Crosstalk Between m6A RNA Methylation and miRNA Biogenesis in Cancer: An Unholy Nexus.

N6-methyladenosine (m6A) is one of the most prevalent internal reversible chemical modification of RNAs in eukaryotes, which has attracted widespread attention recently owing to its regulatory roles in a plethora of normal developmental processes and human diseases like cancer. Deposition of the m6A mark on RNAs is mediated by the dynamic interplay between m6A regulatory proteins such as m6A RNA methyltransferases (m6A writers), m6A RNA demethylases (m6A erasers) and m6A RNA binding proteins (m6A readers). m6A regulators are ectopically expressed in various cancer types, often leading to aberrant expression of tumor-suppressor and oncogenic mRNAs either directly or indirectly via regulating the biogenesis of non-coding RNAs like miRNAs. miRNAs are tiny regulators of gene expression, which often impact various hallmarks of cancer and thus influence tumorigenesis. It is becoming increasingly clear that m6A RNA modification impacts biogenesis and function of miRNAs, and recent studies have interestingly, uncovered many miRNAs whose biogenesis and function are regulated by m6A writers, erasers and readers. In this review, we discuss various mechanisms by which m6A RNA methylation regulates miRNA biogenesis, the functional crosstalk between m6A RNA methylation and miRNAs and how it modulates various aspects of tumorigenesis. The potential of m6A RNA methylation regulated miRNAs as biomarkers and novel therapeutic targets to treat various cancers is also addressed.

Decoding the 'Fifth' Nucleotide: Impact of RNA Pseudouridylation on Gene Expression and Human Disease.

Cellular RNAs, both coding and noncoding are adorned by > 100 chemical modifications, which impact various facets of RNA metabolism and gene expression. Very often derailments in these modifications are associated with a plethora of human diseases. One of the most oldest of such modification is pseudouridylation of RNA, wherein uridine is converted to a pseudouridine (Ψ) via an isomerization reaction. When discovered, Ψ was referred to as the 'fifth nucleotide' and is chemically distinct from uridine and any other known nucleotides. Experimental evidence accumulated over the past six decades, coupled together with the recent technological advances in pseudouridine detection, suggest the presence of pseudouridine on messenger RNA, as well as on diverse classes of non-coding RNA in human cells. RNA pseudouridylation has widespread effects on cellular RNA metabolism and gene expression, primarily via stabilizing RNA conformations and destabilizing interactions with RNA-binding proteins. However, much remains to be understood about the RNA targets and their recognition by the pseudouridylation machinery, the regulation of RNA pseudouridylation, and its crosstalk with other RNA modifications and gene regulatory processes. In this review, we summarize the mechanism and molecular machinery involved in depositing pseudouridine on target RNAs, molecular functions of RNA pseudouridylation, tools to detect pseudouridines, the role of RNA pseudouridylation in human diseases like cancer, and finally, the potential of pseudouridine to serve as a biomarker and as an attractive therapeutic target.

Agreement of a Novel Artificial Intelligence Software With Optical Coherence Tomography and Manual Grading of the Optic Disc in Glaucoma.

PRCIS: The offline artificial intelligence (AI) on a smartphone-based fundus camera shows good agreement and correlation with the vertical cup-to-disc ratio (vCDR) from the spectral-domain optical coherence tomography (SD-OCT) and manual grading by experts. PURPOSE: The purpose of this study is to assess the agreement of vCDR measured by a new AI software from optic disc images obtained using a validated smartphone-based imaging device, with SD-OCT vCDR measurements, and manual grading by experts on a stereoscopic fundus camera. METHODS: In a prospective, cross-sectional study, participants above 18 years (Glaucoma and normal) underwent a dilated fundus evaluation, followed by optic disc imaging including a 42-degree monoscopic disc-centered image (Remidio NM-FOP-10), a 30-degree stereoscopic disc-centered image (Kowa nonmyd WX-3D desktop fundus camera), and disc analysis (Cirrus SD-OCT). Remidio FOP images were analyzed for vCDR using the new AI software, and Kowa stereoscopic images were manually graded by 3 fellowship-trained glaucoma specialists. RESULTS: We included 473 eyes of 244 participants. The vCDR values from the new AI software showed strong agreement with SD-OCT measurements [95% limits of agreement (LoA)=-0.13 to 0.16]. The agreement with SD-OCT was marginally better in eyes with higher vCDR (95% LoA=-0.15 to 0.12 for vCDR>0.8). Interclass correlation coefficient was 0.90 (95% CI, 0.88-0.91). The vCDR values from AI software showed a good correlation with the manual segmentation by experts (interclass correlation coefficient=0.89, 95% CI, 0.87-0.91) on stereoscopic images (95% LoA=-0.18 to 0.11) with agreement better for eyes with vCDR>0.8 (LoA=-0.12 to 0.08). CONCLUSIONS: The new AI software vCDR measurements had an excellent agreement and correlation with the SD-OCT and manual grading. The ability of the Medios AI to work offline, without requiring cloud-based inferencing, is an added advantage.

