The timely resolution of wound healing is critical for restoring the skin as a protective barrier. The switch from a proinflammatory to a reparative microenvironment must be tightly regulated. ...Interleukin (IL)-6 is a key modulator of the inflammatory and reparative process: it is involved in the differentiation, activation, and proliferation of leukocytes, endothelial cells, keratinocytes, and fibroblasts. This review examines the role of IL-6 in the healing of cutaneous wounds, and how dysregulation of IL-6 signaling can lead to either fibrosis or a failure to heal. The role of an IL-6/TGF-β feedback loop is discussed in the context of fibrogenesis, while IL-6 expression and responses in advanced age, diabetes, and obesity is outlined regarding the development of chronic wounds. Current research on therapies that modulate IL-6 is explored. Here, we consider IL-6's diverse impact on cutaneous wound healing.
X-ray phase-contrast imaging and tomography make use of the refraction of X-rays by the sample in image formation. This provides considerable additional information in the image compared to ...conventional X-ray imaging methods, which rely solely on X-ray absorption by the sample. Phase-contrast imaging highlights edges and internal boundaries of a sample and is thus complementary to absorption contrast, which is more sensitive to the bulk of the sample. Phase-contrast can also be used to image low-density materials, which do not absorb X-rays sufficiently to form a conventional X-ray image. In the context of materials science, X-ray phase-contrast imaging and tomography have particular value in the 2D and 3D characterization of low-density materials, the detection of cracks and voids and the analysis of composites and multiphase materials where the different components have similar X-ray attenuation coefficients. Here we review the use of phase-contrast imaging and tomography for a wide variety of materials science characterization problems using both synchrotron and laboratory sources and further demonstrate the particular benefits of phase contrast in the laboratory setting with a series of case studies.
Scarring is a lifelong consequence of skin injury, with scar stiffness and poor appearance presenting physical and psychological barriers to a return to normal life. Lysyl oxidases are a family of ...enzymes that play a critical role in scar formation and maintenance. Lysyl oxidases stabilize the main component of scar tissue, collagen, and drive scar stiffness and appearance. Here we describe the development and characterisation of an irreversible lysyl oxidase inhibitor, PXS-6302. PXS-6302 is ideally suited for skin treatment, readily penetrating the skin when applied as a cream and abolishing lysyl oxidase activity. In murine models of injury and fibrosis, topical application reduces collagen deposition and cross-linking. Topical application of PXS-6302 after injury also significantly improves scar appearance without reducing tissue strength in porcine injury models. PXS-6302 therefore represents a promising therapeutic to ameliorate scar formation, with potentially broader applications in other fibrotic diseases.
RirA is a global iron regulator in diverse
Alphaproteobacteria
that belongs to the Rrf2 superfamily of transcriptional regulators, which can contain an iron-sulfur (Fe-S) cluster. Under iron-replete ...conditions, RirA contains a 4Fe-4S cluster, enabling high-affinity binding to RirA-regulated operator sequences, thereby causing the repression of cellular iron uptake. Under iron deficiency, one of the cluster irons dissociates, generating an unstable 3Fe-4S form that subsequently degrades to a 2Fe-2S form and then to apo RirA, resulting in loss of high-affinity DNA-binding. The cluster is coordinated by three conserved cysteine residues and an unknown fourth ligand. Considering the lability of one of the irons and the resulting cluster fragility, we hypothesized that the fourth ligand may not be an amino acid residue. To investigate this, we considered that the introduction of an amino acid residue that could coordinate the cluster might stabilize it. A structural model of RirA, based on the Rrf2 family nitrosative stress response regulator NsrR, highlighted residue 8, an Asn in the RirA sequence, as being appropriately positioned to coordinate the cluster. Substitution of Asn8 with Asp, the equivalent, cluster-coordinating residue of NsrR, or with Cys, resulted in proteins that contained a 4Fe-4S cluster, with N8D RirA exhibiting spectroscopic properties very similar to NsrR. The variant proteins retained the ability to bind RirA-regulated DNA, and could still act as repressors of RirA-regulated genes
in vivo
. However, they were significantly more stable than wild-type RirA when exposed to O
2
and/or low iron. Importantly, they exhibited reduced capacity to respond to cellular iron levels, even abolished in the case of the N8D version, and thus were no longer iron sensing. This work demonstrates the importance of cluster fragility for the iron-sensing function of RirA, and more broadly, how a single residue substitution can alter cluster coordination and functional properties in the Rrf2 superfamily of regulators.
