Comparison of early changes in tear film protein profiles after small incision lenticule extraction (SMILE) and femtosecond LASIK (FS-LASIK) surgery.

BACKGROUND: Small incision lenticule extraction (SMILE) and femtosecond laser-assisted in situ keratomileusis (LASIK) are widely used surgical methods to correct myopia with comparable efficacy, predictability, and safety. We examined and compared the early changes of tear protein profiles after SMILE and FS-LASIK surgery in order to find possible differences in the initial corneal healing process. METHODS: SMILE operations for 26 eyes were made with Visumax femtosecond laser. In FS-LASIK surgery for 30 eyes, the flaps were made with Ziemer FEMTO LDV Z6 femtosecond laser and stromal ablation with Wavelight EX500 excimer laser. Tear samples were collected preoperatively, and 1.5 h and 1 month postoperatively using glass microcapillary tubes. Tear protein identification and quantification were performed with sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS). RESULTS: Immediately (1.5 h) after we found differences in 89 proteins after SMILE and in 123 after FS-LASIK operation compared to preoperative protein levels. Of these differentially expressed proteins, 48 proteins were common for both surgery types. There were, however, quantitative differences between SMILE and FS-LASIK. Upregulated proteins were mostly connected to inflammatory response and migration of the cells connected to immune system. One month after the operation protein expressions levels were returned to baseline levels with both surgical methods. CONCLUSIONS: Our study showed that immediate changes in protein profiles after SMILE and FS-LASIK surgeries and differences between the methods are connected to inflammatory process, and the protein levels quickly return to the baseline within 1 month. The differences in protein profiles between the methods are probably associated with the different size of the epithelial wound induced.

Proteomics Analysis of R-Ras Deficiency in Oxygen Induced Retinopathy.

Small GTPase R-Ras regulates vascular permeability in angiogenesis. In the eye, abnormal angiogenesis and hyperpermeability are the leading causes of vision loss in several ischemic retinal diseases such as proliferative diabetic retinopathy (PDR), retinal vein occlusion (RVO), and retinopathy of prematurity (ROP). Oxygen-induced retinopathy (OIR) is the most widely used experimental model for these ischemic retinopathies. To shed more light on how the R-Ras regulates vascular permeability in pathological angiogenesis, we performed a comprehensive (>2900 proteins) characterization of OIR in R-Ras knockout (KO) and wild-type (WT) mice by sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics. OIR and age-matched normoxic control retinas were collected at P13, P17, and P42 from R-Ras KO and WT mice and were subjected to SWATH-MS and data analysis. The most significant difference between the R-Ras KO and WT retinas was an accumulation of plasma proteins. The pathological vascular hyperpermeability during OIR in the R-Ras KO retina took place very early, P13. This led to simultaneous hypoxic cell injury/death (ferroptosis), glycolytic metabolism as well compensatory mechanisms to counter the pathological leakage from angiogenic blood vessels in the OIR retina of R-Ras deficient mice.

Clinical Tear Fluid Proteomics-A Novel Tool in Glaucoma Research.

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.

Antibacterial and Oxidative Stress-Protective Effects of Five Monoterpenes from Softwood.

Volatile organic compounds (VOC) affect the quality of indoor air. Terpenes and especially monoterpenes are the main molecules emitted from softwood material (coniferous species), which is widely used in construction. The corneal epithelium is one of the first human membranes to encounter VOCs in the air. Moreover, the industrial use of pleasant-scented monoterpenes in cosmetics, food, and detergents exposes people to monoterpenes in their daily lives. In the present study, the health effective properties of five monoterpenes from softwood were tested; cytotoxicity and oxidative stress-protective effects of α- and ß-pinenes, R- and S-limonene, and 3-carene were tested in a human corneal epithelial (HCE) cell model system and with two additional in vitro antioxidant tests: oxygen radical absorbance capacity (ORAC) and hydrogen peroxide (H2O2) scavenging. Antibacterial efficacies were tested with two bioluminescent bacterial biosensor strains (Escherichia coli K12+pcGLS11 and Staphylococcus aureus RN4220+pAT19) and with minimum inhibitory concentration (MIC) test against Escherichia coli. Only very high concentrations of monoterpenes (0.3-0.5 mg/mL) demonstrated cytotoxicity against HCE cells. Contrary to the original hypothesis, monoterpenes did not exhibit strong antioxidant properties in tested concentrations. However, biosensors and MIC tests indicated clear antibacterial activities for all tested monoterpenes.