Progressive Vessel Density Reduction on OCT Angiography in Glaucoma Eyes with Disc Hemorrhages.

PURPOSE: To identify longitudinal changes in peripapillary and parafoveal vessel density (VD) measured by OCT angiography (OCTA) in primary open-angle glaucoma (POAG) eyes with disc hemorrhages (DHs). DESIGN: Prospective cohort study conducted from August 2016 through August 2020 PARTICIPANTS: Eighteen Asian-Indian participants with POAG (18 eyes) who sought treatment at the clinic with a single DH in the peripapillary region were recruited consecutively. METHODS: The study was conducted at a tertiary eye care center. All participants who were recruited underwent a baseline OCT and OCTA, which were repeated every 4 to 6 months. MAIN OUTCOME MEASURES: Peripapillary VD and retinal nerve fiber layer (RNFL) thickness, parafoveal VD and ganglion cell-inner plexiform layer (GCIPL) thickness in the DH sector, and the corresponding mirror-image sector across the horizontal meridian (control) were evaluated over time using linear mixed-effects models. RESULTS: The baseline average RNFL thickness was 79 ± 9 µm. Mean duration of follow-up was 2.6 ± 0.7 years. In the DH sector, all VD and structural parameters showed a significant negative slope (P < 0.01). In the control sector, the slopes of the structural parameters (RNFL and GCIPL thickness) were not significant (P > 0.05), but the rate of change of the peripapillary and parafoveal VDs were significant (P < 0.01). The rate of change of peripapillary VD was greater in the DH sector compared with the non-DH sector (-2.86 ± 0.6%/year vs. -1.71 ± 0.7%/year; P < 0.01). However, the parafoveal VD slopes did not differ significantly between DH and control sectors (-2.9 ± 0.17%/year vs. -2.8 ± 0.8%/year; P = 0.51). CONCLUSIONS: Eyes with POAG harboring a DH showed not only progressive RNFL and GCIPL loss in the DH sector, but also progressive peripapillary and parafoveal VD reduction in the DH and non-DH regions as documented on OCTA.

Anticancer potential of rhizome extract and a labdane diterpenoid from Curcuma mutabilis plant endemic to Western Ghats of India.

Zingiberaceae plants are well known for their use in ethnomedicine. Curcuma mutabilis Skornick., M. Sabu & Prasanthk., is an endemic Zingiberaceae species from Western Ghats of Kerala, India. Here, we report for the first time, the anticancer potential of petroleum ether extract from C. mutabilis rhizome (CMRP) and a novel labdane diterpenoid, (E)-14, 15-epoxylabda-8(17), 12-dien-16-al (Cm epoxide) isolated from it. CMRP was found to be a mixture of potent bioactive compounds including Cm epoxide. Both the extract and the compound displayed superior antiproliferative activity against several human cancer cell lines, without any display of cytotoxicity towards normal human cells such as peripheral blood derived lymphocytes and erythrocytes. CMRP treatment resulted in phosphatidylserine externalization, increase in the levels of intracellular ROS, Ca2+, loss of mitochondrial membrane potential as well as fragmentation of genomic DNA. Analyses of transcript profiling and immunostained western blots of extract-treated cancer cells confirmed induction of apoptosis by both intrinsic and extrinsic pathways. The purified compound, Cm epoxide, was also found to induce apoptosis in many human cancer cell types tested. Both CMRP and the Cm epoxide were found to be pharmacologically safe in terms of acute toxicity assessment using Swiss albino mice model. Further, molecular docking interactions of Cm epoxide with selected proteins involved in cell survival and death were also indicative of its druggability. Overall, our findings reveal that the endemic C. mutabilis rhizome extract and the compound Cm epoxide isolated from it are potential candidates for development of future cancer chemotherapeutics.