The regulator RirA senses Fe through its 4Fe-4S cluster, which may have only three Cys ligands from the protein. Addition of a likely fourth Asp ligand stabilised the cluster, rendering it unable to sense Fe and locked into its DNA-binding state.
A critical early phase for any synchrotron beamline involves detailed testing, characterization and commissioning; this is especially true of a beamline as ambitious and complex as the Imaging & ...Medical Beamline (IMBL) at the Australian Synchrotron. IMBL staff and expert users have been performing precise experiments aimed at quantitative characterization of the primary polychromatic and monochromatic X‐ray beams, with particular emphasis placed on the wiggler insertion devices (IDs), the primary‐slit system and any in vacuo and ex vacuo filters. The findings from these studies will be described herein. These results will benefit IMBL and other users in the future, especially those for whom detailed knowledge of the X‐ray beam spectrum (or `quality') and flux density is important. This information is critical for radiotherapy and radiobiology users, who ultimately need to know (to better than 5%) what X‐ray dose or dose rate is being delivered to their samples. Various correction factors associated with ionization‐chamber (IC) dosimetry have been accounted for, e.g. ion recombination, electron‐loss effects. A new and innovative approach has been developed in this regard, which can provide confirmation of key parameter values such as the magnetic field in the wiggler and the effective thickness of key filters. IMBL commenced operation in December 2008 with an Advanced Photon Source (APS) wiggler as the (interim) ID. A superconducting multi‐pole wiggler was installed and operational in January 2013. Results are obtained for both of these IDs and useful comparisons are made. A comprehensive model of the IMBL has been developed, embodied in a new computer program named spec.exe, which has been validated against a variety of experimental measurements. Having demonstrated the reliability and robustness of the model, it is then possible to use it in a practical and predictive manner. It is hoped that spec.exe will prove to be a useful resource for synchrotron science in general, and for hard X‐ray beamlines, whether they are based on bending magnets or insertion devices, in particular. In due course, it is planned to make spec.exe freely available to other synchrotron scientists.
A comprehensive model of the Imaging and Medical Beamline at the Australian Synchrotron has been developed, validated against a variety of experimental measurements, and its versatility, reliability and robustness demonstrated. The results presented are of particular relevance in X‐ray imaging, tomography, radiotherapy and dosimetry.
•Keloid fibroblasts are stiffer than normal fibroblasts when cultured on hydrogels that mimic tissue stiffness.•YAP signaling in normal fibroblasts but not keloid fibroblasts is sensitive to both ...biomechanical and biochemical stimuli in vitro.•Constitutive elevated YAP signalling in keloid fibroblasts alters their phenotype to increase α-SMA expression.
Fibroblasts found in keloid tissues are known to present an altered sensitivity to microenvironmental stimuli. However, the impact of changes in extracellular matrix stiffness on phenotypes of normal fibroblasts (NFs) and keloid fibroblasts (KFs) is poorly understood.
Investigation the impact of matrix stiffness on NFs and KFs mainly via detecting yes-associated protein (YAP) expression.
We used fibronectin-coated polyacrylamide hydrogel substrates with a range from physiological to pathological stiffness values with or without TGF-β (fibrogenic inducer). Atomic force microscopy was used to measure the stiffness of fibroblasts. Cellular mechanoresponses were screened by immunocytochemistry, Western blot and Luminex assay.