In vitro stem cell modelling demonstrates a proof-of-concept for excess functional mutant TIMP3 as the cause of Sorsby fundus dystrophy.

Sorsby fundus dystrophy (SFD) is a rare autosomal dominant disease of the macula that leads to bilateral loss of central vision and is caused by mutations in the TIMP3 gene. However, the mechanisms by which TIMP3 mutations cause SFD are poorly understood. Here, we generated human induced pluripotent stem cell-derived retinal pigmented epithelial (hiPSC-RPE) cells from three SFD patients carrying TIMP3 p.(Ser204Cys) and three non-affected controls to study disease-related structural and functional differences in the RPE. SFD-hiPSC-RPE exhibited characteristic RPE structure and physiology but showed significantly reduced transepithelial electrical resistance associated with enriched expression of cytoskeletal remodelling proteins. SFD-hiPSC-RPE exhibited basolateral accumulation of TIMP3 monomers, despite no change in TIMP3 gene expression. TIMP3 dimers were observed in both SFD and control hiPSC-RPE, suggesting that mutant TIMP3 dimerisation does not drive SFD pathology. Furthermore, mutant TIMP3 retained matrix metalloproteinase activity. Proteomic profiling showed increased expression of ECM proteins, endothelial cell interactions and angiogenesis-related pathways in SFD-hiPSC-RPE. By contrast, there were no changes in VEGF secretion. However, SFD-hiPSC-RPE secreted higher levels of monocyte chemoattractant protein 1, PDGF and angiogenin. Our findings provide a proof-of-concept that SFD patient-derived hiPSC-RPE mimic mature RPE cells and support the hypothesis that excess accumulation of mutant TIMP3, rather than an absence or deficiency of functional TIMP3, drives ECM and angiogenesis-related changes in SFD. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Early changes in tear film protein profiles after femtosecond LASIK surgery.

BACKGROUND: Femtosecond laser-assisted in situ keratomileusis (LASIK) has proven to be an efficacious, predictable, and safe procedure for the correction of refractive errors. We examined the early tear protein changes of patients undergoing LASIK surgery in order to better understand the mechanisms and proteins related to laser corneal surgery and initial recovery. METHODS: Corneal flaps were created with Ziemer FEMTO LDV Z6 I femtosecond laser and stroma was ablated using Wavelight EX500 excimer laser. Tear samples were collected preoperatively as well as 1.5 h and 1 month after LASIK treatment using glass microcapillary tubes. Relative quantification of tear proteins was performed with sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS). RESULTS: SWATH-MS revealed that 158 proteins had altered expression levels 1.5 h after the operation. Two-thirds of these proteins, mostly connected to migration and inflammation response, returned to preoperative levels within the first postoperative month. The other proteins, which did not return to baseline levels, included proteins connected to for example epithelial barrier function. We also identified several proteins, which correlated with surgical variables, such as the amount of correction, flap thickness and flap diameter. CONCLUSIONS: The present study showed that an uneventful femtosecond LASIK refractive surgery induced a significant immune cell migration and inflammation-associated changes in tear proteomics profile quickly after the operation, but the expression of most proteins recovered almost completely to the preoperative levels within the first month. The individual proteins identified in our study are potential targets for the follow-up and modification of LASIK-induced biochemical processes.

Bioactive Properties of the Aqueous Extracts of Endophytic Fungi Associated with Scots Pine (Pinus sylvestris) Roots.

