in a controlled manner into the surrounding tissue.

Bone is a highly organized tissue in which each structural level influences the macroscopic and microscopic mechanical behavior. In particular, the quantity, quality, and distribution of the different bone components, i.e. collagen matrix and hydroxyapatite crystals, are associated with bone strength or fragility. Common spectroscopic techniques used to assess bone composition have resolutions limited to the micrometer range. In this study, our aims were two-fold: i) to develop and validate the AFM-IR methodology for skeletal tissues and ii) to apply the methodology to sheep cancellous bone with the objective to obtain novel findings on the composition and structure of trabecular packets.To develop the methodology, we assessed spatial and temporal reproducibility using a known homogeneous material (polymethylmethacrylate, PMMA). We verified that the major peak positions were similar and not shifted when compared to traditional Fourier Transform Infrared imaging (FTIRI). When AFM-IR was applied to sheep cancellous bone, the mineral-to-matrix ratio increased and the acid phosphate substitution ratio decreased as a function of tissue maturity. The resolution of the technique enabled visualization of different stages of the bone maturation process, particularly newly-formed osteoid prior to mineralization. We also observed alternating patterns of IR parameters in line and imaging measurements, suggesting the apposition of layers of alternating structure and / or composition that were not visible with traditional spectroscopic methods. In conclusion, nanoscale IR spectroscopy demonstrates novel compositional and structural changes within trabecular packets in cancellous bone. Based on these results, AFM-IR is a valuable tool to investigate cancellous bone at the nanoscale and, more generally, to analyze small dynamic areas that are invisible to traditional spectroscopic methods.

The main barrier to permeability in human skin resides in the stratum corneum (SC), a layered structure consisting of anucleated, flattened cells (corneocytes) embedded in a heterogeneous lamellar lipid matrix. While lipid structures and packing propensities in the SC and in SC models have been extensively investigated, only limited data are available concerning the kinetics and mechanism of formation of lamellar phases and particular lipid packing motifs. In our prior investigation, kinetic IR spectroscopy measurements probed the temporal sequence of phase separation leading to ordered structures in a three component SC model of equimolar structurally heterogeneous ceramide[NS], chain perdeuterated stearic acid, and cholesterol. In the current work, the phase separation kinetic effects of specific fatty acid chain lengths with a synthetic structurally homogeneous ceramide[NS] in similar ternary mixtures are examined. These are compared with a mixture containing ceramide[NS] with an unsaturated acid chain. The kinetic events are sensitive to the difference in chain lengths between the ceramide acid chain and the fatty acid as well as to the presence of unsaturation in the former. The observed kinetic behaviors span a wide range of phase separation times, ranging from the formation of a solid solution stable for at least 200 h, to a system in which an orthorhombic fatty acid structure is essentially completely formed within the time resolution of the experiment (15 min). The data seem to offer some features of a spinodal phase separation at relatively short times. Overall the approach offers a possible means for addressing several unanswered questions pertinent to skin pharmacology, such as the roles of a wide variety of ceramide and fatty acid species and the design of therapeutic interventions for repair of pathological conditions of the SC.

A variety of strategies for the synthesis of solution processable graphene sheets has been developed so far. However, no approach has been reported to directly produce highly conductive, low-oxygen-containing graphene sheets without relying on toxic reagents and metal containing compounds and without generating toxic by-products. With an aim of developing such an eco-friendly approach, for the first time, this work studied solution phase oxidation of graphite particles and reversible graphite intercalation compounds using molecular oxygen and piranha etching solutions. We found that the synergy of the piranha etching solution and the intercalated molecular oxygen enables controlled oxidation of graphite particles assisted by microwave heating. The controlled oxidation leads to the rapid and direct generation of highly conductive, “clean”, and low oxygen containing graphene sheets without releasing toxic gases or aromatic by-products as detected by gas chromatography-mass spectrometry (GC-MS). These highly conductive graphene sheets have unique molecular structures, different from both graphene oxide and pristine graphene sheets. It is even different from chemically reduced graphene oxide, while combining many of its merits. They can be dispersed in both aqueous and common organic solvents without surfactants/stabilizers, producing “clean” solution phase graphene sheets. “Paper-like” graphene films are generated via simple filtration, resulting in films with a conductivity of 2.3 × 104 S m−1, the highest conductivity observed so far for graphene films assembled via vacuum filtration from solution processable graphene sheets. After 2 hours of low temperature annealing at 300 °C, the conductivity further increased to 7.4 × 104 S m−1. This eco-friendly and rapid approach for the production of highly conductive and “clean” solution-phase graphene sheets would enable a broad spectrum of applications at low cost.

