Small RNA Sequencing Reveals a Distinct MicroRNA Signature between Glucocorticoid Responder and Glucocorticoid Non-Responder Primary Human Trabecular Meshwork Cells after Dexamethasone Treatment.

Glucocorticoids (GCs) are known to regulate several physiological processes and are the mainstay in the management of inflammatory eye diseases. The long-term use of GC causes raised intraocular pressure (IOP) or ocular hypertension (OHT) in about 30-50% of the susceptible individuals depending on the route of administration, and can lead to steroid-induced secondary glaucoma. The present study aims to understand the role of microRNAs (miRNAs) in differential glucocorticoid (GC) responsiveness in human trabecular meshwork (HTM) cells using small RNA sequencing. The human organ-cultured anterior segment (HOCAS) model was used to identify whether donor eyes were from GC-responders (GC-R; n = 4) or GC-non-responders (GC-NR; n = 4) following treatment with either 100 nM dexamethasone (DEX) or ethanol (ETH) for 7 days. The total RNA was extracted from cultured HTM cells with known GC responsiveness, and the differentially expressed miRNAs (DEMIRs) were compared among the following five groups: Group #1: ETH vs. DEX-treated GC-R; #2: ETH vs. DEX-treated GC-NR; #3: overlapping DEGs between Group #1 and #2; #4: Unique DEMIRs of GC-R; #5: Unique DEMIRs of GC-NR; and validated by RT-qPCR. There were 13 and 21 DEMIRs identified in Group #1 and Group #2, respectively. Seven miRNAs were common miRNAs dysregulated in both GC-R and GC-NR (Group #3). This analysis allowed the identification of DEMIRs that were unique to GC-R (6 miRNAs) and GC-NR (14 miRNAs) HTM cells, respectively. Ingenuity Pathway Analysis identified enriched pathways and biological processes associated with differential GC responsiveness in HTM cells. This is the first study to reveal a unique miRNA signature between GC-R and GC-NR HTM cells, which raises the possibility of developing new molecular targets for the management of steroid-OHT/glaucoma.

A Comparative Genome-Wide Transcriptome Analysis of Glucocorticoid Responder and Non-Responder Primary Human Trabecular Meshwork Cells.

Aim: To investigate genes and pathways involved in differential glucocorticoid (GC) responsiveness in human trabecular meshwork (HTM) cells using RNA sequencing. Methods: Using paired human donor eyes, human organ-cultured anterior segment (HOCAS) was established in one eye to characterize GC responsiveness based on intra ocular pressure (IOP) change and, in the other eye, primary HTM cell culture was established. For RNA sequencing, total RNA extracted from GC-responder (GC-R) and non-responder (GC-NR) cells after dexamethasone (DEX) or ethanol (ETH) treatment for 7d was used. Differentially expressed genes (DEGs) were compared among five groups and validated. Results: In total, 616 and 216 genes were identified as significantly dysregulated in Group #1 and #2 (#1: ETH vs. DEX-treated GC-R; #2: ETH vs. DEX-treated GC-NR), respectively. Around 80 genes were commonly dysregulated in Group #3 (overlapping DEGs between #1 and #2), whereas 536 and 136 genes were uniquely expressed in GC-R (#4) and GC-NR HTM (#5) cells, respectively. Pathway analysis revealed that WNT signaling, drug metabolism cytochrome p450, cell adhesion, TGF-ß signaling, and MAPK signaling were associated with GC responsiveness. Conclusion: This is the first study reporting distinct gene signatures and their associated pathways for GC-R and GC-NR HTM cells. WNT and MAPK signaling are potential therapeutic targets for the management of GC-induced glaucoma.

Short and long-term effect of dexamethasone on the transcriptome profile of primary human trabecular meshwork cells in vitro.

