Untargeted metabolomics in the aqueous humor reveals the involvement of TAAR pathway in glaucoma.

Understanding the metabolic dysfunctions and underlying complex pathological mechanisms of neurodegeneration in glaucoma could help discover disease pathways, identify novel biomarkers, and rationalize newer therapeutics. Therefore, we aimed to investigate the local metabolomic alterations in the aqueous humor and plasma of primary glaucomatous patients. This study cohort comprised primary open-angle glaucoma (POAG), primary angle-closure glaucoma (PACG), and cataract control groups. Aqueous humor and plasma samples were collected from patients undergoing trabeculectomy or cataract surgery and subjected to high-resolution mass spectrometry (HRMS) analysis. Spectral information was processed, and the acquired data were subjected to uni-variate as well as multi-variate statistical analyses using MetaboAnalyst ver5.0. To further understand the localized metabolic abnormalities in glaucoma, metabolites affected in aqueous humor were distinguished from metabolites altered in plasma in this study. Nine and twelve metabolites were found to be significantly altered (p < 0.05, variable importance of projection >1 and log2 fold change ≥0.58/≤ -0.58) in the aqueous humor of PACG and POAG patients, respectively. The galactose and amino acid metabolic pathways were locally affected in the PACG and POAG groups, respectively. Based on the observation of the previous findings, gene expression profiles of trace amine-associated receptor-1 (TAAR-1) were studied in rat ocular tissues. The pharmacodynamics of TAAR-1 were explored in rabbits using topical administration of its agonist, ß-phenyl-ethylamine (ß-PEA). TAAR-1 was expressed in the rat's iris-ciliary body, optic nerve, lens, and cornea. ß-PEA elicited a mydriatic response in rabbit eyes, without altering intraocular pressure. Targeted analysis of ß-PEA levels in the aqueous humor of POAG patients showed an insignificant elevation. This study provides new insights regarding alterations in both localized and systemic metabolites in primary glaucomatous patients. This study also demonstrated the propensity of ß-PEA to cause an adrenergic response through the TAAR-1 pathway.

Label-Free Proteomics of Oral Mucosa Tissue to Identify Potential Biomarkers That Can Flag Predilection of Precancerous Lesions to Oral Cell Carcinoma: A Preliminary Study.

Oral squamous cell carcinomas are mostly preceded by precancerous lesions such as leukoplakia and erythroplakia. Our study is aimed at identifying potential biomarker proteins in precancerous lesions of leukoplakia and erythroplakia that can flag their transformation to oral cancer. Four biological replicate samples from clinical phenotypes of healthy control, leukoplakia, erythroplakia, and oral carcinoma were annotated based on clinical screening and histopathological evaluation of buccal mucosa tissue. Differentially expressed proteins were delineated using a label-free quantitative proteomic experiment done on an Orbitrap Fusion Tribrid mass spectrometer in three technical replicate sets of samples. Raw files were processed using MaxQuant version 2.0.1.0, and downstream analysis was done via Perseus version 1.6.15.0. Validation included functional annotation based on biological processes and pathways using the ClueGO plug-in of Cytoscape. Hierarchical clustering and principal component analysis were performed using the ClustVis tool. Across control, leukoplakia, and cancer, L-lactate dehydrogenase A chain, plectin, and WD repeat-containing protein 1 were upregulated, whereas thioredoxin 1 and spectrin alpha chain, nonerythrocytic 1 were downregulated. Across control, erythroplakia, and cancer, L-lactate dehydrogenase A chain was upregulated whereas aldehyde dehydrogenase 2, peroxiredoxin 1, heat shock 70 kDa protein 1B, and spectrin alpha chain, nonerythrocytic 1 were downregulated. We found that proteins involved in leukoplakia were associated with alteration in cytoskeletal disruption and glycolysis, while in erythroplakia, they were associated with alteration in response to oxidative stress and glycolysis across phenotypes. Hierarchical clustering subgrouped half of precancerous samples under the main branch of the control and the remaining half under carcinoma. Similarly, principal component analysis identified segregated clusters of control, precancerous lesions, and cancer, but erythroplakia phenotypes, in particular, overlapped more with the cancer cluster. Qualitative and quantitative protein signatures across control, precancer, and cancer phenotypes explain possible functional outcomes that dictate malignant transformation to oral carcinoma.

Retinal Pigment Epithelium in Human Donor Eyes Contains Higher Levels of Bisretinoids Including A2E in Periphery than Macula.