To demonstrate the efficacy of infrared (IR) spectroscopic imaging for evaluation of lateral diffusion in stratum corneum (SC) and for elucidation of intermolecular interactions between exogenous agents and SC constituents.In separate experiments, acyl chain perdeuterated oleic acid (OA-d) and deuterated dimethyl sulfoxide (DMSO-d) were applied to the surface of isolated human SC. The lateral distribution of permeant concentrations was monitored using the time-dependence of IR images. Diffusion coefficients (D) were estimated from Fick's second law. Interactions between the exogenous agents and the SC were tracked from changes in CD2 and Amide I stretching frequencies.Networked glyphs served as the major pathway for lateral distribution of OA-d. In glyph-poor regions, D values from 0.3-1 × 10(-8) cm(2)/s bracketed the OA-d data and apparently decreased with time. Although diffusion of DMSO-d is relatively fast compared to our experimental measurement time, the results suggest values of ~10(-7) cm(2)/s. OA-d spectral changes suggest penetration into the ordered lipids of the SC; DMSO-d penetration results in perturbation of SC keratin structure.IR imaging provides concentration profiles, diffusion coefficients, and unique molecular level information about structural changes in the endogenous SC constituents and exogenous agents upon their mutual interaction. Transport along glyphs is the dominant mode of distribution for OA-d.

Plant-derived oils consisting of triglycerides and small amounts of free fatty acids (FFAs) are commonly used in skincare regimens. FFAs are known to disrupt skin barrier function. The objective of this study was to mechanistically study the effects of FFAs, triglycerides and their mixtures on skin barrier function. The effects of oleic acid (OA), glyceryl trioleate (GT) and OA/GT mixtures on skin barrier were assessed in vivo through measurement of transepidermal water loss (TEWL) and fluorescein dye penetration before and after a single application. OA's effects on stratum corneum (SC) lipid order in vivo were measured with infrared spectroscopy through application of perdeuterated OA (OA-d34 ). Studies of the interaction of OA and GT with skin lipids included imaging the distribution of OA-d34 and GT ex vivo with IR microspectroscopy and thermodynamic analysis of mixtures in aqueous monolayers. The oil mixtures increased both TEWL and fluorescein penetration 24 h after a single application in an OA dose-dependent manner, with the highest increase from treatment with pure OA. OA-d34 penetrated into skin and disordered SC lipids. Furthermore, the ex vivo IR imaging studies showed that OA-d34 permeated to the dermal/epidermal junction while GT remained in the SC. The monolayer experiments showed preferential interspecies interactions between OA and SC lipids, while the mixing between GT and SC lipids was not thermodynamically preferred. The FFA component of plant oils may disrupt skin barrier function. The affinity between plant oil components and SC lipids likely determines the extent of their penetration and clinically measurable effects on skin barrier functions.

Acid phosphate substitution into mineralized tissues is an important determinant of their mechanical properties and their response to treatment. This study identifies and validates Fourier transform infrared spectroscopic imaging (FTIRI) spectral parameters that provide information on the acid phosphate (HPO4) substitution into hydroxyapatite in developing mineralized tissues. Curve fitting and Fourier self-deconvolution were used to identify subband positions in model compounds (with and without HPO4). The intensity of subbands at 1127 and 1110 cm(-1) correlated with the acid phosphate content in these models. Peak height ratios of these subbands to the ν3 vibration at 1096 cm(-1) found in stoichiometric apatite were evaluated in the model compounds and mixtures thereof. FTIRI spectra of bones and teeth at different developmental ages were analyzed using these spectral parameters. Factor analysis (a chemometric technique) was also conducted on the tissue samples and resulted in factor loadings with spectral features corresponding to the HPO4 vibrations described above. Images of both factor correlation coefficients and the peak height ratios 1127/1096 and 1112/1096 cm(-1) demonstrated higher acid phosphate content in younger vs. more mature regions in the same specimen. Maps of the distribution of acid phosphate content will be useful for characterizing the extent of new bone formation, the areas of potential decreased strength, and the effects of therapies such as those used in metabolic bone diseases (osteoporosis, chronic kidney disease) on mineral composition. Because of the wider range of values obtained with the 1127/1096 cm(-1) parameter compared to the 1110/1096 cm(-1) parameter and the smaller scatter in the slope, it is suggested that this ratio should be the parameter of choice.