Spondias pinnata (L.f.) Kurz Leaf Extract Derived Zinc Oxide Nanoparticles Induce Dual Modes of Apoptotic-Necrotic Death in HCT 116 and K562 Cells.

This study evaluates the effects of phyto-derived zinc oxide nanoparticles (ZnONPs) on human cancer cells, colon carcinoma HCT 116, and chronic myelogenous leukemic K562, along with normal lymphocytes/erythrocytes. The commercial, chemically synthesized ZnONPs (cZnONPs) were also assessed in parallel. Using an eco-friendly approach devoid of harmful chemicals, biogenic nanoparticles were synthesized from aqueous leaf extract of Spondias pinnata (SpLZnONPs) by a sol-gel method. Optical, structural, and elemental characterization of both particle types were carried out deploying UV-Vis/photoluminescence spectroscopy, FTIR, XRD, FESEM, HRTEM, and EDX. Both SpLZnONPs and cZnONPs displayed hexagonal wurtzite structure with particle sizes averaging 30 and 48.5 nm, respectively. SpLZnONPs were found to be cytotoxic to both cancer cell types while cZnONPs exhibited toxicity only against HCT 116 cells. Interestingly, the cytomorphological changes and analysis of DNA laddering pattern observed in these treated cells were indicative of simultaneous induction of dual modes of death involving apoptosis and necrosis. Flow cytometric analysis of cell-cycle distribution, clonogenic, wound healing, and comet assays provided evidences of the antiproliferative potential of the tested nanoparticles. Apoptosis induction via oxidative stress-mediated Ca2+ release, ROS generation, loss of mitochondrial membrane potential, and externalization of phosphatidylserine was also determined biochemically. Relative expression of apoptotic genes was quantified using RT-qPCR and Western blot analysis. Mitotic index analysis, MTT, and hemolytic assays on lymphocytes and erythrocytes clearly revealed the absence of any deleterious effect(s) of SpLZnONPs in these cells compared with the toxicity of the chemically derived cZnONPs, thereby attesting to the biocompatibility and selective action of the biogenic nanoparticles.

A Comparison of the Visual Field Parameters of SITA Faster and SITA Standard Strategies in Glaucoma.

PURPOSE: To compare the visual field (VF) parameters of the new Swedish Interactive Thresholding Algorithm (SITA), SITA Faster (SFR) with that of SITA Standard (SS) on the Humphrey Field Analyzer. METHODS: Ninety-seven eyes of 97 subjects (63 glaucoma, 26 glaucoma suspects, and 8 normal eyes) underwent VF examination with SFR and SS strategies on the same day in random order. Agreement in VF parameters between SFR and SS strategies was assessed by Bland and Altman plots. In addition, some subjects underwent a second VF examination with SFR strategy to evaluate its test-retest variability. RESULTS: The median test duration of SS strategy was 6 minutes 14 seconds, whereas SFR was 2 minutes 49 seconds (55% shorter, P<0.001). Median mean deviation (-7.3 vs. -7.6 dB, P=0.73) and VF index (88 vs. 88%, P=0.32) were similar between the 2 strategies, whereas pattern standard deviation was significantly higher (4.8 vs. 4.7 dB, P=0.01) with SS strategy. Overall average threshold sensitivity and Garway-Heath sector-wise threshold sensitivities were similar between the 2 strategies except for the nasal sector where SFR strategy had higher sensitivity (26 vs. 25 dB, P=0.02). Bland-Altman plots showed the mean difference in all VF parameters between the SS and SFR strategies were small (ranging from -1.0 dB for the nasal sector to -0.01 dB for superotemporal sector sensitivity). The test-retest variability of VF parameters with SFR strategy was low. CONCLUSIONS: VF parameters with SFR showed good agreement with that of SS strategy. This, combined with low test-retest variability, suggests that SFR can be considered for diagnosis and monitoring of glaucoma.

