MiR-18a-5p Targets Connective Tissue Growth Factor Expression and Inhibits Transforming Growth Factor β2-Induced Trabecular Meshwork Cell Contractility

Increased trabecular meshwork (TM) cell and tissue contractility is a driver of the reduced outflow facility and elevation of intraocular pressure (IOP) associated with primary open-angle glaucoma (POAG). Connective tissue growth factor (CTGF) is an established mediator of TM cell contractility, and its expression is increased in POAG due to transforming growth factor β 2 (TGFβ2) signalling. Inhibiting CTGF upregulation using microRNA (miRNA) mimetics could represent a new treatment option for POAG. A combination of in silico predictive tools and a literature review identified a panel of putative CTGF-targeting miRNAs. Treatment of primary human TM cells with 5 ng/mL TGFβ2 for 24 h identified miR-18a-5p as a consistent responder, being upregulated in cells from five different human donors. Transfection of primary donor TM cells with 20 nM synthetic miR-18a-5p mimic reduced TGFβ2-induced CTGF protein expression, and stable lentiviral-mediated overexpression of this miRNA reduced TGFβ2-induced contraction of collagen gels. Together, these findings identify miR-18a-5p as a mediator of the TGFβ2 response and a candidate therapeutic agent for glaucoma via its ability to inhibit CTGF-associated increased TM contractility

Laminin N-terminus α31 is upregulated in invasive ductal breast cancer and changes the mode of tumour invasion

AbstractLaminin N-terminus α31 (LaNt α31) is an alternative splice isoform derived from the laminin α3 gene. The LaNt α31 protein is enriched around the terminal duct lobular units in normal breast tissue. In the skin and cornea the protein influences epithelial cell migration and tissue remodelling. However, LaNt α31 has never been investigated in a tumour environment. Here we analysed LaNt α31 in invasive ductal carcinoma and determined its contribution to breast carcinoma invasion. LaNt α31 expression and distribution were analysed by immunohistochemistry in human breast tissue biopsy sections and tissue microarrays covering 232 breast cancer samples. This analysis revealed LaNt α31 to be upregulated in 56 % of invasive ductal carcinoma specimens compared with matched normal tissue, and further increased in nodal metastasis compared with the tumour mass in 45 % of samples. 65.8 % of triple negative cases displayed medium to high LaNt α31 expression. To study LaNt α31 function, an adenoviral system was used to induce expression in MCF-7 and MDA-MB-231 cells. Metabolic activity, 2D cell migration, and invasion into collagen hydrogels were not significantly different between LaNt α31 overexpressing cells and control treated cells. However, LaNt α31 overexpressing MDA-MB-231 cells displayed a striking change in their mode of invasion into laminin-containing Matrigel; changing from multicellular streaming to individual cellular-invasion. In agreement with these results, 66.7% of the tumours with the highest LaNt α31 expression were non-cohesive. Together these findings indicate that breast cancer-associated changes in LaNt α31 expression could directly contribute to tumour invasiveness, and that this little-studied protein may become a therapeutic target.</jats:p

Laminin N-terminus α31 is upregulated in invasive ductal breast cancer and changes the mode of tumour invasion