Acne vulgaris is a disease of pilosebaceous units with multifactorial pathogenesis, including hyperkeratinization, increased sebum secretion, and inflammation. Recently, it was suggested that acne subjects may have also impaired skin barrier. We hypothesized that excess unsaturated free fatty acids (UFFA) present in the sebum may cause barrier impairment associated with increased follicular stratum corneum (SC) thickening and inflammation seen in acne. Therefore, epidermal and sebaceous lipid profiles from acne and healthy subjects were analyzed and an in vitro epidermal tissue model was developed to validate this hypothesis. Significantly increased levels of free fatty acids (p

Transition-metal-catalyzed alkylation reactions of arenes have become a central transformation in organic synthesis. Herein, we report the first general strategy for alkylation of arenes with styrenes and alcohols catalyzed by carbon-based materials, exploiting the unique property of graphenes to produce valuable diarylalkane products in high yields and excellent regioselectivity. The protocol is characterized by a wide substrate scope and excellent functional group tolerance. Notably, this process constitutes the first general application of graphenes to promote direct C–C bond formation utilizing polar functional groups anchored on the GO surface, thus opening the door for an array of functional group alkylations using benign and readily available graphene materials. Mechanistic studies suggest that the reaction proceeds via a tandem catalysis mechanism in which both of the coupling partners are activated by interaction with the GO surface.

An extremely simple and rapid (seconds) approach is reported to directly synthesize gram quantities of P-doped graphitic porous carbon materials with controlled P bond configuration. For the first time, it is demonstrated that the P-doped carbon materials can be used as a selective metal free catalyst for aerobic oxidation reactions. The work function of P-doped carbon materials, its connectivity to the P bond configuration, and the correlation with its catalytic efficiency are studied and established. In direct contrast to N-doped graphene, the P-doped carbon materials with higher work function show high activity in catalytic aerobic oxidation. The selectivity trend for the electron donating and withdrawing properties of the functional groups attached to the aromatic ring of benzylic alcohols is also different from other metal free carbon based catalysts. A unique catalytic mechanism is demonstrated, which differs from both GO and N-doped graphene obtained by high temperature nitrification. The unique and unexpected catalytic pathway endows the P-doped materials with not only good catalytic efficiency but also recyclability. This, combined with a rapid, energy saving approach that permits fabrication on a large scale, suggests that the P-doped porous materials are promising materials for “green catalysis” due to their higher theoretical surface area, sustainability, environmental friendliness and low cost.

For effective topical delivery, a drug must cross the stratum corneum (SC) barrier into viable tissue. The use of permeation enhancers is a widespread approach for barrier modification. In the current study, flufenamic acid (FluA), a non-steroidal anti-inflammatory drug, is a model agent for investigating the influence of hydrophobic versus hydrophilic enhancers. In separate experiments, FluA in octanol or propylene glycol/ethanol (75/25) is applied to the SC for varying times followed by confocal Raman microscopic mapping of drug and enhancer penetration and spatial distribution. Deuterated versions of the enhancers permit us to spectroscopically distinguish the exogenous chemicals from the endogenous SC lipids without affecting penetration parameters. The FluA pathway is tracked by the CC stretching mode at ∼1618cm(-1). Discrete, small inclusions of both enhancers are observed throughout the SC. High concentrations of FluA are co-localized with octanol domains which appear to provide a pathway to the viable epidermis for the drug. In contrast, FluA concentrates in the upper SC when using the hydrophilic agent and endogenous lipids appear unperturbed in regions outside the enhancer pockets. The ability to examine perturbations to endogenous ultrastructure and molecular structure in skin while tracking penetration pathways provides insight into delivery mechanisms.