Purpose: With age, human retinal pigment epithelium (RPE) accumulates bisretinoid fluorophores that may impact cellular function and contribute to age-related macular degeneration (AMD). Bisretinoids are comprised of a central pyridinium, dihydropyridinium, or cyclohexadiene ring. The pyridinium bisretinoid A2E has been extensively studied, and its quantity in the macula has been questioned. Age-changes and distributions of other bisretinoids are not well characterized. We measured levels of three bisretinoids and oxidized A2E in macula and periphery in human donor eyes of different ages. Methods: Eyes (N = 139 donors, 61 women and 78 men, aged 40-80 years) were dissected into 8 mm diameter macular and temporal periphery punches. Using liquid chromatography - electrospray ionization - mass spectrometry (LC-ESI-MS) and an authentic synthesized standard, we quantified A2E (ng). Using LC-ESI-MS and a 50-eye-extract of A2E, we semiquantified A2E and 3 other compounds (eye extract equivalent units [EEEUs): A2-glycerophosphoethanolamine (A2GPE), dihydropyridine phosphatidyl ethanolamine (A2DHPE), and monofuranA2E (MFA2E). Results: A2E quantities in ng and EEEUs were highly correlated (r = 0.97, P < 0.001). From 262 eyes, 5 to 9-fold higher levels were observed in the peripheral retina than in the macula for all assayed compounds. A2E, A2DHPE, and MFA2E increased with age, whereas A2GPE remained unaffected. No significant right-left or male-female differences were detected. Conclusions: Significantly higher levels were observed in the periphery than in the macula for all assayed compounds signifying biologic differences between these regions. Levels of oxidized A2E parallel native A2E and not the distribution of retinal illuminance. Data will assist with the interpretion of clinical trial outcomes of agents targeting bisretinoid-related pathways.

Elevated histamine levels in aqueous humor of patients with glaucoma.

Purpose: Neurotransmitters (NTs) are the key mediators of essential ocular functions, such as processing the visual functions of the retina, maintaining homeostasis of aqueous humor, and regulating ocular blood flow. This study aims to determine variations in the levels of L-glutamate and γ-aminobutyric acid (GABA), histaminergic, adrenergic, cholinergic, and serotonergic NTs in patients with primary glaucoma versus patients with cataract. Methods: This case-control study involved three age-matched groups of patients with primary open angle glaucoma (POAG, n = 14), primary angle closure glaucoma (PACG, n = 21), and cataract (control, n = 19). Patients' aqueous humor and plasma were collected, snap frozen at -80 °C, and subjected to ultrasensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis for quantification of NTs. Results: Baseline intraocular pressure and the cup-to-disc ratio were found to be statistically significantly elevated in the POAG and PACG groups compared to the cataract control group. In aqueous humor, histamine was found to be statistically significantly elevated (5-fold, p<0.0001), whereas 1-methyl histamine was statistically significantly decreased (p<0.05) in POAG compared to the control group. A statistically significant increase in L-glutamate and GABA was observed among both patient groups with glaucoma compared to the cataract control group. Adrenaline was found to be elevated only in the PACG group (2.7-fold, p<0.05). No statistically significant difference was observed among the plasma NT levels between the groups. Conclusions: This study demonstrated the prominent role of the histaminergic system apart from autonomic mechanisms in the progression of glaucoma. Elevated L-glutamate and GABA could be due to retinal ganglionic cell death. Further studies are required to evaluate the effects of histamine on Müller cell dysfunction.

Evaluation of the Presence and Functional Importance of Nucleoside Transporters in Lacrimal Gland for Tear Disposition of Intravenously Injected Substrate in Rabbits.

Purpose: Purpose of the current study was to assess the presence and functionality of the nucleoside transporters in the lacrimal gland for the tear disposition of its substrate given intravenously in rabbits.Materials and Methods: Rabbits were divided into two groups - control and blocker pretreated. The blocker pretreated group received 5 mg/kg of dipyridamole 30 min before ribavirin (substrate), which was given at a dose of 2.5 mg/kg. All the treatments were given intravenously. Blood and tear samples were collected at 5, 15, 30, 60, 90, 120, 180, 240, 300 and 360 min (n = 4; each time point) after substrate administration. Tear samples were collected on Schirmer's strips, and plasma was separated immediately after blood collection. All the samples were stored at -80°C until analysis by LC-MS/MS.Results: Plasma ribavirin concentration for blocker pretreated group showed significantly (p < .05) higher levels at 5, 15, 30, 60, 120, 180 and 300 min as compared to the control group. Similarly, tear ribavirin concentration for blocker pretreated group also showed a significant (p < .05) increase at 5, 15, 60, 90, 180, 240 and 300 min compared to the control group. Plasma and tear AUC(0-6) for blocker pretreated group was 1.7 (p < .001) and 2.42 (p < .001) folds higher in a significant manner as compared to the control group, respectively. Percentage penetration of ribavirin from plasma to tears was also different between control and blocker pretreated group. Permeation ratio of ribavirin from plasma to tear for blocker pretreated group was found to be 1.4-folds higher in a significant (p < .05) manner.Conclusion: It is evident from the results that nucleoside transporters are present in lacrimal gland. The blocker treatment induced increase in tear transport of ribavirin indicates the possibility of the presence of nucleoside transporters on the apical side of lacrimal acinar cells in the uptake position.