Chemical composition, antioxidant and antiproliferative activities of essential oil from rhizome and leaves of Curcuma mutabilis Skornick., M. Sabu & Prasanthk., endemic to Western Ghats of India.

Hydro-distilled essential oils, from fresh rhizomes and leaves of Curcuma mutabilis Skornick., M.Sabu & Prasanthk., characterized by GC-MS revealed the presence of thirty three and twenty three compounds therein respectively. Whilst estrone methyl ether (3-Methoxyestra-1,3,5(10)-trien-17-one) was the major component in rhizome oil (47.35%), sesquiterpene hydrocarbons predominated as the major group (63.92%) in leaf oil with a higher preponderance of ß-caryophyllene (25.48%), ß-farnesene (19.47%) and α-humulene (11.01%). Weak antioxidant activities observed in these oils determined by DPPH and ABTS methods were apparently influenced both by the oil composition and the assay conditions. Rhizome oil showed higher antiproliferative activity than leaf oil against leukemic K562 (IC50-6.8µg/mL) and colorectal HCT116 (IC50-8.5µg/mL) cancer cell lines. This first report reveals composition and biological activities of essential oils from C. mutabilis.

Choroidal Microvascular Dropout in Pseudoexfoliation Glaucoma.

Purpose: To compare the prevalence of choroidal microvasculature dropout (CMvD) in pseudoexfoliation glaucoma (PXG) and disease severity-matched primary open-angle glaucoma (POAG) eyes. Methods: In a cross-sectional study, 39 eyes with PXG (33 patients) and 39 glaucoma severity-matched POAG eyes (34 patients) underwent visual fields, optical coherence tomography and optical coherence tomography angiography examination. Peripapillary vessel density (VD) was evaluated from the radial peripapillary capillary slab, parafoveal VD was measured on the superficial vascular plexus slab of the macula, and CMvD was evaluated on the choroidal slabs of the optic disc scan. Results: The PXG and POAG groups were similar with respect to average mean deviation on visual fields (-12.1 vs. -12.0 decibel, P = 0.96) and average peripapillary retinal nerve fiber layer thickness on optical coherence tomography (71 vs. 74 µ, P = 0.29). Average peripapillary superficial VD (49.7% vs. 51.3%, P = 0.35) and parafoveal VD (44.8% vs. 45.8%, P = 0.33) were similar between the PXG and POAG groups. CMvD was seen in 18 PXG and 31 POAG eyes (46.2% vs. 79.5%, P = 0.002). On multivariate analysis that accounted for the severity of glaucoma, the odds of CMvD was significantly lower in the PXG group when compared with the POAG group (odds ratio: 0.18-0.21, P < 0.01). Conclusions: The prevalence of CMvD was significantly lower in the PXG eyes when compared with the POAG eyes.

Manilkara zapota (L.) P. Royen Leaf Extract Derived Silver Nanoparticles Induce Apoptosis in Human Colorectal Carcinoma Cells Without Affecting Human Lymphocytes or Erythrocytes.