Despite the continuing interest in various plant and natural products, only a small portion of the biologically active compounds from nature has been discovered and exploited. In this study, antioxidant and antibacterial properties of aqueous fractions of three endophytic fungi isolated from the roots of 8-year-old Scots pines (Pinus sylvestris) growing on a drained peatland were investigated. The endophytic fungi species were Acephala applanata, Phialocephala fortinii, and Humicolopsis cephalosporioides/Coniochaeta mutabilis. The bioactivities were examined using hydrogen peroxide scavenging and oxygen radical absorbance capacity tests as well as sensitive Escherichia coli-based biosensors, which produce a luminescent signal in the presence of substances with oxidative or genotoxic properties. In addition, cell models for Parkinson's disease, age-related macular degeneration, and osteoarthritis were used to evaluate the potential for pharmaceutical applications. The aqueous extracts of fungi and 19 out of 42 fractions were found to be active in one or more of the tests used. However, no activity was found in the age-related macular degeneration and osteoarthritis cell model tests. Additionally, bioactivity data was connected with metabolites putatively annotated, and out of 330 metabolites, 177 were interesting in view of the bioactivities investigated. A majority of these were peptides and all three fungal species shared a highly similar metabolome. We propose that Scots pine endophytic fungi are a rich source of interesting metabolites, and synergistic effects may cause the bioactivities, as they were found to vary after the fractionation process.

Age-associated changes in human tear proteome.

BACKGROUND: Prevalence of many eye and ocular surface diseases increases with age. While the clinical characteristics and pathophysiologic mechanisms of these conditions are often either known or extensively studied, the effects of normal aging on tear film and ocular surface have not been as widely researched. METHODS: In order to examine the effects of aging on tear fluid proteomics, tear fluid samples were collected preoperatively from 115 subjects undergoing strabismus or refractive surgery using glass microcapillary tubes. In addition to their refractive error or strabismus, the subjects did not have any other current, known eye diseases. The non-pooled samples were analysed using NanoLC-TripleTOF implementing a sequential window acquisition of all theoretical fragment ion spectra mass spectrometry, resulting in quantified data of 849 proteins. RESULTS: According to correlation results, 17 tear proteins correlated significantly with increased age and many of these proteins were connected to inflammation, immune response and cell death. According to enrichment analysis, growth and survival of cells decreased while immune response and inflammation increased with aging. We also discovered several well-known, activated and inhibited upstream regulators, e.g. NF-κB, which has been previously connected to aging in numerous previous studies. CONCLUSIONS: Overall, the results show the common age-dependent alterations in tear fluid protein profile, which demonstrate similar age-associated alterations of biological functions previously shown in other tissue and sample types.

Comparison of iTRAQ and SWATH in a clinical study with multiple time points.

BACKGROUND: Advances in mass spectrometry have accelerated biomarker discovery in many areas of medicine. The purpose of this study was to compare two mass spectrometry (MS) methods, isobaric tags for relative and absolute quantitation (iTRAQ) and sequential window acquisition of all theoretical fragment ion spectra (SWATH), for analytical efficiency in biomarker discovery when there are multiple methodological constraints such as limited sample size and several time points for each patient to be analyzed. METHODS: A total of 140 tear samples were collected from 28 glaucoma patients at 5 time points in a glaucoma drug switch study. Samples were analyzed with iTRAQ and SWATH methods using NanoLC-MSTOF mass spectrometry. RESULTS: We discovered that even though iTRAQ is faster than SWATH with respect to analysis time per sample, it loses in sensitivity, reliability and robustness. While SWATH analysis yielded complete data of 456 proteins in all samples, with iTRAQ we were able to quantify 477 proteins in total but on average only 125 proteins were quantified in a sample. 283 proteins were common in the datasets produced by the two methods. Repeatability of the methods was assessed by calculating percent relative standard deviation (% RSD) between replicate MS analyses: SWATH was more repeatable (56% of proteins < 20% RSD), compared to iTRAQ (43% of proteins < 20% RSD). Despite the overall benefits of SWATH, both methods showed less than 1 log fold change difference in the expression of 74% common proteins. In addition, comparison to MS/MS peptide results using 8 isotopically labeled peptide standards, SWATH and iTRAQ showed similar results in terms of accuracy. Moreover, both methods detected similar trends in a longitudinal analysis of protein expression of two known tear biomarkers. CONCLUSIONS: Overall, we conclude that SWATH should be preferred for biomarker discovery studies when analyzing limited volumes of clinical samples collected at multiple time points. TRIAL REGISTERATION: The study was approved by the Ethics Committee at Tampere University Hospital and was registered in EU clinical trials register (EudraCT Number: 2010-021039-14).

