Accurate and rapid assessment of the healing status of a wound in a simple and non-invasive manner would enable clinicians to diagnose wounds in real time and promptly adjust treatments to hasten the resolution of non-healing wounds. Histologic and biochemical characterization of biopsied wound tissue, which is currently the only reliable method for wound assessment, is invasive, complex to interpret and slow. Here we demonstrate the use of Raman microspectroscopy coupled with multivariate spectral analysis as a simple, non-invasive method to biochemically characterize healing wounds in mice and to accurately identify different phases of healing of wounds at different time-points. Raman spectra were collected from ‘splinted’ full thickness dermal wounds in mice at 4 time-points (0, 1, 5 and 7 days) corresponding to different phases of wound healing, as verified by histopathology. Spectra were deconvolved using multivariate factor analysis (MFA) into 3 ‘factor score spectra’(that act as spectral signatures for different stages of healing) that were successfully correlated with spectra of prominent pure wound bed constituents (i.e. collagen, lipids, fibrin, fibronectin, etc.) using non-negative least squares (NNLS) fitting. We show that the factor loadings (weights) of spectra that belonged to wounds at different time-points provide a quantitative measure of wound healing progress in terms of key parameters such as inflammation and granulation. Wounds at similar stages of healing were characterized by clusters of loading values and slowly healing wounds amongst them were successfully identified as ‘outliers’. Overall, our results demonstrate that Raman spectroscopy can be used as a non-invasive technique to provide insight into the status of normally-healing and slow-to-heal wounds, and that it may find use as a complementary tool for real-time, in situ biochemical characterization in wound healing studies and clinical diagnosis.
The development of versatile methods that provide spatial and temporal control over the presentation of physical and biochemical cues on wound beds can lead to new therapeutic approaches that expedite wound healing by favorably influencing cellular behaviors. Towards that goal, we report that native chemical functional groups presented by wound beds can be utilized for direct covalent attachment of polymeric microbeads. Specifically, we demonstrated the covalent attachment of maleimide-functionalized and catechol-functionalized microbeads, made of either polystyrene (non-degradable) or poly(lactic-co-glycolic acid) ((PLGA), degradable), to sulfhydryl and amine groups present on porcine dermis used here as an ex vivo model wound bed. A pronounced increase (10–70 fold) in the density and persistence of the covalently reactive microbeads was observed relative to microbeads that adsorb via non-covalent interactions. Complementary characterization of the surface chemistry of the ex vivo wound beds using Raman microspectroscopy provides support for our conclusion that the increased adherence of the maleimide-functionalized beads results from their covalent bond formation with sulfhydryl groups on the wound bed. The attachment of maleimide-functionalized microbeads to wounds created in live wild-type and diabetic mice led to observations of differential immobilization of microbeads on them and were consistent with anticipated differences in the presentation of sulfhydryl groups on the two different wound types. Finally, the incorporation of maleimide-functionalized microbeads in wounds created in wild-type mice did not impair the rate of wound closure relative to an untreated wound. Overall, the results presented in this paper enable a general and facile approach to the engineering of wound beds in which microbeads are covalently immobilized to wound beds. Such immobilized microbeads could be used in future studies to release bioactive factors (e.g., antimicrobial agents or growth factors) and/or introduce topographical cues that promote cell behaviors underlying healing and wound closure.
Aspartate aminotransferases (EC 2.6.1.1) catalyse the conversion of aspartate and-ketoglutarate to oxaloacetate and glutamate in a reversible manner. Thus, the aspartate aminotransferase of Plasmodium falciparum (PfAspAT) plays a central role in the transamination of amino acids. Recent findings suggest that PfAspAT may also play a pivotal role in energy metabolism and the de novo biosynthesis of pyrimidines. While therapeutics based upon the inhibition of other proteins in these pathways are already used in the treatment of malaria, the advent of multidrug-resistant strains has limited their efficacy. The presence of PfAspAT in these pathways may offer additional opportunities for the development of novel therapeutics. In order to gain a deeper understanding of the function and role of PfAspAT, it has been expressed and purified to homogeneity. The successful crystallization of PfAspAT, the collection of a 2.8 Å diffraction data set and initial attempts to solve the structure using molecular replacement are reported.
Antimicrobial surfaces with covalently attached biocidal functionalities only kill microbes that come into direct contact with the surfaces (contact-killing surfaces). Herein, the activity of contact-killing surfaces is shown to be enhanced by using gradients in the concentration of soluble chemoattractants (CAs) to attract bacteria to the surfaces. Two natural and nonbiocidal CAs (aspartate and glucose) were used to attract bacteria to model surfaces decorated with quaternary ammonium groups (known to kill bacteria that come into contact with them). These results demonstrate the killing of Escherichia coli and Salmonella typhimurium, two common pathogens, at levels 10- to 20-times greater than that of the native surfaces alone. This approach is general and provides new strategies for the design of active or dynamic contact-killing surfaces with enhanced antimicrobial activities.
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