The type I cGMP-dependent protein kinases serve essential physiological functions, including smooth muscle relaxation, cardiac remodeling, and platelet aggregation. These enzymes form homodimers through their N-terminal dimerization domains, a feature implicated in regulating their cooperative activation. Previous investigations into the activation mechanisms of PKG I isoforms have been largely influenced by structures of the cAMPdependent protein kinase (PKA). Here, we examined PKG Ia activation by cGMP and cAMP by engineering a monomeric form that lacks N-terminal residues 1-53 ( 53). We found that the construct exists as a monomer as assessed by whole-protein mass spectrometry, size-exclusion chromatography, and small-angle X-ray scattering (SAXS). Reconstruction of the SAXS 3D envelope indicates that 53 has a similar shape to the heterodimeric RIa:C complex of PKA. Moreover, we found that the 53 construct is autoinhibited in its cGMP-free state and can bind to and be activated by cGMP in a manner similar to full-length as assessed by surface plasmon resonance spectroscopy (SPR). However we found that the 53 variant does not exhibit cooperative activation, and its cyclic nucleotide selectivity is diminished. These findings support a model in which, despite structural similarities, PKG Ia activation is distinct from PKA, and its cooperativity is driven by in trans interactions between protomers.

Ocular surface diseases are becoming more prevalent worldwide. Reasons for this include the ongoing population ageing and increasing use of digital displays, although ophthalmologists have a wide selection of tools, which can be implemented in the evaluation of the ocular surface health, methods, which enable the in-depth study of biological functions are gaining more interest. These new approaches are needed, since the individual responses to ocular surface diseases and treatments can vary from person to person, and the correlations between clinical signs and symptoms are often low. Modern mass spectrometry (MS) methods can produce information on hundreds of tear proteins, which in turn can provide valuable information on the biological effects occurring on the ocular surface. In this review article, we will provide an overview of the different aspects, which are part of a successful tear proteomics study design and equip readers with a better understanding of the methods most suited for their MSbased tear proteomics study in the field of ophthalmology and ocular surface.

Tear fluid forms the outermost layer of the ocular surface and its characteristics and composition have been connected to various ocular surface diseases. As tear proteomics enables the non-invasive investigation of protein levels in the tear fluid, it has become an increasingly popular approach in ocular surface and systemic disease studies. Glaucoma, which is a set of multifactorial diseases affecting mainly the optic nerve and retinal ganglion cells, has also been studied using tear proteomics. In this condition, the complete set of pathophysiological changes occurring in the eye is not yet fully understood, and biomarkers for early diagnosis and accurate treatment selection are needed. More in-depth analyses of glaucoma tear proteomics have started to emerge only more recently with the implementation of LC-MS/MS and other modern technologies. The aim of this review was to examine the published data of the tear protein changes occurring during glaucoma, its topical treatment, and surgical interventions.

Purpose To report the unforeseen complication of total obstruction of a glaucoma drainage implant (GDI) tube lumen by white deposit material and to present a preliminary report identifying the composition of this material. Methods Two subjects with a high IOP due to total obstruction of a GDI tube were reviewed. Both patients had a long history with brinzolamide and timolol maleate eye drops. The GDI tube was swept with a 5-0 polypropylene suture stent in order to open the tube. The intraluminal solid sample was successfully collected from the implant tube in one patient. High-performance liquid chromatography-mass spectrometry (HPLC-MS) was used to determine the origin of the intraluminal sample. Results Intraluminal deposits containing components of antiglaucoma drugs e.g., timolol and brinzolamide are a rare cause of total obstruction of GDI tubes. Conclusions Our study describes a new cause of total obstruction GDI tubes. The long-term use of timolol maleate and brinzolamide and their presence in the intraluminal solid sample collected from the blocked GDI tube suggest that the glaucoma medication may have a role in the pathogenesis. However, the exact mechanism is unknown and requires further studies.

Purpose: To establish what preoperative clinical factors and tear protein levels influence the trabeculectomy outcome. Methods: In this prospective study, we followed 80 glaucomatous eyes for 5 years after trabeculectomy. Preoperatively, tear fluid samples and clinical data were collected from all patients. Postoperative statuses, i.e., qualified success (QS) in case of 5‐fluorouracil (5FU), needling or restart of glaucoma medication and failure (F) in case of reoperation, were recorded. The patients' postoperative statuses were then compared against the preoperative tear proteomics and clinical variables with Wilcoxon rank sum test. Proteomic pathway analyses were performed using Ingenuity Pathway Analysis (IPA) software. Results: Within 1 year of the surgery, 15 (19%) QS + F patients were detected. Patients with early failure were younger than those with a successful early outcome (64.7 ± 9.5 vs 70.1 ± 7.6 years, respectively, p = 0.04) and according to pathway analysis results, had increased activation of immune responses prior to surgery (all p‐values <0.05). Overall, 24 (31%) QS + F cases occurred during the 5‐year follow‐up. Pathway analysis revealed that these patients had increased levels of apoptosis prior to surgery (activation z‐score = 2.2, p‐value = 0.02). Conclusions: These results further emphasize the importance of observing the preoperative state of the ocular surface. The tear proteomic profiles reveal biological functions that are associated with the trabeculectomy outcome.