Laminins are complex extracellular macromolecules that are major players in the control of a variety of core cell processes, including regulating rates of cell proliferation, differentiation, adhesion, and migration. Laminins, and related extracellular matrix components, have essential roles in tissue homeostasis; however, during wound healing, the same proteins are critical players in re-epithelialization and angiogenesis. Understanding how these proteins influence cell behavior in these different conditions holds great potential in identifying new strategies to enhance normal wound closure or to treat chronic/nonhealing wounds. Laminin-derived bioactive peptides and, more recently, laminin-peptide conjugated scaffolds, have been designed to improve tissue regeneration after injuries. These peptides have been shown to be effective in decreasing inflammation and granulation tissue, and in promoting re-epithelialization, angiogenesis, and cell migration. Although there is now a wealth of knowledge concerning laminin form and function, there are still areas of some controversy. These include the relative contribution of two laminin-based adhesive devices (focal contacts and hemidesmosomes) to the re-epithelialization process, the impact and implications of laminin proteolytic processing, and the importance of laminin polymer formation on cell behavior. In addition, the roles in wound healing of the laminin-related proteins, netrins, and LaNts are still to be fully defined. The future of laminin-based therapeutics potentially lies in the bioengineering of specific substrates to support laminin deposition for expansion of autologous cells for graft formation and transplantation. Significant recent advances suggest that this goal is within sight.

PURPOSE. Laminin N-terminus (LaNt) a31 is a relatively unstudied protein derived from the laminin a3 gene but structurally similar to netrins. LaNt a31 has, to date, been investigated only in two-dimensional (2D) keratinocyte culture where it influences cell migration and adhesion, processes integral to wound repair. Here we investigated LaNt a31 distribution in ocular surface epithelium, during limbal stem cell activation, and corneal wound healing.METHODS. Human, mouse, and pig eyes, ex vivo limbal explant cultures, and alkali burn wounds were processed for immunohistochemistry with antibodies against LaNt a31 along with progenitor cell-associated proteins. LaNt a31 expression was induced via adenoviral transduction into primary epithelial cells isolated from limbal explants, and cell spreading and migration were analyzed using live imaging.RESULTS. LaNt a31 localized to the basal layer of the conjunctival, limbal, and corneal epithelial cells. However, staining was nonuniform with apparent subpopulation enrichment, and some suprabasal reactivity was also noted. This LaNt a31 distribution largely matched that of keratin 15, epidermal growth factor receptor, and transformation-related protein 63a (p63a), and displayed similar increases in expression in activated limbal explants. During active alkali burn wound repair, LaNt a31 displayed increased expression in limbal regions and loss of basal restriction within the cornea. Distribution returned to predominately basal cell restricted once the wounded epithelium matured. Cultured corneal epithelial cells expressing LaNt a31 displayed increased 2D area and reduced migration, suggesting a functional link between this protein and key wound repair activities.CONCLUSIONS. These data place LaNt a31 in position to influence laminin-dependent processes including wound repair and stem cell activation.

Measuring the organization of the cellular cytoskeleton and the surrounding extracellular matrix (ECM) is currently of wide interest as changes in both local and global alignment can highlight alterations in cellular functions and material properties of the extracellular environment. Different approaches have been developed to quantify these structures, typically based on fiber segmentation or on matrix representation and transformation of the image, each with its own advantages and disadvantages. Here we present AFT − Alignment by Fourier Transform, a workflow to quantify the alignment of fibrillar features in microscopy images exploiting 2D Fast Fourier Transforms (FFT). Using pre-existing datasets of cell and ECM images, we demonstrate our approach and compare and contrast this workflow with two other well-known ImageJ algorithms to quantify image feature alignment. These comparisons reveal that AFT has a number of advantages due to its grid-based FFT approach. 1) Flexibility in defining the window and neighborhood sizes allows for performing a parameter search to determine an optimal length scale to carry out alignment metrics. This approach can thus easily accommodate different image resolutions and biological systems. 2) The length scale of decay in alignment can be extracted by comparing neighborhood sizes, revealing the overall distance that features remain anisotropic. 3) The approach is ambivalent to the signal source, thus making it applicable for a wide range of imaging modalities and is dependent on fewer input parameters than segmentation methods. 4) Finally, compared to segmentation methods, this algorithm is computationally inexpensive, as high-resolution images can be evaluated in less than a second on a standard desktop computer. This makes it feasible to screen numerous experimental perturbations or examine large images over long length scales. Implementation is made available in both MATLAB and Python for wider accessibility, with example datasets for single images and batch processing. Additionally, we include an approach to automatically search parameters for optimum window and neighborhood sizes, as well as to measure the decay in alignment over progressively increasing length scales.

Application of sunscreen is a widely used mechanism for protecting skin from the harmful effects of UV light. However, protection can only be achieved through effective application, and areas that are routinely missed are likely at increased risk of UV damage. Here we sought to determine if specific areas of the face are missed during routine sunscreen application, and whether provision of public health information is sufficient to improve coverage. To investigate this, 57 participants were imaged with a UV sensitive camera before and after sunscreen application: first visit; minimal pre-instruction, second visit; provided with a public health information statement. Images were scored using a custom automated image analysis process designed to identify areas of high UV reflectance, i.e. missed during sunscreen application, and analysed for 5% significance. Analyses revealed eyelid and periorbital regions to be disproportionately missed during routine sunscreen application (median 14% missed in eyelid region vs 7% in rest of face, p<0.01). Provision of health information caused a significant improvement in coverage to eyelid areas in general however, the medial canthal area was still frequently missed. These data reveal that a public health announcement-type intervention could be effective at improving coverage of high risk areas of the face, however high risk areas are likely to remain unprotected therefore other mechanisms of sun protection should be widely promoted such as UV blocking sunglasses.

16Laminin N-terminus 31 (LaNt 31) is an alternative splice isoform derived from the laminin 17 3 gene. The LaNt 31 protein is enriched around the terminal duct lobular units in normal 18 129 HBreD145Su02; US Biomax, Rockville, Maryland, USA). Sections were dewaxed and 130 processed using a Leica Bond autostainer with Bond™ Polymer Refine Detection system 131 (Leica Biosystems, Wetzlar, Germany). Briefly, following dewaxing, antigen retrieval was 132 performed by incubating with a Tris/EDTA (pH 9 solution) solution for 20 mins at 60 °C, then 133 endogenous peroxidases were blocked for 5 mins at room temperature with Bond hydrogen 134 peroxide solution. Sections were incubated with primary or isotype-matched control 135 antibodies at room temperature for 30 mins in Bond primary Ab solution (Tris-buffered 136 saline, TBS, containing surfactant and protein stabilizer), then secondary anti-mouse IgG 137 antibodies (<10 μg mL -1 ) with 10 % v/v animal serum in TBS were added for 15 mins at room 138 temperature. DAB (66 mM) chromogen substrate was added for 20 mins at room