Joshua Glass

The tear film is a multi-layer fluid that covers the corneal and conjunctival epithelia of the eye and provides lubrication, nutrients, and protection from the outside environment. Tear fluid contains a high concentration of proteins and has thus been recognized as a potential source of biomarkers for ocular disorders due to its proximity to disease sites on the ocular surface and the non-invasive nature of its collection. This is particularly true in the case of dry eye disease, which directly impacts the tear film and its components. Proteomic analysis of tear fluid is challenging mainly due to the wide dynamic range of proteins and the small sample volumes. However, recent advancements in mass spectrometry have revolutionized the field of proteomics enabling unprecedented depth, speed, and accuracy, even with small sample volumes. In this study using the Orbitrap Fusion Tribrid mass spectrometer, we compared four different mass spectrometry workflows for the proteomic analysis of tear fluid collected via Schirmer strips. We were able to establish a method of in-strip protein digestion that identified >3000 proteins in human tear samples from 11 healthy subjects. Our method offers a significant improvement in the number of proteins identified compared to previously reported methods without pooling samples.

Long-term hyperglycemia-mediated oxidative stress and inflammation lead to the blood-retinal barrier (BRB) dysfunction and increased vascular permeability associated with diabetic retinopathy (DR). Interleukin-6 (IL-6) is one of the primary mediators of retinal vascular inflammation. IL-6 signaling through its membrane-bound IL-6 receptor is known as classical signaling, and through a soluble IL-6 receptor (sIL-6R) is known as trans-signaling. Increasing evidence suggests that classical signaling is primarily anti-inflammatory, whereas trans-signaling induces the pro-inflammatory effects of IL-6. The purpose of this study was to compare the effects of these two pathways on paracellular permeability and expression of genes involved in inter-endothelial junctions in human retinal endothelial cells (HRECs). IL-6 trans-signaling activation caused significant disruption to paracellular integrity, with increased paracellular permeability, and was associated with significant changes in gene expression related to adherens, tight, and gap junctions. IL-6 classical signaling did not alter paracellular resistance in HRECs and had no distinct effects on gene expression. In conclusion, IL-6 trans-signaling, but not classical signaling, is a major mediator of the increased paracellular permeability characteristic of inner BRB breakdown in diabetic retinopathy. This study also identified potential inter-endothelial junction genes involved in the IL-6 trans-signaling mediated regulation of paracellular permeability in HRECs.

The precise molecular mechanisms of diabetic retinopathy (DR) pathogenesis are unclear, and treatment options are limited. There is an urgent need to discover and develop novel therapeutic targets for the treatment of this disease. Glycosylation is a post-translational modification that plays a critical role in determining protein structure, function, and stability. Recent studies have found that serum glycoproteomic changes are associated with the presence or progression of several inflammatory diseases. However, very little is known about the glycoproteomic changes associated with DR. In this study, glycoproteomic profiling of the serum of diabetic patients with and without DR was performed. A total of 15 glycopeptides from 11 glycoproteins were found to be significantly altered (5 upregulated and 10 downregulated) within the serum glycoproteome of DR patients. These glycoproteins are known to be involved in the maintenance of the extracellular matrix and complement system through peptidolytic activity or regulation.