Jimmy Ka-Wai Cheung

Tear lipids are crucial for tear film stability and ocular surface health. Changes in tear composition could be associated with ocular and systemic diseases such as meibomian gland dysfunction and dyslipidemia. Profiling tear lipids may help biomarker discovery for disease diagnosis and management. However, common tear sampling methods present distinct limitations: Schirmer's strips frequently cause ocular irritation and discomfort due to their large contact area with the eye surface, while microcapillary tube collection could yield low reproducibility due to operator variability, especially when performed by different personnel. These limitations might compromise the accuracy and consistency of lipidomic data. This study introduces a minimally invasive phenol red thread (PRT)-based sampling method optimized for tear lipidomics. The thin structure of PRT minimizes the risk of ocular irritation and allows rapid and gentle tear collection. This user-friendly and easy-to-perform method is suitable for subjects with reduced tear volume or lower tolerance for foreign body sensation, and it enables more reproducible sample collection by reducing operator-dependent variability. Tear lipids collected by PRT were extracted using an optimized methanol/methyl tert-butyl ether (MTBE) phase separation protocol and analyzed by high-resolution LC-Orbitrap-IQX MS/MS with LipidSearch software. The workflow identified more than 700 unique tear lipid species, each characterized by specific fatty-acid-derived product ions. These results indicate that PRT-based sampling provides robust lipid recovery for tear lipidomic analysis. This minimally invasive and reproducible approach offers a practical platform for clinical and experimental tear lipid research. Ultimately, this could also facilitate biomarker discovery and disease monitoring.

Tear lipids are crucial for tear film stability and ocular surface health. Changes in tear composition could be associated with ocular and systemic diseases such as meibomian gland dysfunction and dyslipidemia. Profiling tear lipids may help biomarker discovery for disease diagnosis and management. However, common tear sampling methods present distinct limitations: Schirmer's strips frequently cause ocular irritation and discomfort due to their large contact area with the eye surface, while microcapillary tube collection could yield low reproducibility due to operator variability, especially when performed by different personnel. These limitations might compromise the accuracy and consistency of lipidomic data. This study introduces a minimally invasive phenol red thread (PRT)-based sampling method optimized for tear lipidomics. The thin structure of PRT minimizes the risk of ocular irritation and allows rapid and gentle tear collection. This user-friendly and easy-to-perform method is suitable for subjects with reduced tear volume or lower tolerance for foreign body sensation, and it enables more reproducible sample collection by reducing operator-dependent variability. Tear lipids collected by PRT were extracted using an optimized methanol/methyl tert-butyl ether (MTBE) phase separation protocol and analyzed by high-resolution LC-Orbitrap-IQX MS/MS with LipidSearch software. The workflow identified more than 700 unique tear lipid species, each characterized by specific fatty-acid-derived product ions. These results indicate that PRT-based sampling provides robust lipid recovery for tear lipidomic analysis. This minimally invasive and reproducible approach offers a practical platform for clinical and experimental tear lipid research. Ultimately, this could also facilitate biomarker discovery and disease monitoring.

The vitreous humor (VH) is a transparent gelatin-like substance that occupies two-thirds of the eyeball and undergoes the most significant changes during eye elongation. Quantitative proteomics on the normal growth period in the VH could provide new insights into understanding its progression mechanism in the early stages of myopia. In this study, a data-independent acquisition (SWATH-MS) was combined with targeted LC-ESI-MS/MS to identify and quantify the relative protein changes in the vitreous during the normal growth period (4, 7, 14, 21 and 28 days old) in the chick model. Chicks were raised under normal growing conditions (12/12 h Dark/light cycle) for 28 days, where ocular measurements, including refractive and biometric measurements, were performed on days 4 (baseline), 7, 14, 21 and 28 (n = 6 chicks at each time point). Extracted vitreous proteins from individual animals were digested and pooled into a left eye pool and a right pool at each time point for protein analysis. The vitreous proteome for chicks was generated using an information-dependent acquisition (IDA) method by combining injections from individual time points. Using individual pool samples, SWATH-MS was employed to quantify proteins between each time point. DEPs were subsequently confirmed in separate batches of animals individually on random eyes (n = 4) using MRMHR between day 7 and day 14. Refraction and vitreous chamber depth (VCD) were found to be significantly changed (p < 0.05, n = 6 at each time point) during the period. A comprehensive vitreous protein ion library was built with 1576 non-redundant proteins (22987 distinct peptides) identified at a 1% false discovery rate (FDR). A total of 12 up-regulated and 26 down-regulated proteins were found across all time points compared to day 7 using SWATH-MS. Several DEPs, such as alpha-fetoprotein, the cadherin family group, neurocan, and reelin, involved in structural and growth-related pathways, were validated for the first time using MRMHR under this experimental condition. This study provided the first comprehensive spectral library of the vitreous for chicks during normal growth as well as a list of potential growth-related protein biomarker candidates using SWATH-MS and MRMHR during the emmetropization period.

