phenol-red-thread-based-sampling-procedure-for-untargeted-tear-fluid

Phenol Red Thread-based Sampling Procedure for Untargeted Tear Fluid Lipidomics in Biomarker Discovery

Tear lipids are crucial for tear film stability and ocular surface health. Changes in tear composition could be associated with ocular and systemic ...

The Hong Kong Polytechnic University

Centre for Eye and Vision Research (CEVR)

To investigate the mechanism for the production of paralytic shellfish toxins (PST) in toxic dinoflagellates, with a 2D-gel based approach, we had made two sets of proteomic comparisons: (a) between a toxic  (AC-T) and a phylogenetically closely related non-toxic strain (AC-N), (b) between toxic AC-T grown in a medium with 10% normal amount of phosphate (AC-T-10%P) known to induce higher toxicity and AC-T grown in normal medium. We found that photosynthesis and energy production related proteins were up-regulated in AC-T when compared to AC-N. However, the same group of proteins was down-regulated in AC-T-10%P when compared to normal AC-T. Examining the relationship of photosynthesis and toxin content of AC-T upon continuous photoperiod experiment revealed that while growth and associated toxin content increased after 8 days of continuous light, toxin content maintained constant when cells were shifted from continuous light to continuous dark for 3 days. This emphasized the cruciality of light availability on toxin biosynthesis in AC-T, while another light-independent mechanism may be responsible for higher toxicity in AC-T-10%P compared to normal AC-T. Taken all together, it is believed that the interplay between "illumination", "photosynthesis", "phosphate availability", and "toxin production" is much more complicated than what we had previously anticipated.

Production of Paralytic Shellfish Toxins (PSTs) in Toxic  is Intertwined with Photosynthesis and Energy Production.

As a sequel to our previous report of the existence of species-specific protein/peptide expression profiles (PEPs) acquired by mass spectrometry in some dinoflagellates, we established, with the help of a plasma-membrane-impermeable labeling agent, a surface amphiesmal protein extraction method (SAPE) to label and capture species-specific surface proteins (SSSPs) as well as saxitoxins-producing-species-specific surface proteins (Stx-SSPs) that face the extracellular space (i.e., SSSPs and Stx-SSPs). Five selected toxic dinoflagellates, ,  and  were used in this study. Transcriptomic databases of these five species were also constructed. With the aid of liquid chromatography linked-tandem mass spectrometry (LC-MS/MS) and the transcriptomic databases of these species, extracellularly facing membrane proteomes of the five different species were identified. Within these proteomes, 16 extracellular-facing and functionally significant transport proteins were found. Furthermore, 10 SSSPs and 6 Stx-SSPs were identified as amphiesmal proteins but not facing outward to the extracellular environment. We also found SSSPs and Stx-SSPs in the proteomes. The potential functional correlation of these proteins towards the production of saxitoxins in dinoflagellates and the degree of species specificity were discussed accordingly.

Finding Species-Specific Extracellular Surface-Facing Proteomes in Toxic Dinoflagellates.

Dry eye syndrome (DES) affects millions of people worldwide. However, as the cellular responses of the corneal epithelium under hyperosmotic stress remain unclear, this study investigated the proteomic changes between human corneal epithelial cells (HCECs) cultured with isosmotic and hyperosmotic media. Under hyperosmotic stress, HCECs increased expressions of sodium-coupled neutral amino acid transporter (SNAT2), glutaminase (GLS-1), and a few isoforms of heat shock protein and aldo-keto reductase family 1. The expressions of SNAT2 and GLS-1 were increased after 6 h of exposure to hyperosmotic stress but not by glutamine deprivation. The hyperosmotic stress increased intracellular levels of glutamine, mitochondrial superoxide, and mitochondrial membrane potential and induced mitochondrial fission in HCECs. Thus, the intracellular level of glutamine was elevated in the hyperosmotic stressed HCECs via the upregulation of SNAT2. Glutamine can act as an osmolyte to regulate the osmolarity of HCECs or be converted to glutamate by GLS-1 for the tricarboxylic acid cycle and oxidative phosphorylation to maintain ATP production under the hyperosmotic stress-induced mitochondrial fission. Thus, the increases in the expressions of SNAT2 and GLS-1 are key osmoregulations in HCECs upon the hyperosmotic stress and may act as corneal biomarkers for monitoring DES progression.

Upregulations of SNAT2 and GLS-1 Are Key Osmoregulatory Responses of Human Corneal Epithelial Cells to Hyperosmotic Stress.

The corneal epithelium serves as the front barrier against environmental stimuli and pathogens on the ocular surface. A comprehensive protein profile of the corneal epithelium would be crucial for understanding the molecular mechanisms that are related to corneal disease. This work demonstrated a library-free data-independent acquisition (DIA) approach across different mass spectrometers and proteomic software to build a comprehensive proteomic dataset for human corneal epithelial cells (HCECs). With the combinational use of different data-independent acquisition technologies of multiple mass spectrometers, including Sciex ZenoTOF 7600 (DIA-SWATH), Bruker TimsTOF Pro2 (DIA-PASEF), and ThermoFisher Orbitrap Fusion Lumos (DIA-HRMS1), protein identification and quantification were performed with superior sensitivity and resolution. By using a library-free DIA approach, this study constructed a more diverse and unbiased proteomic profile of human corneal epithelial cells (HCECs), comprising 11,954 protein groups (1% FDR). This represents the largest corneal proteome reported to date. All raw proteomic data were deposited to ProteomeXchange Consortium via Proteomics Identifications database (PRIDE) with the dataset identifier accession number PXD059451. Our findings hold the potential to enhance future understanding of corneal pathologies and transformative therapeutics.

Article	Year
Production of Paralytic Shellfish Toxins (PSTs) in Toxic is Intertwined with Photosynthesis and Energy Production. Toxins\| PubMed ID: 32727048	2020
Finding Species-Specific Extracellular Surface-Facing Proteomes in Toxic Dinoflagellates. Toxins\| PubMed ID: 34564629	2021
Upregulations of SNAT2 and GLS-1 Are Key Osmoregulatory Responses of Human Corneal Epithelial Cells to Hyperosmotic Stress. Journal of proteome research\| PubMed ID: 40360154	2025
A Comprehensive Proteome of Human Corneal Epithelial Cells Constructed by Cross-platform DIA-Mass Spectrometry. Scientific data\| PubMed ID: 40410165	2025

Kenrick Kai-Yuen Chan

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