In the quest of identifying newer molecular targets for the management of glucocorticoid-induced ocular hypertension (GC-OHT) and glaucoma (GCG), several microarray studies have attempted to investigate the genome-wide transcriptome profiling of primary human trabecular meshwork (TM) cells in response to dexamethasone (DEX). However, no studies are reported so far to demonstrate the temporal changes in the expression of genes in the cultured human TM cells in response to DEX treatment. Therefore, in the present study, the time-dependent changes in the genome-wide expression of genes in primary human TM cells after short (16 hours: 16 h) and long exposure (7 days: 7 d) of DEX was investigated using RNA sequencing. There were 199 (118 up-regulated; 81 down-regulated) and 525 (119 up-regulated; 406 down-regulated) DEGs in 16 h and 7 d treatment groups respectively. The unique genes identified in 16 h and 7 d treatment groups were 152 and 478 respectively. This study found a distinct gene signature and pathways between two treatment regimes. Longer exposure of DEX treatment showed a dys-regulation of Wnt and Rap1 signaling and so highlighted potential therapeutic targets for pharmacological management of GC-OHT/glaucoma.

Dysregulated expression of microRNAs in aqueous humor from intraocular tuberculosis patients.

BACKGROUND: Systemic Mycobacterium tuberculosis (Mtb) infection alters microRNA's expression that controls cellular processes and modulates host defense mechanisms. However, the role of miRNAs in intraocular tuberculosis (IOTB) remains unknown. Therefore, this study aims to identify dysregulated miRNAs in the aqueous humor (AH) of patients with IOTB. METHODS: AH from intraocular tuberculosis patients (n = 2) and cataract controls (n = 2) were used for small RNA deep sequencing using HiSeq Illumina sequencing platform. Differentially expressed miRNAs and their targets were identified by the bioinformatics approach, and their regulatory functions were predicted by pathway enrichment analysis. The expression of selected miRNAs and their binding targets were further validated by real-time quantitative PCR (RT-qPCR). RESULTS: In total, we identified 56 differentially expressed miRNAs in the AH of intraocular tuberculosis (IOTB) patients compared to controls. We selected four significantly dysregulated miRNAs (miR-423-5p, miR-328-3p, miR-21-5p, and miR-16-5p) based on the RT-qPCR validation and predicted their gene targets. We developed a miRNA-targets regulatory network by combining pathways of interest and genes associated with TB. We identified that these four miRNAs might play an important role in IOTB pathogenesis via tuberculosis-associated pathways; PI3K-Akt signaling, autophagy and MAPK pathway. CONCLUSIONS: For the first time, this study identifies the dysregulation of four miRNAs in the AH of IOTB patients using the ultra-low input small-RNA sequencing approach. Further target prediction and validation identify the role of these miRNAs in tuberculosis pathogenesis via tuberculosis-related pathways. This study identifies miRNAs as potentially ideal biomarkers in the aqueous humor of IOTB patients.

Characterization of antibiotic resistance and virulence genes of ocular methicillin-resistant Staphylococcus aureus strains through complete genome analysis.

Virulence-factor encoding genes (VFGs) and antimicrobial resistance genes (ARGs) of ocular Methicillin-Resistant Staphylococcus aureus (MRSA), are the reason behind the common cause of severe and untreatable ocular infection and are largely unknown. The unavailability of the complete genome sequence of ocular MRSA strains hinders the unambiguous determination of ARGs and VRGs role in disease pathogenesis and their genomic location. To fulfill this critical need, we achieved the high-quality complete genome of four ocular MRSA strains (AMRF3 - AMRF6) by combining MinION nanopore sequencing technology, followed by polishing with Illumina sequence reads. We obtained a single chromosome and a plasmid in each strain. Sequence typing revealed that AMRF3 and AMRF5 strains harbored ST772, whereas AMRF4 and AMRF6 harbored ST 2066. All plasmids carried heavy metal cadmium resistance genes cadC and cadD, while cadA was detected only in the plasmid pSaa6159 of AMRF4 and AMRF6 strains. Further, pSaa6159 contains a complete Tn552 transposon with beta-lactamase genes, blaI, blaR1, and blaZ. Interestingly, pSaa6159 in AMRF6 carried five copies of Tn552 transposon. Several exotoxins and enterotoxins were identified across ocular MRSA strains and ST2066 strains found to be not carried any enterotoxins; this finding suggests that these two strains are exotoxigenic. Besides, ST2066 strains carried serine proteases (splA, splB, splD, splE and spIF) and exotoxin (seb and set 21) for their virulence, while ST772 carried antimicrobial resistance genes (blaZ, dfrG, msrA, mphC and fosB) and enterotoxin sec for virulence, suggesting sequence type-specific resistance and virulence. Also, we identified many VFGs and ARGs, that provided multi-drug resistance, enterotoxigenic, exotoxigenic, biofilm-forming, host tissue adhesion and immune response evasion in ocular MRSA strains. Thus, our study provides a better insight into the genomes of ocular MRSA strains that would provide more effective treatment strategies for ocular MRSA infection.