Ocular kinetics and safety of intravitreally injected angiotensin converting enzyme inhibitor lisinopril.

BACKGROUND AND OBJECTIVES: The study investigated the intravitreal safety and vitreous disposition of lisinopril, an angiotensin converting enzyme inhibitor in rabbits for its projected use in retinopathy. METHODS: For the safety study, following the baseline ERG recording and fundus photography, 40 µg/50 µl of lisinopril sterile injection was injected unilaterally in the rabbit eyes (n = 4), where other eye served as a control. The electroretinogram and fundus images were obtained at 24, 48, 72 and 168 h following the intravitreal injection. For pharmacokinetics evaluation of the lisinopril, one eye of each rabbit (n = 4) received an intravitreal injection of lisinopril (40 µg/50 µl). The concentration of lisinopril in the ocular tissues, humours, plasma, lung, kidney and liver were measured through ESI-LC-MS/MS. RESULTS: Upon the electroretinography studies, no significant difference was observed in the ERG pattern in the lisinopril injected eye when compared to the baseline of the respective animals till the 7th day of the study. In the fundus imaging, no morphological changes were observed in the retina of the animal. The concentration of the lisinopril was found to be above to the IC50 in the retina-choroid till 36 h. The concentration found in the plasma and body tissues were many folds less than the IC50 of the lisinopril. CONCLUSIONS: Intravitreal injection of 40 µg/50 µl of lisinopril found to be safe in the rabbit eye as evidenced by the electroretinography and fundus imaging studies. The average half-life of lisinopril is 12.6 h and the above-mentioned dose able to sustain its IC50 value till the 36 h.

Serum Mortalin Correlated with α-Synuclein as Serum Markers in Parkinson's Disease: A Pilot Study.

Mortalin, a mitochondrial chaperone, plays a crucial role in reducing toxicity of Lewy bodies. Earlier studies had reported that Mortalin level gets downregulated in astrocytes and other brain tissue samples in Parkinson's disease (PD). This study aims to estimate the Mortalin concentration in serum and correlate with α-synuclein (α-Syn) in PD. The concentration of Mortalin and α-Syn in serum samples of 38 PD patients and 33 control group (CG) individuals was quantified by surface plasmon resonance. The receiver operating characteristic curves were plotted to develop it as blood-based protein marker. The expression of Mortalin in serum was validated by western blot. The Mortalin level was found to be declined in PD patients (1.98 ± 0.53 ng/µL) in comparison with CG individuals (3.13 ± 0.48 ng/µL), whereas α-Syn level was found to be elevated in PD patients (38.20 ± 4.22 ng/µL) than CG individuals (34.31 ± 3.23 ng/µL) in serum. The statistical analysis revealed the negative correlation between Mortalin and α-Syn. This preliminary study summarized that Mortalin plays a significant role in PD with negative correlation with α-Syn. This study provides a new paradigm for the development of Mortalin as a potent serum protein marker for diagnosis of PD.

Involvement of nucleoside transporters in the transcorneal permeation of topically instilled substrates in rabbits in-vivo.