Plant-derived synthesis of silver nanoparticles (AgNPs) has found wide biomedical applications including cancer cure. This report deals with biosynthesis of silver nanoparticles (MZLAgNPs) employing leaf extracts of Manilkara zapota (L.) under optimized conditions. Characterization of MZLAgNPs using UV-Vis spectroscopy, FTIR, XRD, and FESEM analyses revealed that the particles were predominantly spherical averaging 24 nm in size. Their cellular effects were assessed by MTT assay, fluorescence, and scanning electron microscopy of cells stained with propidium iodide, acridine orange/ethidium bromide, and annexin V-FITC to visualize signs of apoptosis. Evaluation of cell proliferation by clonogenic assay, wound healing ability by scratch assay and cell cycle distribution by flow-cytometry was also carried out. Apoptosis-related gene expressions were analyzed by RTq-PCR and western blot analysis. MZLAgNPs selectively inhibited growth of colorectal carcinoma HCT116, HeLa, and non-small lung carcinoma A549 cells, dose-dependently with IC50 concentrations of 8, 16, and 29 µg/mL respectively, following 72-h treatment, without affecting growth of normal human lymphocytes and erythrocytes. Apoptosis induction was observed by fluorescence and scanning electron microscopy. Overproduction of reactive oxygen species (ROS), reduction of mitochondrial membrane potential, upregulation of apoptotic-related genes - PUMA, cas-3, cas-8, cas-9, and BAX, expression of caspase 3, and occurrence of PARP cleavage were observed in MZLAgNPs/cisplatin treated cells. Taken together, our results clearly demonstrate the therapeutic potential of biogenic MZLAgNPs as an effective agent for killing colorectal carcinoma cells by apoptosis induction.

Choroidal Microvascular Dropout in Primary Open-angle Glaucoma Eyes With Disc Hemorrhage.

PURPOSE: The main purpose of this study was to compare the prevalence of choroidal microvascular dropout (CMvD) in primary open-angle glaucoma (POAG) eyes with and without disc hemorrhage (DH). METHODS: In a cross-sectional study, 44 eyes of 44 control subjects, 32 eyes of 32 POAG patients with DH, and 41 eyes of 41 POAG patients without DH underwent visual fields (VFs), optical coherence tomography (OCT) and OCT angiography (OCTA). Presence of CMvD was evaluated on the choroidal OCTA slab. VF defect in the glaucoma eyes were classified into initial nasal defect, initial parafoveal scotoma, and combined nasal and parafoveal defect. RESULTS: CMvD was detected in 17 POAG eyes with DH (53.1%) and 13 POAG eyes without DH (31.7%; P=0.06). On univariate analysis, CMvD in POAG eyes was associated with DH [odds ratio (OR): 2.44, P=0.06] and measures of glaucoma severity: VF mean deviation (OR: 0.85, P=0.02), retinal nerve fiber layer thickness (OR: 0.95, P=0.03), and peripapillary vessel density (OR: 0.94, P=0.09). On multivariate models that accounted for the measures of glaucoma severity, CMvD in POAG eyes was statistically significantly associated with DH (OR≥3, P<0.05). CMvD was more frequently seen in eyes with initial parafoveal scotoma than initial nasal defect both in POAG eyes with DH (P=0.06) and POAG eyes without DH (P<0.001). CONCLUSIONS: Prevalence of CMvD was significantly greater in POAG eyes with DH compared with POAG eyes without DH. CMvD in POAG eyes was also significantly associated with central VF defects and greater severity of glaucomatous damage.

Repeatability and comparability of peripapillary vessel density measurements of high-density and non-high-density optical coherence tomography angiography scans in normal and glaucoma eyes.

AIMS: To compare the peripapillary vessel density (VD) measurements of high-density (HD) and non-HD optical coherence tomography angiography (OCTA) scans in normal and glaucoma eyes, and to evaluate the intrasession repeatability of VD measurements of HD scans. METHODS: In a cross-sectional study, 46 normal (33 subjects) and 89 glaucoma (64 patients) eyes underwent 3 HD and 1 non-HD optic nerve head OCTA scans in the same session. Agreement in VD measurements between HD and non-HD scans was assessed using Bland and Altman analysis. Repeatability of the VD measurements of HD scans was assessed using within-subject coefficient of repeatability (CRw) and variation (CVw). RESULTS: The mean difference in the VDs ranged between 0.7% (temporal sector VD) and 2.0% (inferonasal sector VD), with HD scans showing significantly greater VD values than non-HD scans. The 95% limits of agreement (LoA) in glaucoma eyes ranged between -2.0% and 5.0% for whole enface VD and between -4.8% and 9.6% for superotemporal VD. CRw (%) and CVw (%) of VD measurements of HD scans ranged from 3.0 to 4.9 and from 2.0 to 3.1 in normal eyes. The same ranged from 3.2 to 6.7 and from 2.6 to 4.8, respectively, in glaucoma eyes. CONCLUSIONS: VD of HD scans was higher than that of non-HD scans. The wide 95% LoA indicates that the VD measurements of HD and non-HD scans cannot be used interchangeably. Test-retest repeatability of VDs on HD scans was as high as 6%. These results should be considered while using OCTA for longitudinal evaluation of glaucoma.