Intraluminal deposits: A rare cause of glaucoma drainage implant total obstruction.

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.

Proteomics of Deep Cervical Lymph Nodes After Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) damages the glymphatic-lymphatic system. We hypothesized that brain injury associated with trauma results in the enrichment of brain-relevant proteins in deep cervical lymph nodes (DCLNs), the end station of meningeal lymphatic vessels, and that some of these proteins will present mechanistic tissue biomarkers for TBI. Proteomes of rat DCLNs were investigated in the left DCLN (ipsilateral to injury) and right DCLN at 6.5 months after severe TBI induced by lateral fluid percussion injury or after sham operation. DCLN proteomes were identified using sequential window acquisition of all theoretical mass spectra. Group comparisons, together with functional protein annotation analyses, were used to identify regulated protein candidates for further validation and pathway analyses. Validation of a selected candidate was assessed using enzyme-linked immunosorbent assay. Analysis comparing post-TBI animals with sham-operated controls revealed 25 upregulated and 16 downregulated proteins in the ipsilateral DCLN and 20 upregulated and 28 downregulated proteins in the contralateral DCLN of post-TBI animals. Protein class and function analyses highlighted the dysregulation of enzymes and binding proteins. Pathway analysis indicated an increase in autophagy. Biomarker analysis suggested that a subgroup of post-TBI animals had an increase in zonula occludens-1 coexpressed with proteins linked to molecular transport and amyloid precursor protein. We propose here that, after TBI, a subgroup of animals exhibit dysregulation of the TBI-relevant protein interactome in DCLNs, and that DCLNs might thus serve as an interesting biomarker source in future studies aiming to elucidate pathological brain functioning.

Looking deeper into ocular surface health: an introduction to clinical tear proteomics analysis.

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 MS-based tear proteomics study in the field of ophthalmology and ocular surface.

Ocular surface health of the Finnish elderly population.

PURPOSE: The aim of this study was to describe the clinical ocular surface characteristics in a population-based sample of Finnish elderly people. METHODS: This cross-sectional study included 601 subjects (335 females, 266 males) born between the years 1933-1956 and living in Savitaipale, Finland. Ocular surface health was evaluated using a comprehensive set of diagnostic tests. Previous dry eye (DE) diagnosis and history of drug treatment of DE were also recorded. Differences between sexes were estimated with Wilcoxon rank sum test and Fisher's exact test. RESULTS: Overall, 10% and 33% of people displayed signs of DE and ocular surface disease (OSD), respectively, and 30% had been previously diagnosed with DE and 36% used some form of drugs for DE. Men displayed more severe signs of meibomian gland dysfunction, blepharitis and conjunctival redness (p < 0.001), while women had higher scores in corneal staining (p = 0.005) and OSD Index (p < 0.001). CONCLUSION: Signs of OSD and DE are common among the Finnish elderly population. However, the diagnosis is affected by the diagnostic criteria used and significant differences exist between sexes. Although women were more frequently diagnosed with DE and OSD and experienced more ocular surface irritation, men had more often lid and meibomian gland-related issues. The current diagnostic criteria of DE pose a risk of misclassifying men, who commonly display less severe symptoms in comparison with women yet exhibit more severe clinical signs associated especially with the lid margin.

The effect of successful trabeculectomy on the ocular surface and tear proteomics-a prospective cohort study with 1-year follow-up.

PURPOSE: To report changes in the ocular surface and tear proteomics after discontinuation of chronic glaucoma medication. METHODS: Patients requiring trabeculectomy were recruited from the glaucoma clinic of Tampere University Hospital, Finland. Fifty-seven patients with previous history of anti-glaucomatous eye drops (8.1 ± 6.8 years) and having undergone a successful trabeculectomy were included in this report. Outcomes of interest were conjunctival redness grading, tear secretion (Schirmer I) and tear film proteomics (SWATH-MS) in addition to thorough clinical examination. The protocol included five time points: preoperative visit and postoperative visits at month 1, 3, 6 and 12. All parameters measured were compared to the corresponding preoperative levels of each individual eye. RESULTS: Conjunctival redness and irritation were significantly reduced during follow-up, while tear production remained unchanged. Protein profiles of the tear film indicated significant changes in the ocular surface. Lipid transport was increased while several pro-inflammatory proteins were consistently decreased after the surgery. CONCLUSION: Clinical signs as well as the proteomics results indicated that the trabeculectomy and resulting cessation of topical glaucoma medication were very beneficial to the ocular surface. The state of the conjunctiva improved throughout the 1-year follow-up while the levels of pro-inflammatory proteins decreased and lipid transport-associated functions were increased.

