Customized corneal cross-linking

3nononePersonalized accelerated crosslinking nomograms for the management of corneal ectasia were conceived after comparative analysis of demarcation lines and cell viability observed after customized accelerated epithelium-off crosslinking CXL treatments by spectral domain corneal OCT and scanning laser in vivo confocal microscopy matching all the clinical and instrumental data with mathematical models. Accelerated high-fluence Topography-guided CXL at 30 mW/cm2 UV-Power and Accelerated epithelium-off CXL with 9 and 15 mW/cm2 UV-A power with standardized Fluence of 5.4 J/cm2 were safe and effective demostrating a keratocytes apoptosis and demarcation line depth between 280 and 340 μm. The 30 mW ACXL showed a penetration with continuous and pulsed light between 150 and 200 μm. No endothelial damage was reported in any case. In vivo morphological studies demonstrated that Accelerated CXL allow a pachymetry-guided cutomization of CXL maintainig the standard Fluence of 5.4 J/cm2 and a total treatment time under 20 min. Moreover a pachymetry-guided ACXL nomogram (M nomogram) developed by Mazzotta C and Friedman M matching the physical and mathematical calculations with the miscostructural IVCM and OCT observations of demarcation lines depths allow an efficacous CXL management of primary and iatrogenic ectatic corneas also allowing a safe management of thin ectatic corneas.noneMazzotta C.; Rechichi M.; Ferrise M.Mazzotta, C.; Rechichi, M.; Ferrise, M

Serial deletion reveals structural basis and stability for the core enzyme activity of human glutaminase 1 isoforms: relevance to excitotoxic neurodegeneration

Abstract Background Glutaminase 1 is a phosphate-activated metabolic enzyme that catalyzes the first step of glutaminolysis, which converts glutamine into glutamate. Glutamate is the major neurotransmitter of excitatory synapses, executing important physiological functions in the central nervous system. There are two isoforms of glutaminase 1, KGA and GAC, both of which are generated through alternative splicing from the same gene. KGA and GAC both transcribe 1–14 exons in the N-terminal, but each has its unique C-terminal in the coding sequence. We have previously identified that KGA and GAC are differentially regulated during inflammatory stimulation and HIV infection. Furthermore, glutaminase 1 has been linked to brain diseases such as amyotrophic lateral sclerosis, Alzheimer’s disease, and hepatic encephalopathy. Core enzyme structure of KGA and GAC has been published recently. However, how other coding sequences affect their functional enzyme activity remains unclear. Methods We cloned and performed serial deletions of human full-length KGA and GAC from the N-terminal and the C-terminal at an interval of approximately 100 amino acids (AAs). Prokaryotic expressions of the mutant glutaminase 1 protein and a glutaminase enzyme activity assay were used to determine if KGA and GAC have similar efficiency and efficacy to convert glutamine into glutamate. Results When 110 AAs or 218 AAs were deleted from the N-terminal or when the unique portions of KGA and GAC that are beyond the 550 AA were deleted from the C-terminal, KGA and GAC retained enzyme activity comparable to the full length proteins. In contrast, deletion of 310 AAs or more from N-terminal or deletion of 450 AAs or more from C-terminal resulted in complete loss of enzyme activity for KGA/GAC. Consistently, when both N- and C-terminal of the KGA and GAC were removed, creating a truncated protein that expressed the central 219 AA - 550 AA, the protein retained enzyme activity. Furthermore, expression of the core 219 AA - 550 AA coding sequence in cells increased extracellular glutamate concentrations to levels comparable to those of full-length KGA and GAC expressions, suggesting that the core enzyme activity of the protein lies within the central 219 AA - 550 AA. Full-length KGA and GAC retained enzyme activities when kept at 4 °C. In contrast, 219 AA - 550 AA truncated protein lost glutaminase activities more readily compared with full-length KGA and GAC, suggesting that the N-terminal and C-terminal coding regions are required for the stability KGA and GAC. Conclusions Glutaminase isoforms KGA and GAC have similar efficacy to catalyze the conversion of glutamine to glutamate. The core enzyme activity of glutaminase 1 protein is within the central 219 AA - 550 AA. The N-terminal and C-terminal coding regions of KGA and GAC help maintain the long-term activities of the enzymes