The deposition of extracellular matrix (ECM) produced by osteoblasts is one of the first steps in bone formation. Composition and structure of the ECM influence the development and strength of bone, as well as the onset of its mineralization. Since ECM is secreted onto the surface where the cells attach, Fourier transform infrared (FT-IR) attenuated total reflection (ATR), as a surface sensitive technique, provides a useful tool for its investigation, as ECM instead of the cells is predominantly detected by the IR beam. The purpose of the present study was to develop the FT-IR ATR technique so that real-time measurements of the ECM produced by MC3T3-E1 osteoblasts could be obtained in situ. Measurements were performed using polarized incident IR light to apply a procedure for solvent compensation which reduces the influence of culture medium on the evaluation of the amide I and II bands. The formation of ECM took place in a flow-through chamber under dynamic conditions by applying a constant flow of culture medium and was tracked over a time period of two weeks by evaluation of the integrated absorbance of amide I and II bands reflecting the amount and isotropic arrangement of amide bonds in the ECM. Cultures without ascorbic acid had a reduced protein concentration that enabled the analysis of cell-mediated matrix accumulation. Presence and proliferation of cells after two weeks of permanent flow-through of culture medium was shown by cell counting exhibiting a 67% increase in cell number as well as by crystal violet and live/dead staining. These results demonstrate the application of FT-IR ATR spectroscopy for monitoring matrix formation.

Recent technological advances have led to an increase in the use of confocal Raman microscopy to tackle biophysical and pharmacological issues in topical delivery and dermatology. Advantages of the technique include the ability to optically section intact, full thickness skin without the need to microtome or otherwise physically segment the skin. In addition, both exogenous agents and perturbations to endogenous structure can be monitored without the introduction of probe molecules. In the current work, flufenamic acid (FluA), a non-steroidal anti-inflammatory drug, is a model agent for investigating the influence of lipophilic versus hydrophilic penetration enhancers on the outer barrier layer of skin. For topical delivery to be effective, a drug must cross the stratum corneum (SC) barrier into viable tissue. In separate experiments, FluA in octanol or propylene glycol/ethanol (75/25) is applied to skin for varying times followed by confocal Raman microscopic mapping of drug and enhancer spatial distribution. The FluA pathway is tracked by the C=C stretching mode at 1618 cm−1. The exogenous enhancers are spectroscopically differentiated from the endogenous lipids by utilizing deuterated versions of the enhancers which do not affect penetration parameters. Discrete pockets (5-20 μm) of both enhancers are observed throughout the SC. High concentrations of FluA are co-localized with octanol inclusions which appear to provide a pathway to the viable epidermis for the drug. In contrast, FluA concentrates in the upper SC when using the hydrophilic agent and endogenous lipids are unperturbed in regions outside the enhancer pockets. The ability to examine perturbations to endogenous ultrastructure and molecular structure in skin while tracking penetration pathways provides insight into delivery mechanisms.

The barrier function of skin is primarily provided by the lamellar lipid matrix of the stratum corneum (SC), which has been shown in previous infrared (IR) and related studies to consist predominantly of ordered lipids packed in orthorhombic and hexagonal domains. In the current work, we investigate the effects of the anionic surfactant, sodium dodecyl sulphate (SDS), on SC lipid packing and phase behaviour, using FT-IR spectroscopy. The use of acyl chain perdeuterated SDS allows unequivocal spectroscopic detection of both endogenous lipid and exogenous material in intact tissue. IR spectra were acquired as a function of temperature from isolated human SC exposed to SDS for various incubation periods at 34� C. SDS is found to enter the SC and is observed to be in a more ordered state in the SC than in solution, indicating that the SDS interacts with the ordered SC lipids. The results reveal that SDS reduces the amount of orthorhombic phase in the SC and increases the amount of hexagonally packed lipid at physiologically relevant temperatures. In addition, a small decrease in the lipid Tm (acyl chain melting temperature) is observed. Furthermore, these SDS-induced changes were found to be strongly dependent on the time of exposure.