Comparison of Capillary and Schirmer Strip Tear Fluid Sampling Methods Using SWATH-MS Proteomics Approach.

Purpose: The purpose of this study was to examine the protein profile differences between capillary and Schirmer strip tear fluid samples. Methods: Both capillary and Schirmer strip tear samples were collected from 31 healthy participants at the same visit, and the samples were analyzed with nanoflow liquid chromatography coupled with time-of-flight mass spectrometer (NanoLC-MSTOF), implementing a sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS). Sample type-specific and combined spectral libraries were used to evaluate the differences between the sample types in protein expression levels and biological functions. Results: In proportion, more extracellular proteins connected to immune response were quantified from the capillary samples while Schirmer strip samples contained more intracellular proteins. The sample types yielded similar counts of quantified proteins when a combined spectral library including both sample types was implemented. The differential expression analysis between the sample types identified proteins increased in the capillary samples (e.g., immunoglobulins) and Schirmer strip samples (e.g., heat-shock proteins, annexins, and S100 proteins). Conclusions: Tear proteomics data originating from the same participants vary depending on whether the sample is collected with capillary or Schirmer strip, although there is also overlap between the two sample types when a combined spectral library is implemented in the SWATH-MS analysis. In discovery-based proteomics research of tear fluid, appropriate sampling method should be chosen carefully based on the research focus. Translational Relevance: Currently, there is no consensus on how the tear fluid sampling methods affect the resulting proteomics data, and hence, identification of the most suitable sampling methods for clinical researchers with varying research interests is important.

Patient stratification in clinical glaucoma trials using the individual tear proteome.

Glaucoma patients are prone to concomitant ocular surface diseases; however, switching from preserved to preservative-free medication can often alleviate these symptoms. The objective of this study was to examine how the adverse effects and tear proteome change for glaucoma patients (n = 28) during a 12-month drug switch from preserved latanoprost (Xalatan) to preservative-free tafluprost (Taflotan). We hypothesized that patient stratification could help identify novel recovery patterns in both tear proteomics and clinical data. In order to accomplish patient stratification, we implemented sequential window acquisition of all theoretical mass spectrometry (SWATH-MS) as a tool for quantitative analysis of individual tear protein profiles. During each visit (baseline and four follow-up visits), the patients' tears were sampled and the state of their ocular surface was evaluated clinically. Altogether 785 proteins were quantified from each tear sample using SWATH strategy and as these protein expression levels were compared between baseline and 12-month follow-up, three distinct patient groups were identified. We evaluated how these patient groups differed in their protein expression levels at baseline and discovered that the patients with increased levels of pro-inflammatory proteins and decreased levels of protective proteins benefitted most from the medication switch.

Integrative proteomics in prostate cancer uncovers robustness against genomic and transcriptomic aberrations during disease progression.

To understand functional consequences of genetic and transcriptional aberrations in prostate cancer, the proteomic changes during disease formation and progression need to be revealed. Here we report high-throughput mass spectrometry on clinical tissue samples of benign prostatic hyperplasia (BPH), untreated primary prostate cancer (PC) and castration resistant prostate cancer (CRPC). Each sample group shows a distinct protein profile. By integrative analysis we show that, especially in CRPC, gene copy number, DNA methylation, and RNA expression levels do not reliably predict proteomic changes. Instead, we uncover previously unrecognized molecular and pathway events, for example, several miRNA target correlations present at protein but not at mRNA level. Notably, we identify two metabolic shifts in the citric acid cycle (TCA cycle) during prostate cancer development and progression. Our proteogenomic analysis uncovers robustness against genomic and transcriptomic aberrations during prostate cancer progression, and significantly extends understanding of prostate cancer disease mechanisms.