Myopia control efficacy and long-term safety of the Breath-O-Correct orthokeratology (OK) lens was evaluated in a 2-year randomized, single vision (SV) spectacle lens-controlled, single-blind clinical trial combining clinical and tear proteomics data. A total of 71 children (43 OK, 9.8 ± 1.3 years; 28 SV, 9.5 ± 1.4 years) completed the 2-year study. Axial length (AL), cycloplegic refraction, clinical safety parameters (best-corrected visual acuity, central cornea thickness, corneal endothelial health, ocular surface disease index), and quantitative tear proteomics were evaluated by masked examiners. Mean 2-year-normalized AL elongations in the OK and SV groups differed significantly (p = 0.03) and were 0.37 ± 0.37 mm and 0.60 ± 0.41 mm, respectively. OK-mediated myopia control efficacy was 37.1%. No significant difference was found in clinical safety parameters of both groups (p > 0.10), except for a thinner central corneal thickness in the OK group (p = 0.01). Proteomics revealed modest OK lens-mediated effects on immune response proteins, including an increased abundance of haptoglobin at 6 and 12 months and a decreased abundance of two proteins (neutrophil defensin 3 and histone 4) at 6 months. The changes were further validated using a high-resolution multiple-reaction monitoring (MRM^HR) mass spectrometry. In summary, the Breath-O-Correct OK lens significantly reduced AL elongation in schoolchildren without adverse clinical effects or subclinical inflammatory responses.

Diabetic retinopathy (DR) was identified as a leading cause of blindness and vision impairment in 2020. In addition to vasculopathy, DR has been found to involve retinal neurons, including amacrine cells and retinal ganglion cells. Despite possessing features that are susceptible to diabetic conditions, photoreceptor cells have received relatively little attention with respect to the development of DR. Until recently, studies have suggested that photoreceptors secret proinflammatory molecules and produce reactive oxygen species that contribute to the development of DR. However, the effect of hyperglycemia on photoreceptors and its underlying mechanism remains elusive. In this study, the direct effect of high glucose on photoreceptor cells was investigated using a 661w photoreceptor-like cell line. A data-independent sequential window acquisition of all theoretical mass spectra (SWATH)-based proteomic approach was employed to study changes induced by high glucose in the proteomic profile of the cells. The results indicated that high glucose induced a significant increase in apoptosis and ROS levels in the 661w cells, with mitochondrial dysfunction among the major affected canonical pathways. The involvement of mitochondrial dysfunction was further supported by increased mitochondrial fission and reduced mitochondrial bioenergetics. Collectively, these findings provide a biological basis for a possible role of photoreceptors in the pathogenesis of DR.

This study used isotope-coded protein label (ICPL) quantitative proteomics and bioinformatics analysis to examine changes in vitreous protein content and associated pathways during lens-induced eye growth. First, the vitreous protein profile of normal 7-day old chicks was characterized by nano-liquid chromatography electrospray ionization tandem mass spectrometry. A total of 341 unique proteins were identified. Next, myopia and hyperopia were induced in the same chick by attaching −10D lenses to the right eye and +10D lenses to the left eye, for 3 and 7 days. Protein expression in lens-induced ametropic eyes was analyzed using the ICPL approach coupled to LCMS. Four proteins (cystatin, apolipoprotein A1, ovotransferrin, and purpurin) were significantly up-regulated in the vitreous after 3 days of wearing −10D lenses relative to +10D lens contralateral eyes. The differences in protein expression were less pronounced after 7 days when the eyes approached full compensation. In a different group of chicks, western blot confirmed the up-regulation of apolipoprotein A1 and ovotransferrin in the myopic vitreous relative to both contralateral lens-free eyes and hyperopic eyes in separate animals wearing +10D lenses. Bioinformatics analysis suggested oxidative stress and lipid metabolism as pathways involved in compensated ocular elongation.