Bone strength depends on both bone quantity and quality. The former is routinely estimated in clinical settings through bone mineral density measurements but not the latter. Bone quality encompasses the structural and material properties of bone. Although its importance is appreciated, its contribution in determining bone strength has been difficult to precisely quantify partly because it is multifactorial and requires investigation of all bone hierarchical levels. Fourier transform infrared spectroscopy provides one way to explore these levels.The purposes of our review were to (1) provide a brief overview of Fourier transform infrared spectroscopy as a way to establish bone quality, (2) review the major bone material parameters determined from Fourier transform infrared spectroscopy, and (3) review the role of Fourier transform infrared microspectroscopic analysis in establishing bone quality.We used the ISI Web of Knowledge database initially to identify articles containing the Boolean term "infrared" AND "bone." We then focused on articles on infrared spectroscopy in bone-related journals.Infrared spectroscopy provides information on bone material properties. Their microspectroscopic versions allow one to establish these properties as a function of anatomic location, mineralization extent, and bone metabolic activity. It provides answers pertaining to the contribution of mineral to matrix ratio, mineral maturity, mineral carbonate substitution, and collagen crosslinks to bone strength. Alterations of bone material properties have been identified in disease (especially osteoporosis) not attainable by other techniques.Infrared spectroscopic analysis is a powerful tool for establishing the important material properties contributing to bone strength and thus has helped better understand changes in fragile bone.

Water is an integral part of collagen's triple helical and higher order structure. Studies of model triple helical peptides have revealed the presence of repetitive intrachain, interchain, and intermolecular water bridges (Bella et al., Structure 1995, 15, 893-906). In addition, an extended cylinder of hydration is thought to be responsible for collagen fiber assembly. Confocal Raman spectroscopy and dynamic vapor sorption (DVS) measurements of human Type I collagen and pigskin dermis were performed to probe relative humidity (RH)-dependent differences in the nature and level of collagen hydration. Raman spectra were also acquired as a function of time for both Type I collagen and pigskin dermis samples upon exchange of a 100% RH H(2) O to deuterium oxide (D(2) O) environment. Alterations in Amide I and III modes were consistent with anticipated changes in hydrogen bonding strength as RH increased and upon H → D exchange. Of note is the identification of a Raman spectral marker (band at 938 cm(-1) ) which appears to be sensitive to alterations in collagen-bound water. Analysis of DVS isotherms provided a quantitative measure of adsorbed and absorbed water vapor consistent with the Raman results. The development of a Raman spectral marker of collagen hydration in intact tissue is relevant to diverse fields of study ranging from the evaluation of therapeutics for wound healing to hydration of aging skin.

Infrared reflection–absorption spectroscopy (IRRAS) of lipid/protein monolayer films in situ at the air/water interface provides unique molecular structure and orientation information from the film constituents. The technique is thus well suited for studies of lipid/protein interaction in a physiologically relevant environment. Initially, the nature of the IRRAS experiment is described and the molecular structure information that may be obtained is recapitulated. Subsequently, several types of applications, including the determination of lipid chain conformation and tilt as well as elucidation of protein secondary structure are reviewed. The current article attempts to provide the reader with an understanding of the current capabilities of IRRAS instrumentation and the type of results that have been achieved to date from IRRAS studies of lipids, proteins, and lipid/protein films of progressively increasing complexity. Finally, possible extensions of the technology are briefly considered.

A surface functionalization methodology for the development of ZnO nanotips biosensors that can be integrated with microelectronics was developed. Two types of long chain carboxylic acids linkers were employed for the functionalization of 0.5 mum thick MOCVD-grown ZnO nanotip films with single-stranded DNA (ssDNA), followed by hybridization with complementary ssDNA tagged with fluorescein. The ZnO functionalization strategy was developed for the fabrication of ZnO nanotips-linker-biomolecule films integrated with bulk acoustic wave (BAW) biosensors, and it involved three main steps. First, 16-(2-pyridyldithiol)hexadecanoic acid or N-(15-carboxypentadecanoyloxy)succinimide, both bifunctional C16 carboxylic acids, were bound to ZnO nanotip films through the COOH group, leaving at the opposite end of the alkyl chain a thiol group protected as a 2-pyridyl disulfide, or a carboxylic group protected as a N-succinimide, respectively. In the second step, ssDNA was covalently linked to each type of ZnO-linker film: the 2-pyridyl disulfide end group was substituted with 16 bases 5'-thiol-modified DNA (SH-ssDNA), and the N-succinimide ester end group was substituted with 16 bases 5'-amino-modified DNA (NH(2)-ssDNA). In the third step, the DNA-functionalized ZnO nanotip films were hybridized with complementary 5'-fluorescein ssDNA. The surface-modified ZnO nanotip films were characterized after each step by FT-IR-ATR, fluorescence emission spectroscopy, and fluorescence microscopy. This functionalization approach allows sequential reactions on the surface and, in principle, can be extended to numerous other molecules and biomolecules.