The objective of the current study was to characterize and evaluate the functional importance of the Nucleoside Transporters (NTs) in the cornea of the rabbits. Reverse transcriptase polymerase chain reaction (RT-PCR) was used for the molecular characterization of the NTs. Their functionality was evaluated using two substrates, ribavirin and cytarabine. Dipyridamole was used as a blocker for the study. All the treatments were given topically. Molecular characterization of NTs revealed presence of ent1, ent2, ent3 and cnt3 in the cornea. The concentration vs time profile for cytarabine in Aqueous Humor (AH) exhibited a statistically significant (p<0.05) drop at 1h with blocker pretreatment. The mean AUC0-2 between the treatments was also differing in a significant (p<0.05) manner. The concentration vs time profile for ribavirin in AH also showed a significant (p<0.05) decrease in its concentration at 1h with blocker pretreatment. Dipyridamole was able to block ribavirin's entry with as low as 40nM concentration while complete blockade was achieved at 8mM and above. When cytarabine and ribavirin were co-administered, ribavirin at a concentration of 6.5mM significantly inhibited (p<0.05) the transcorneal permeation of cytarabine up to 80%. To conclude, this study showed the presence and functional importance of NTs in the transcorneal uptake of nucleoside substrates. This study further revealed the presence of concentration dependent competitive inhibition among substrates for their transcorneal permeation.

Vitreous Amyloidosis: Ocular, Systemic, and Genetic Insights.

PURPOSE: To report the unique clinical and surgical characteristics encountered in eyes with vitreous amyloidosis. Systemic evaluation and visual outcome after vitrectomy are discussed. A novel mutation in the transthyretin gene (TTR) in Indian patients with familial amyloid polyneuropathy (FAP) is described. DESIGN: Retrospective, observational study. PARTICIPANTS: Ten eyes of 5 patients from 2 pedigrees with a diagnosis of vitreous amyloidosis. METHODS: Detailed history, pedigree charting, systemic and ocular examination of 10 eyes (5 patients from 2 pedigrees) were carried out. Tests were performed to rule out vitreitis, retinal vasculitis, vitreous hemorrhage, and systemic amyloidosis. Genetic analysis to identify the mutation was performed in 1 patient. Vitreous biopsy, followed by 25-gauge pars plana vitrectomy, was performed in the same sitting in all cases. Samples were sent for Congo red staining and polarized microscopy. Patients were followed up on days 1, 7, and 28 and then every 2 months. Visual acuity assessment, intraocular pressure measurement, and fundus examination were performed each time. MAIN OUTCOME MEASURES: Mutations in TTR and postoperative visual acuity. RESULTS: Mean age at presentation was 32 years, with a 3:2 male-to-female distribution. Family history was positive in all patients. Nine eyes had pseudopodia lentis, whereas all 10 had glass wool-like vitreous. Glaucoma developed in 1 patient (2 eyes). Waxy paper-like vitreous with firm vitreous adhesions beyond major arcades and along retinal vessels was noted during surgery in all eyes. Congo red staining and apple green birefringence demonstrated vitreous amyloidosis. The mean preoperative best-corrected visual acuity (BCVA) was 1.39±0.64 logarithm of the minimum angle of resolution (logMAR), whereas the postoperative BCVA improved to 0.17±0.07 logMAR (P = 0.004). Gene sequencing revealed a phenylalanine→isoleucine mutation in the 33rd position of exon 2 of TTR in 1 patient of 1 pedigree, confirming the diagnosis of FAP. Two patients subsequently were found to have sensorimotor autonomic neuropathy, whereas 2 others had subclinical autonomic dysfunction. CONCLUSIONS: The clinical clues, management strategy, surgical characteristics, vitrectomy outcomes, and significance of systemic evaluation in vitreous amyloidosis are highlighted. A novel single mutation (Phe33Ile) in a case of FAP with vitreous amyloidosis from India is reported.

Comparative Proteomic Analysis of Advanced Ovarian Cancer Tissue to Identify Potential Biomarkers of Responders and Nonresponders to First-Line Chemotherapy of Carboplatin and Paclitaxel.

Conventional treatment for advanced ovarian cancer is an initial debulking surgery followed by chemotherapy combination of carboplatin and paclitaxel. Despite initial high response, three-fourths of these women experience disease recurrence with a dismal prognosis. Patients with advanced-stage ovarian cancer who underwent cytoreductive surgery were enrolled and tissue samples were collected. Post surgery, these patients were started on chemotherapy and followed up till the end of the cycle. Fluorescence-based differential in-gel expression coupled with mass spectrometric analysis was used for discovery phase of experiments, and real-time polymerase chain reaction, Western blotting, and pathway analysis were performed for expression and functional validation of differentially expressed proteins. While aldehyde reductase, hnRNP, cyclophilin A, heat shock protein-27, and actin are upregulated in responders, prohibitin, enoyl-coA hydratase, peroxiredoxin, and fibrin-ß are upregulated in the nonresponders. The expressions of some of these proteins correlated with increased apoptotic activity in responders and decreased apoptotic activity in nonresponders. Therefore, the proteins qualify as potential biomarkers to predict chemotherapy response.