Choroidal Microvascular Dropout in Primary Angle Closure Glaucoma.

PURPOSE: To determine the prevalence and factors associated with the presence of choroidal microvascular dropout (CMvD) in primary angle-closure glaucoma (PACG) eyes compared to primary open-angle glaucoma (POAG) eyes. DESIGN: Cross-sectional study. METHODS: Thirty-six POAG eyes (36 patients) and 28 PACG eyes (28 patients) underwent optical coherence tomography angiography (OCTA). Presence of CMvD was evaluated on choroidal OCTA slabs. Visual field (VF) defects in the glaucoma eyes were classified into initial nasal defect (IND), initial parafoveal scotoma (IPFS), and combined nasal and parafoveal defect, and the association between type of VF defect and CMvD was evaluated. RESULTS: CMvD was detected in 21 POAG (58.3%) and 10 PACG (35.7%) eyes (P = .07). CMvD in POAG eyes was associated with pretreatment intraocular pressure (odds ratio [OR] = 0.91/mm Hg higher intraocular pressure, P = .06), VF mean deviation (MD, OR = 0.75/dB higher MD, P = .007), retinal nerve fiber layer thickness (OR = 0.92/µm increase in thickness, P = .02), and peripapillary vessel density (OR = 0.80/unit increase in density, P = .01). CMvD in PACG eyes was associated only with VF MD (OR = 0.90/dB higher MD, P = .05). When analyzed in the entire cohort of glaucoma patients (64 eyes), CMvD was significantly associated with POAG (OR > 3.5, P < .05) after accounting for glaucoma severity. CMvD was seen in 6 of 7 eyes with IPFS and 1 of 13 with IND in the POAG group (P < .05) and 1 of 2 eyes with IPFS and 0 of 10 with IND in the PACG group (P < .05). CONCLUSIONS: Prevalence of CMvD was significantly lower in PACG compared to POAG. As in POAG, CMvD in PACG was associated with advanced VF damage and with IPFS on VF.

Mutational analyses of regulatory genes, mexR, nalC, nalD and mexZ of mexAB-oprM and mexXY operons, in efflux pump hyperexpressing multidrug-resistant clinical isolates of Pseudomonas aeruginosa.

The present study deals with membrane-bound efflux pumps, MexAB-OprM and MexXY and their respective regulatory genes mexR, nalC, nalD and mexZ in multidrug resistant (MDR) Pseudomonas aeruginosa. Following antibiotic sensitivity testing and detection of various beta-lactamases, hyperexpression of efflux pump genes, mexB and mexY in the isolates was investigated using semi-quantitative and real-time reverse transcription-PCR. Amplicons from regulatory genes were sequenced and subjected to mutational and phylogenetic analysis. Twenty-nine clinical isolates of P. aeruginosa were obtained from a total of 144 MDR gram-negative bacteria collected from Kerala State, South India. All strains were found to be resistant to ampicillin and nalidixic acid with 13.8, 44.8 and 31% testing positive for extended-spectrum beta-lactamases, metallo-beta-lactamases and AmpC producers respectively. Increased mexB and mexY transcription was detected respectively in 10.3 and 20.7% of the isolates in comparison with P. aeruginosa reference strain, PAO (MTCC). Co-expression of MexY was also observed in MexB overproducers. Various synonymous/and non-synonymous mutations in regulatory gene sequences of efflux pump operons were detected. In the strain designate Pa16, mexR was found to harbour four novel point mutations with one transversion and three transitions which included a substitution of an ochre codon with that for serine. The gene also displayed a novel mutation involving insertion of a cysteine at the 444th base position, followed by an opal codon. The genetic divergence and homogeneity of the concatenated (mexR, nalC and nalD) regulatory gene sequences of mexAB-oprM operon was apparent in the phylogram generated with similar sequences retrieved from public database.