The repair of cutaneous wounds in the adult body involves a complex series of spatially and temporally organized processes to prevent infection and restore homeostasis. Three characteristic phases of wound repair (inflammation, proliferation including re-epithelialization and remodelling) overlap in time and space. We have utilized a human skin wound-healing model to correlate changes in genotype and pheno-type with infrared (IR) and confocal Raman spectroscopic images during the re-epithelialization of excisional wounds. The experimental protocols validated as IR images clearly delineate the keratin-rich migrating epithelial tongue from the collagen-rich wound bed. Multivariate statistical analysis of IR datasets acquired 6 days post-wounding reveal subtle spectral differences that map to distinct spatial distributions, which are correlated with immunofluorescent staining patterns of different keratin types. Images computed within collagen-rich regions expose complementary spatial patterns and identify elastin in the wound bed. The temporal sequence of events is explored through a comparison of gene array analysis with confocal Raman microscopy. Our approach demonstrates the feasibility of acquiring detailed molecular structure information from the various proteins and their subclasses involved in the wound-healing process.

Effective innate host defense requires early recognition of pathogens. Surfactant protein D (SP-D), shown to play a role in host defense, binds to the lipopolysaccharide (LPS) component of Gram-negative bacterial membranes. Binding takes place via the carbohydrate recognition domain (CRD) of SP-D. Recombinant trimeric neck+CRDs (NCRD) have proven valuable in biophysical studies of specific interactions. Although X-ray crystallography has provided atomic level information on NCRD binding to carbohydrates and other ligands, molecular level information about interactions between SP-D and biological ligands under physiologically relevant conditions is lacking. Infrared reflection-absorption spectroscopy (IRRAS) provides molecular structure information from films at the air/water interface where protein adsorption to LPS monolayers serves as a model for protein-lipid interaction. In the current studies, we examine the adsorption of NCRDs to Rd 1 LPS monolayers using surface pressure measurements and IRRAS. Measurements of surface pressure, Amide I band intensities, and LPS acyl chain conformational ordering, along with the introduction of EDTA, permit discrimination of Ca (2+)-mediated binding from nonspecific protein adsorption. The findings support the concept of specific binding between the CRD and heptoses in the core region of LPS. In addition, a novel simulation method that accurately predicts the IR Amide I contour from X-ray coordinates of NCRD SP-D is applied and coupled to quantitative IRRAS equations providing information on protein orientation. Marked differences in orientation are found when the NCRD binds to LPS compared to nonspecific adsorption. The geometry suggests that all three CRDs are simultaneously bound to LPS under conditions that support the Ca (2+)-mediated interaction.

The four-component system acyl chain perdeuterated 1,2-dipalmitoylphosphatidylcholine (DPPC)/1,2-dipalmitoylphosphatidylglycerol/ (DPPG)/pulmonary surfactant protein SP-C/cholesterol provides a useful model for in vitro biophysical studies of the reversible monolayer to multilayer transition that occurs during compression--expansion cycles in the lung. Monolayer films of this mixture (with chain perdeuterated DPPC-d62) at the air/water interface have been transferred to solid substrates under conditions of continuously varying surface pressure, an approach termed COVASP (continuously varying surface pressures) (Langmuir 2007, 23, 4958). The thermodynamic properties of the Langmuir films have been examined with pressure-area isotherms, while the molecular properties of the film constituents in the transferred films in the monolayer and multilayer phases have been examined with IR spectroscopy. Quantitative intensity measurements of the DPPC-d62, DPPG, and SP-C components in each phase reveal that the DPPG and SP-C constituents are relatively enriched in the multilayer compared with the DPPC-d62, although all three species are present in both phases. Some molecular structure information is available from the surface-pressure-induced variation in IR parameters. The DPPC-d62 exhibits slightly increased conformational order in the multilayer phase as detected from decreases in the CD2 stretching frequencies upon compression, while the lipid phosphate residues become dehydrated, as deduced from increases in the 1245 cm-1 symmetric PO2- stretching frequency. A small increase is observed in the protein amide I frequency; possible interpretations of these changes are presented. The current observations are compared with ideas contained in the "squeeze-out hypothesis" (Handbook of Physiology, The Respiratory System; American Physiological Society Press: Bethesda, MD, 1986; Vol. III, p 247) and in the "liquid crystalline collapse" model (Biophys. J. 2003, 84, 3792). Within the limitation of the current procedures, the data contain elements from both these descriptions of the monolayer transformation. Extensions and possible limitations of the COVASP-IR method are discussed.