SWATH-MS Proteomic Analysis of Oxygen-Induced Retinopathy Reveals Novel Potential Therapeutic Targets.

Purpose: Oxygen-induced retinopathy (OIR) is the most widely used model for ischemic retinopathies such as retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and retinal vein occlusion (RVO). The purpose of this study was to perform the most comprehensive characterization of OIR by a recently developed technique, sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics. Methods: Control and OIR retina samples collected from various time points were subjected to SWATH-MS and detailed data analysis. Immunohistochemistry from mouse retinas as well as neovascular membranes from human PDR and RVO patients were used for the detection of the localization of the proteins showing altered expression in the retina and to address their relevance to human ischemic retinopathies. Results: We report the most extensive proteomic profiling of OIR to date by quantifying almost 3000 unique proteins and their expression differences between control and OIR retinas. Crystallins were the most prominent proteins induced by hypoxia in the retina, while angiogenesis related proteins such as Filamin A and nonmuscle myosin IIA stand out at the peak of angiogenesis. Majority of the changes in protein expression return to normal at P42, but there is evidence to suggest that proteins involved in neurotransmission remain at reduced level. Conclusions: The results reveal new potential therapeutic targets to address hypoxia-induced pathological angiogenesis taking place in number of retinal diseases. The extensive proteomic profiling combined with pathway analysis also identifies novel molecular networks that could contribute to the pathogenesis of retinal diseases.

Topical fluorometholone treatment and desiccating stress change inflammatory protein expression in tears.

PURPOSE: It was hypothesized that tear protein biomarkers could predict the effects of topical steroid treatment and desiccating stress in patients with dry eye disease (DED). To test this concept, a randomized, double-masked, controlled clinical trial with 41 patients was conducted. METHODS: The patients were treated topically with either 0.1% fluorometholone (FML) or polyvinyl alcohol (PA). Tear samples were collected using 1 µl glass capillaries at recruitment into the study and after a 3-week treatment period, both before and after 2 h exposure to desiccating stress, in a controlled environment chamber. Relative quantification of tear proteins was conducted by NanoLC-MSTOF using sequential window acquisition of all theoretical mass spectra (SWATH). Ocular surface integrity (corneal and conjunctival staining and conjunctival hyperemia) was selected as the key DED-related sign and analyzed with proteomic data. Analysis of covariance (ANCOVA) and linear models were used to analyze the data with R. RESULTS: 758 proteins were identified and relatively quantified from each tear sample. Analysis revealed 9 differentially expressed proteins between FML and PA treatments after 3 weeks and 7 after desiccating stress (P < 0.05). We also identified several differentially expressed proteins at the initial collection, which could be used to predict changes of conjunctival and corneal staining and conjunctival hyperemia after FML treatment and after desiccating stress. These proteins include complement C3 (C3) and calmodulin like 5 (CALML5), which could also differentiate the severity of DED at baseline. CONCLUSIONS: The identified proteins could be further used as biomarkers to identify patients most benefiting from FML treatment.

Comparative proteomic analysis of human embryonic stem cell-derived and primary human retinal pigment epithelium.

Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) provide an unlimited cell source for retinal cell replacement therapies. Clinical trials using hESC-RPE to treat diseases such as age-related macular degeneration (AMD) are currently underway. Human ESC-RPE cells have been thoroughly characterized at the gene level but their protein expression profile has not been studied at larger scale. In this study, proteomic analysis was used to compare hESC-RPE cells differentiated from two independent hESC lines, to primary human RPE (hRPE) using Isobaric tags for relative quantitation (iTRAQ). 1041 common proteins were present in both hESC-RPE cells and native hRPE with majority of the proteins similarly regulated. The hESC-RPE proteome reflected that of normal hRPE with a large number of metabolic, mitochondrial, cytoskeletal, and transport proteins expressed. No signs of increased stress, apoptosis, immune response, proliferation, or retinal degeneration related changes were noted in hESC-RPE, while important RPE specific proteins involved in key RPE functions such as visual cycle and phagocytosis, could be detected in the hESC-RPE. Overall, the results indicated that the proteome of the hESC-RPE cells closely resembled that of their native counterparts.