Effect of S-methylisothiourea in acetaminophen-induced hepatotoxicity in rat.

Nitric oxide synthesized from inducible nitric oxide synthase (iNOS) plays role in acetaminophen (APAP)-induced liver damage. The present study was undertaken to evaluate the effect of iNOS inhibitor S-methylisothiourea (SMT) in APAP-induced hepatotoxicity in rats (1 g/kg, i.p.). SMT was (10, 30, and 100 mg/kg; i.p.) given 30 min before and 3 h after APAP administration. At 6 and 24 h, blood was collected to measure alanine transaminase (ALT), aspartate transaminase (AST), and nitrate plus nitrite (NOx) levels in serum. At 48 h, animals were sacrificed, and blood and liver tissues were collected for biochemical estimation. SMT reduced significantly the serum ALT, AST, and NOx levels at 24 and 48 h and liver NOx levels at 48 h as compared with APAP-treated control. The amount of peroxynitrite measured by rhodamine assay was significantly reduced by SMT, as compared with APAP-treated control group. SMT treatment (30 mg/kg) has significantly reduced the lipid peroxidation and protein carbonyl levels, increased SOD and catalase, and reduced glutathione and total thiol levels significantly as compared with APAP-treated control. SMT 30 mg/kg dose has protected animals from APAP-induced hypotension and reduced iNOS gene expression. Hepatocytes were isolated from animals, and effect of SMT on apoptosis, MTP, and ROS generation was studied, and their increased value in APAP intoxicated group was found to be significantly decreased by SMT (30 mg/kg) at 24 and 48 h. In conclusion, nitric oxide produced from iNOS plays important role in toxicity at late hours (24 to 48 h), and SMT inhibits iNOS and reduces oxidative and nitrosative stress.

Eicosapentaenoic acid-induced endothelium-dependent and -independent relaxation of sheep pulmonary artery.

It is known that long chain polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), have beneficial effects on cardiovascular function including pulmonary hypertension. The purpose of the present study was to examine the mechanisms involved in EPA-induced relaxation of sheep isolated pulmonary artery by measuring isometric tension. Nitric oxide (NO) derived from constitutive nitric oxide synthase (cNOS) was measured by Greiss method in the presence of the inducible nitric oxide synthase (iNOS) selective inhibitor N-[[3-(aminomethyl) phenyl]methyl]-ethanimidamide, dihydrochloride (1400 W). EPA (10(-)(7)-10(-)(4)M) caused concentration-dependent relaxation of sheep pulmonary artery with a pD(2) of 5.56+/-0.09 and E(max) of 87.40+/-3.10% (n=9). N(G)-nitro-L-arginine methyl ester (L-NAME) 100 microM significantly attenuated (E(max) 41.95+/-6.70%; n=8) EPA-induced relaxation of endothelium intact arterial rings. Similarly, endothelium denudation markedly inhibited (E(max) 17.60+/-1.21%; n=4) EPA-induced relaxation. EPA (30 microM) significantly increased the cNOS-derived NO release (10.17+/-0.96; n=8 versus control 7.43+/-0.78 pmol/mg tissue wet wt./h; n=7) in endothelium intact vessels. However, EPA-stimulated NO release was markedly blunted by either 100 microM L-NAME (7.07+/-0.54 pmol/mg tissue wet wt./h; n=8) or endothelium removal (6.97+/-0.87 pmol/mg tissue wet wt./h; n=17). In endothelium-denuded K(+) (80 mM)-depolarized arterial rings, EPA (30 microM) significantly inhibited CaCl(2)-induced contractions (E(max) 42.77+/-5.90% versus control 94.78+/-9.82%; n=5). The fatty acid also inhibited nifedipine (1 microM)-insensitive 5-HT-induced contractions in this vessel (E(max) 70.57+/-4.88% versus control 161.50+/-17.46%; n=5). In conclusion, EPA relaxes sheep pulmonary artery primarily through endothelium-dependent NO release, and the residual endothelium-independent relaxation may result from inhibition of Ca(2+)-influx through L-type calcium channels, as well as 5-HT-stimulated intracellular Ca(2+) release.