Three seminal studies published between 1930 and 1955 played a critical role in defining a received version of South African Jewish history. All three helped towards a self—definition of a community looking for a usable and respectable past in an age of anxiety and vulnerability. Shaped in significant measure by these texts, a collective memory emerged which incorporated a questionable understanding of the community's origins, development and character. Critical dimensions of the South African Jewish experience were ignored or distorted in this drive towards acceptance and respectability.

Polyphenolic resveratrol has been identified as a potent antioxidant acting as both a free radical scavenger and an inhibitor of enzyme oxidative activity. However, the reactive propensity of resveratrol also limits its use in topical formulations. A transient derivative of resveratrol, resveratrol triphosphate, has been designed to provide a means for the delayed delivery of the active compound in skin tissue where endogenous enzymes capable of dephosphorylation reside. Confocal Raman microscopy studies of intact pigskin biopsies treated with modified resveratrol provided information about the spatial distribution and time-dependence of permeation and conversion to the native active form. Conversion to the active form was not observed when skin samples were exposed to steam, a procedure that likely inactivates endogenous skin enzymes. In addition, treatment with the triphosphate compared to the parent compound revealed a more homogeneous distribution of resveratrol throughout the stratum corneum and viable epidermis when the former was applied. Thus, the bioavailability of resveratrol in the epidermis appears to be enhanced upon application of the pro-molecule compared to resveratrol.

Autoimmunity is a complex trait disease where the environment influences susceptibility to disease by unclear mechanisms. T cell receptor clustering and signaling at the immune synapse, T cell proliferation, CTLA-4 endocytosis, T(H)1 differentiation, and autoimmunity are negatively regulated by beta1,6GlcNAc-branched N-glycans attached to cell surface glycoproteins. Beta1,6GlcNAc-branched N-glycan expression in T cells is dependent on metabolite supply to UDP-GlcNAc biosynthesis (hexosamine pathway) and in turn to Golgi N-acetylglucosaminyltransferases Mgat1, -2, -4, and -5. In Jurkat T cells, beta1,6GlcNAc-branching in N-glycans is stimulated by metabolites supplying the hexosamine pathway including glucose, GlcNAc, acetoacetate, glutamine, ammonia, or uridine but not by control metabolites mannosamine, galactose, mannose, succinate, or pyruvate. Hexosamine supplementation in vitro and in vivo also increases beta1,6GlcNAc-branched N-glycans in naïve mouse T cells and suppresses T cell receptor signaling, T cell proliferation, CTLA-4 endocytosis, T(H)1 differentiation, experimental autoimmune encephalomyelitis, and autoimmune diabetes in non-obese diabetic mice. Our results indicate that metabolite flux through the hexosamine and N-glycan pathways conditionally regulates autoimmunity by modulating multiple T cell functionalities downstream of beta1,6GlcNAc-branched N-glycans. This suggests metabolic therapy as a potential treatment for autoimmune disease.