KFs are stiffer than NFs with greater expression of α-SMA. In NFs, YAP nuclear translocation was induced by increasing matrix stiffness as well as by stimulation with TGF-β. In contrast, KFs showed higher baseline levels of nuclear YAP that was not responsive to matrix stiffness or TGF-β. TGF-β1 induced p-SMAD3 in both KFs and NFs, demonstrating the pathway was functional and not hyperactivated in KFs. Moreover, blebbistatin suppressed α-SMA expression and cellular stiffness in KFs, linking the elevated YAP signaling to keloid phenotype.
These data suggest that whilst normal skin fibroblasts respond to matrix stiffness in vitro, keloid fibroblasts have elevated activation of mechanotransduction signaling insensitive to the microenvironment. This elevated signaling appears linked to the expression of α-SMA, suggesting a direct link to disease pathogenesis. These findings suggest changes to keloid fibroblast phenotype related to mechanotransduction contribute to disease and may be a useful therapeutic target.
The molecular chaperone Hsp90 protects deregulated signaling proteins that are vital for tumor growth and survival. Tumors generally display sensitivity and selectivity toward Hsp90 inhibitors; ...however, the molecular mechanism underlying this phenotype remains undefined. We report that the mitotic checkpoint kinase Mps1 phosphorylates a conserved threonine residue in the amino-domain of Hsp90. This, in turn, regulates chaperone function by reducing Hsp90 ATPase activity while fostering Hsp90 association with kinase clients, including Mps1. Phosphorylation of Hsp90 is also essential for the mitotic checkpoint because it confers Mps1 stability and activity. We identified Cdc14 as the phosphatase that dephosphorylates Hsp90 and disrupts its interaction with Mps1. This causes Mps1 degradation, thus providing a mechanism for its inactivation. Finally, Hsp90 phosphorylation sensitizes cells to its inhibitors, and elevated Mps1 levels confer renal cell carcinoma selectivity to Hsp90 drugs. Mps1 expression level can potentially serve as a predictive indicator of tumor response to Hsp90 inhibitors.
Display omitted
•Mps1 phosphorylates a conserved threonine in Hsp90 and regulates its function•Hsp90 phosphorylation by Mps1 is essential for the mitotic checkpoint•Cdc14 phosphatase dephosphorylates Hsp90 and disrupts the Mps1-Hsp90 complex•Mps1 phosphorylation of Hsp90 confers tumor selectivity to its inhibitors
The mitotic checkpoint kinase Mps1 is required for accurate chromosome segregation. Woodford et al. show that Mps1 is a client of the molecular chaperone Hsp90 and directly phosphorylates a conserved threonine residue in the N-domain of the chaperone. This phosphorylation regulates Hsp90 function and is essential for the mitotic checkpoint. Additionally, Mps1 phosphorylation of Hsp90 sensitizes cells to Hsp90 inhibitors, and elevated Mps1 levels confer tumor selectivity on Hsp90 drugs. The phosphatase Cdc14 dephosphorylates Hsp90 and disrupts its interaction with Mps1.
The dynamic network of chaperone interactions known as the chaperome contributes significantly to the proteotoxic cell response and the malignant phenotype. To bypass the inherent redundancy in the ...network, we have used a microRNA (mir) approach to target multiple members of the chaperome simultaneously. We identified a potent microRNA, miR-570 that could bind the 3'untranslated regions of multiple HSP mRNAs and inhibit HSP synthesis. Transfection of cells with this miR species reduced expression of multiple HSPs, inhibited the heat shock response and reduced tumor cell growth while acted additively in combination with cytotoxic drugs. As overexpression of miR-570 elicited tumor suppressive effects, we inferred that this miR could play a potential role in inhibiting tumorigenesis and cancer cell growth. In accordance with this hypothesis, we determined a significant role for miR-570 in regulating markers of mammary tumor progression, including cell motility and invasion. Our data provide a proof of the principle that the tumor chaperome can be targeted by microRNAs suggesting a potential therapeutic avenue towards cancer therapy.
PDF
