No abstract

Peptides have a role in the inflammatory response, tumor biology, and endocrine processes, presenting them as appealing biomarker candidates. However, peptide extraction efficacy for clinical profiling remains a pivotal technological challenge, as maximum coverage of the plasma peptidome is limited by a range of factors including the inherent complexity of human plasma and the lower concentration of peptides compared to abundant proteins. The aim of this study was to evaluate commonly employed peptide extraction methodologies in terms of total number of peptides detected and the mass range of peptides observed by MALDI. Despite showing coelution of proteins, solid-phase extraction (SPE) methods exhibited superior plasma peptide recovery than ultrafiltration, acetonitrile (ACN) precipitation, or size-exclusion chromatography methods under conditions employed in the study. Not surprisingly, in line with studies challenging the veracity of many peptide biomarker studies, the majority of identified peptides eluted from SPE methods corresponded to proteolytic truncations of the most abundant plasma proteins. The prefractionation of plasma with acetonitrile precipitation prior to SPE provided distinct ion signal profiles and is worthy of further study. In conclusion, this study favors the use of SPE in peptide extraction protocols for increased biomarker coverage and diversity from the plasma peptidome.

Pseudomonas aeruginosa has emerged as an important causative agent of bacterial keratitis, a rapidly progressive ocular condition that may result in blindness. Secretory mucin forms the main constituent of the precorneal tear film, a three-layer film on the ocular surface protecting the underlying corneal epithelium from potential pathogens. The purpose of the present study was to compare mucin degradation mechanisms between ocular P. aeruginosa strains. Mucin degradation was assessed by agarose electrophoresis, lectin blotting, and size exclusion chromatography. The results indicate that certain P. aeruginosa strains (Paer12, ATCC 15442, 6294, and Paer25) had depleted mucin from the culture supernatant and that this was contingent on the inherent ability of these isolates to produce proteases. Non-protease-producing strains (Paer1 and Paer3) did not appreciably degrade mucin. Further, galactosidase, N-acetylglucosaminidase, and N-acetylgalactosaminidase activities were detected in some strains, suggesting the operation of further mechanisms of mucin degradation by P. aeruginosa. Mucin degradation by P. aeruginosa also seemed to be for the acquisition of nutrients, as a growth advantage was observed in mucin-depleting strains over nondepleting strains in the long term. It is postulated that the degradation of mucin serves to collapse the mucin barrier and its associated network containing antibacterial tear components and to provide energy for sustained bacterial growth.Microbial keratitis is a highly destructive and invasive infection that may result in corneal ulceration and loss of vision if prompt treatment is not administered (11). Pseudomonas aeruginosa has been isolated from most microbial keratitis patients, in some cases as the predominant bacterium (7, 56) and in others as the main gram-negative bacterium (44). It has also been associated with ocular disease resulting from contact lens wear, namely, contact lens acute red eye (CLARE) (24). This noninvasive ocular condition, characterized by a host inflammatory response, is presumably initiated by bacterial lipopolysaccharide (47). Contact lens wear seems to reduce tear exchange (39), where mucus is the predominant component of the tear film. Delayed tear clearance in certain contact lens wearers may prove opportune for the initiation of pathogenesis such as microbial keratitis.In the eye, mucus is the predominant component of the tear film (8, 37, 43), a three-layered film on the surface of the eye forming the first barrier between the outside environment and the underlying corneal epithelial cells. It is constituted primarily of secretory MUC5AC mucin secreted from conjunctival goblet cells (28), while membrane-bound mucins, MUC1 and MUC4, are present on corneal and conjunctival epithelial cells (1), with recent evidence that a soluble form of MUC4 is present in human tears (41). Ocular mucin is polydisperse, consisting of species with different size, charge, and glycosylation patterns (3, 17, 23) that contribute to the structure and function of mucus...