X-ray crystallography of collagen model peptides has provided high-resolution structures of the basic triple-helical conformation and its water-mediated hydration network. Vibrational spectroscopy provides a useful bridge for transferring the structural information from X-ray diffraction to collagen in its native environment. The vibrational mode most useful for this purpose is the amide I mode (mostly peptide bond C=O stretch) near 1650 cm-1. The current study refines and extends the range of utility of a novel simulation method that accurately predicts the infrared (IR) amide I spectral contour from the three-dimensional structure of a protein or peptide. The approach is demonstrated through accurate simulation of the experimental amide I contour in solution for both a standard triple helix, (Pro-Pro-Gly)10, and a second peptide with a Gly --> Ala substitution in the middle of the chain that models the effect of a mutation in the native collagen sequence. Monitoring the major amide I peak as a function of temperature gives sharp thermal transitions for both peptides, similar to those obtained by circular dichroism spectroscopy, and the Fourier transform infrared (FTIR) spectra of the unfolded states were compared with polyproline II. The simulation studies were extended to model early stages of thermal denaturation of (Pro-Pro-Gly)10. Dihedral angle changes suggested by molecular dynamics simulations were made in a stepwise fashion to generate peptide unwinding from each end, which emulates the effect of increasing temperature. Simulated bands from these new structures were then compared to the experimental bands obtained as temperature was increased. The similarity between the simulated and experimental IR spectra lends credence to the simulation method and paves the way for a variety of applications.

The outermost region of the epidermis, the stratum corneum (SC), provides an essential barrier to water loss and protects against exogenous substances. The functional integrity of the SC depends on a complex maturation and exfoliation process, which is often perturbed in skin diseases. The maturation of corneocytes isolated from different depths in healthy human SC was investigated using infrared (IR) spectroscopic imaging and Raman microscopy. Both IR and Raman spectral quality of individual corneocytes was high and revealed depth-dependent variations in molecular composition. Spectral changes were identified as arising from alterations in the concentration of the major constituents of natural moisturizing factor (NMF), important in maintaining SC hydration. A significant decrease in the concentration of NMF was observed for corneocytes isolated from superficial compared to deeper SC layers (layer 3 vs. layer 11, respectively). An IR parameter that measures the relative NMF concentration in corneocytes is introduced. The potential role of vibrational imaging to evaluate corneocyte composition and molecular structure in the treatment of NMF-related diseases is discussed.

The major barrier to permeability in skin resides in the outermost layer of the epidermis, the stratum corneum (SC). The major SC lipid components are ceramides, free fatty acids, and cholesterol. Ternary mixtures containing these constituents are widely used for physicochemical characterization of the barrier. Prior X-ray diffraction and IR spectroscopy studies have revealed the existence of ordered lipid chains packed in orthorhombic subcells. To monitor the kinetics of formation of regions rich in fatty acids, the current study utilizes a modification of the method (J. Phys. Chem. 1992, 96, 10008) developed to monitor component demixing in n-alkane mixtures. The approach is based on changes in the scissoring or rocking mode contours in the IR spectra of (orthorhombically packed) ordered chains. In the current study, equimolar mixtures of ceramides (either non-hydroxy fatty acid sphingosine ceramide or alpha-hydroxy fatty acid sphingosine ceramide) with chain perdeuterated fatty acids (either palmitic or stearic acid) and cholesterol reveal a time evolution of the scissoring contour of the deuterated fatty acid chains following quenching from relatively high temperatures where random mixing occurs. Segregation of domains enriched in the fatty acid component is observed. The kinetics of segregation are sensitive to the quenching temperature and to the chemical composition of the mixture. The kinetic regimes are conveniently catalogued with a power law of the form P=Ktalpha where P is a (measured) property related to domain composition. The time scales for demixing in these experiments are similar to times observed in several studies that have tracked the restoration of the in vivo permeability barrier following nonthermal challenges to SC integrity. Further evidence for the physiological importance of the current measurements is the detection of these phases in native SC. The current work constitutes the first direct, structure-based determination of the kinetics of barrier formation in relevant skin lipid barrier models.

Metabolic bone diseases manifesting fragility fractures (such as osteoporosis) are routinely diagnosed based on bone mineral density (BMD) measurements, and the effect of various therapies also evaluated based on the same outcome. Although useful, it is well recognized that this metric does not fully account for either fracture incidence or the effect of various therapies on fracture incidence, thus, the emergence of bone quality as a contributing factor in the determination of bone strength. Infrared and Raman vibrational spectroscopic techniques are particularly well-suited for the determination of bone quality as they provide quantitative and qualitative information of the mineral and organic matrix bone components, simultaneously. Through the use of microspectroscopic techniques, this information is available in a spatially resolved manner, thus, the outcomes may be easily correlated with outcomes from techniques such as histology, histomorphometry, and nanoindentation, linking metabolic status with material properties.