Role of organic cation transporters in the ocular disposition of its intravenously injected substrate in rabbits: implications for ocular drug therapy.

The present study was conducted to test the hypothesis; OCT may be active from blood-to-vitreous for the uptake of its substrates. Ocular uptake of Tetraethylammonium (TEA) across blood ocular barriers and the tissue distribution was evaluated in vivo in New Zealand albino rabbits after intravenous administration. Quinidine (blocker) pretreatment resulted in a significant (p < 0.05) reduction in the Area Under the Curve (AUC) of TEA in vitreous (4.2 fold) and aqueous humor (1.8 fold) as compared to the control group which supports the role of OCT in uptake transport of its substrate across Blood ocular barrier. The blockade of OCT also affected the elimination of its substrate resulting in increased plasma levels. In most of the tissues, OCT are functionally present from apical to basolateral. The gene expression studies also showed the presence of OCT1, OCTN1 and OCTN2 in various ocular tissues studied. The present findings suggest that OCT are functionally active in blood ocular barriers and involved in the transport of its substrate from blood-to-vitreous humor.

Evaluation of the functional importance of organic cation transporters on the ocular disposition of its intravitreally injected substrate in rabbits.

PURPOSE: To evaluate the functional role of organic cation transporters (OCT) and ocular tissue distribution of intravitreally injected OCT substrate tetraethylammonium (TEA) in presence of OCT blocker (quinidine). METHODS: New Zealand albino rabbits of either sex were used. Intravitreal quinidine pretreatment was made 30 min before the administration of TEA. Modulation of vitreous and ocular tissue kinetics of OCT substrate was evaluated with or without blocker pretreatment. Gamma scintigraphy was also performed to visualize the vitreous residence of (99m)Tc-labelled TEA in the presence and absence of blocker. RESULTS: Intravitreally injected quinidine did not significantly alter the ocular disposition of TEA. TEA showed less significant posterior elimination kinetics and slow anterior elimination which resulted in longer residence time of TEA in eye after intravitreal administration. CONCLUSIONS: Intravitreally injected OCT substrates may follow an anterior elimination pathway and prolonged residence time in vitreous humor. The present study shows that OCT may not be active from vitreous-to-blood route in the blood-retinal barrier.

Development and validation of a highly sensitive LC-MS/MS method for organic cation transporter (OCT) substrate tetraethylammonium (TEA) in rabbits.

Tetraethylammonium is widely used as a probe in organic cation transporters studies. A simple, highly sensitive, and specific method using direct protein precipitation was developed using Hydrophilic Interaction Liquid Chromatography coupled with positive electrospray ionization tandem mass spectrometry for the determination of tetraethylammonium (TEA) in rabbit plasma. Isocratic separation was achieved using a ZIC-HILIC column with acetonitrile and 5mM ammonium acetate in the ratio of 8:2 containing 0.1% formic acid. Acquisition was performed in multiple reaction monitoring mode with the transitions: m/z 130→100 and 130→86 for TEA and m/z 276.1→142.2 for internal standard (homatropine). This method was validated to determine selectivity, linearity, sensitivity, precision, accuracy, recovery and stability. A good linearity was found within a range of 1.53-784.6 ng/mL. The above method has been demonstrated for its capability to estimate the plasma levels of TEA after its topical instillation in rabbit eyes. This method provides an accurate, precise and sensitive tool for determining TEA levels for transporter studies.

Group III PLA2 from the scorpion, Mesobuthus tamulus: cloning and recombinant expression in E. coli

Phospholipases A2 (PLA2) are enzymes that specifically hydrolyze the sn-2 fatty acid acyl bond of phospholipids, producing a free fatty acid and a lyso-phospholipid. We report the cloning and expression of a secretory phospholipase A2 (sPLA2) from Mesobuthus tamulus, Indian red scorpion. The nucleotide sequence codes for a 167 residue enzyme. The open reading frame codes for a 31 amino acid signal peptide followed by a mature portion of the protein. The primary structure shows the calcium binding motif, catalytic residues, 8 highly-conserved cysteines and C-terminal extension which classify it as a group III PLA2. The entire transcript was expressed in Escherichia coli and was purified by metal affinity chromatography under denaturing conditions. The protein was refolded by serial dilutions in the refolding buffer to its active form. Hemolytic assays indicate that the protein adopts a functional conformation. The functional requisites such as optimum pH of 8 and calcium dependency are shown. This report provides a simple but robust methodology for recombinant expression of toxic proteins.