Laminin N-terminus α31 (LaNt α31) is an alternative splice isoform derived from the laminin α3 gene. The LaNt α31 protein is enriched around the terminal duct lobular units in normal breast tissue. In the skin and cornea the protein influences epithelial cell migration and tissue remodelling. However, LaNt α31 has never been investigated in a tumour environment. Here we analysed LaNt α31 in invasive ductal carcinoma and determined its contribution to breast carcinoma invasion. LaNt α31 expression and distribution were analysed by immunohistochemistry in human breast tissue biopsy sections and tissue microarrays covering 232 breast cancer samples. This analysis revealed LaNt α31 to be upregulated in 56% of invasive ductal carcinoma specimens compared with matched normal tissue, and further increased in nodal metastasis compared with the tumour mass in 45% of samples. 65.8% of triple negative cases displayed medium to high LaNt α31 expression. To study LaNt α31 function, an adenoviral system was used to induce expression in MCF-7 and MDA-MB-231 cells. 2D cell migration and invasion into collagen hydrogels were not significantly different between LaNt α31 overexpressing cells and control treated cells. However, LaNt α31 overexpression reduced the proliferation rate of MCF-7 and MDA-MB-231 cells. Moreover, LaNt α31 overexpressing MDA-MB-231 cells displayed a striking change in their mode of invasion into laminin-containing Matrigel; changing from multicellular streaming to individual cellular-invasion. In agreement with these results, 66.7% of the tumours with the highest LaNt α31 expression were non-cohesive. Together these findings indicate that breast cancer-associated changes in LaNt α31 expression could contribute to the processes involved in tumour invasion and may represent a new therapeutic target.</jats:p

Update on Suture Techniques in Corneal Transplantation: A Systematic Review

Effective suturing remains key to achieving successful outcomes in corneal surgery, especially anterior lamellar keratoplasty and full thickness transplantation. Limitations in the technique may result in complications such as wound leak, infection, or high astigmatism post corneal graft. By using a systematic approach, this study reviews articles and conducts content analysis based on update 2020 PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses criteria). The aim of this paper is to summarize the state of the art of corneal suturing techniques for every type of corneal transplant and patient age and also their outcomes regarding astigmatism and complications. Future developments for corneal transplantation will be also discussed. This is important because especially the young surgeon must have knowledge of the implications of every suture performed in order to achieve consistent and predictable post-operative outcomes and also be aware of all the possible complications

Dynamic clamp with StdpC software

Dynamic clamp is a powerful method that allows the introduction of artificial electrical components into target cells to simulate ionic conductances and synaptic inputs. This method is based on a fast cycle of measuring the membrane potential of a cell, calculating the current of a desired simulated component using an appropriate model and injecting this current into the cell. Here we present a dynamic clamp protocol using free, fully integrated, open-source software (StdpC, for spike timing-dependent plasticity clamp). Use of this protocol does not require specialist hardware, costly commercial software, experience in real-time operating systems or a strong programming background. The software enables the configuration and operation of a wide range of complex and fully automated dynamic clamp experiments through an intuitive and powerful interface with a minimal initial lead time of a few hours. After initial configuration, experimental results can be generated within minutes of establishing cell recording

The Relationship Between Mechanical Properties, Ultrastructural Changes, and Intrafibrillar Bond Formation in Corneal UVA/Riboflavin Cross-linking Treatment for Keratoconus

PURPOSE: To determine the relationship between mechanical behavior in cross-linked corneas and changes in the corneal ultrastructure after corneal cross-linking (CXL). METHODS: Porcine corneas were treated following the “Dresden” protocol, the current gold standard for clinical treatment, consisting of dropwise application of 0.1% riboflavin in 20% dextran followed by 30 minutes of ultraviolet-A (UVA) irradiation. The effect of CXL was assessed using uniaxial tensile testing, transmission electron microscopy, and Fourier transform infrared spectroscopy, with results compared against corneas treated with each of the treatment solution components individually. RESULTS: UVA/riboflavin cross-linked corneas displayed 28% ± 17% increase in the material tangent modulus compared with dextran treatment alone, and altered collagen architecture within the first 300 µm of stromal depth consisting of 5% increase in the thickness of collagen fibrils, no significant changes to interfibrillar spacing, and an 8% to 12% decrease in number of fibrils per unit area. Fourier transform infrared spectroscopy confirmed formation of interfibrillar bonds (P = .012) induced by UVA-mediated CXL. CONCLUSIONS: The data support a model wherein collagen fibril diameter and structural density are fundamental parameters in defining tissue stiffening following UVA/riboflavin CXL and provide benchmarks against which modifications to the Dresden CXL protocol can be evaluated