RGS4 controls Gαi3-mediated regulation of Bcl-2 phosphorylation on TGN38-containing intracellular membranes.

Intracellular pools of the heterotrimeric G-protein α-subunit Gαi3 (encoded by GNAI3) have been shown to promote growth factor signaling, while at the same time inhibiting the activation of JNK and autophagic signaling following nutrient starvation. The precise molecular mechanisms linking Gαi3 to both stress and growth factor signaling remain poorly understood. Importantly, JNK-mediated phosphorylation of Bcl-2 was previously found to activate autophagic signaling following nutrient deprivation. Our data shows that activated Gαi3 decreases Bcl-2 phosphorylation, whereas inhibitors of Gαi3, such as RGS4 and AGS3 (also known as GPSM1), markedly increase the levels of phosphorylated Bcl-2. Manipulation of the palmitoylation status and intracellular localization of RGS4 suggests that Gαi3 modulates phosphorylated Bcl-2 levels and autophagic signaling from discreet TGN38 (also known as TGOLN2)-labeled vesicle pools. Consistent with an important role for these molecules in normal tissue responses to nutrient deprivation, increased Gαi signaling within nutrient-starved adrenal glands from RGS4-knockout mice resulted in a dramatic abrogation of autophagic flux, compared to wild-type tissues. Together, these data suggest that the activity of Gαi3 and RGS4 from discreet TGN38-labeled vesicle pools are critical regulators of autophagic signaling that act via their ability to modulate phosphorylation of Bcl-2.

THE CURRENT SURGICAL MANAGEMENT OF LARGE, RECURRENT, OR PERSISTENT MACULAR HOLES.

PURPOSE: To evaluate the current surgical options available for the management of large (>400 µm), recurrent, or persistent macular holes (MHs). METHODS: A review of the literature was performed, focusing on the epidemiology, pathophysiology, diagnosis, and surgical treatments of large, recurrent, or persistent MHs. Based on this review, a comprehensive overview was provided regarding the topic of large, recurrent, or persistent MHs and focused on recent surgical management updates. RESULTS: For large MHs, variations of the inverted internal limiting membrane flap technique demonstrated promising rates of primary hole closure and significant visual acuity improvements. For recurrent or recalcitrant MHs, early repeat vitrectomy with extension of the internal limiting membrane peel remains the most straightforward and optimal surgical technique to achieve secondary closure. Regardless of the surgical approach, the goal of each technique described is to induce or aid in stimulating gliosis within the MH to maximize closure. CONCLUSION: Despite the high success rate of modern MH surgery, large, recurrent, or persistent MHs remain a challenge for retinal surgeons. This review provides a detailed summary on the rationality and efficacy of current surgical options.

Origin-specific epigenetic program correlates with vascular bed-specific differences in Rgs5 expression.

Cells from multiple origins contribute to vascular smooth muscle cell (VSMC) development. Phenotypic heterogeneity of VSMCs is associated with their point of developmental origin; however, the mechanisms driving such differences are unknown. We here examined the mechanisms controlling vascular bed-specific differences in Rgs5 expression during development. Rgs5 levels were similar across different regions of the vasculature in neonatal animals but were >15-fold higher in descending aortas compared with carotid arteries of adult mice. Thus, vessel bed-specific changes in regulation of Rgs5 expression occurred during vessel maturation. Examination of adult Rgs5-LacZ reporter mice revealed lower Rgs5 expression in VSMCs originating from the third (carotid artery) branchial arch compared with those originating in the fourth and sixth (aortic B segment, right subclavian, and ductus arteriosus) branchial arches. Indeed, a mosaic Rgs5 expression pattern, with discreet LacZ boundaries between VSMCs derived from different developmental origins, was observed. Furthermore, Rgs5-LacZ expression was correlated with the site of VSMC origin (splanchic mesoderm ≈ local mesenchyme > somites > proepicardium > mesothelium). Surprisingly, Rgs5 reporter activity in cultured carotid artery- and descending aorta-derived cells did not recapitulate the differences observed in vivo. Consistent with a developmental origin-specific epigenetic mechanism driving the observed expression differences in vivo, the Rgs5 promoter showed increased methylation on CpG dinucleotides in carotid arteries compared with that in descending aortas in adult but not in neonatal mice. In vitro methylation of the Rgs5 promoter confirmed that its activity is sensitive to transcriptional down-regulation by CpG methylation. These data suggest that an origin-dependent epigenetic program regulates vascular bed- and maturation state-dependent regulation of VSMC-specific gene transcription.

A role for protein phosphatase 2A in regulating p38 mitogen activated protein kinase activation and tumor necrosis factor-alpha expression during influenza virus infection.

Influenza viruses of avian origin continue to pose pandemic threats to human health. Some of the H5N1 and H9N2 virus subtypes induce markedly elevated cytokine levels when compared with the seasonal H1N1 virus. We previously showed that H5N1/97 hyperinduces tumor necrosis factor (TNF)-alpha through p38 mitogen activated protein kinase (MAPK). However, the detailed mechanisms of p38MAPK activation and TNF-alpha hyperinduction following influenza virus infections are not known. Negative feedback regulations of cytokine expression play important roles in avoiding overwhelming production of proinflammatory cytokines. Here we hypothesize that protein phosphatases are involved in the regulation of cytokine expressions during influenza virus infection. We investigated the roles of protein phosphatases including MAPK phosphatase-1 (MKP-1) and protein phosphatase type 2A (PP2A) in modulating p38MAPK activation and downstream TNF-alpha expressions in primary human monocyte-derived macrophages (PBMac) infected with H9N2/G1 or H1N1 influenza virus. We demonstrate that H9N2/G1 virus activated p38MAPK and hyperinduced TNF-alpha production in PBMac when compared with H1N1 virus. H9N2/G1 induced PP2A activity in PBMac and, with the treatment of a PP2A inhibitor, p38MAPK phosphorylation and TNF-alpha production were further increased in the virus-infected macrophages. However, H9N2/G1 did not induce the expression of PP2A indicating that the activation of PP2A is not mediated by p38MAPK in virus-infected PBMac. On the other hand, PP2A may not be the targets of H9N2/G1 in the upstream of p38MAPK signaling pathways since H1N1 also induced PP2A activation in primary macrophages. Our results may provide new insights into the control of cytokine dysregulation.

Emerging Evidence of Noncoding RNAs in Bleb Scarring after Glaucoma Filtration Surgery.

PURPOSE: To conduct a narrative review of research articles on the potential anti- and pro-fibrotic mechanisms of noncoding RNAs following glaucoma filtration surgery. METHODS: Keyword searches of PubMed, and Medline databases were conducted for articles discussing post-glaucoma filtration surgeries and noncoding RNA. Additional manual searches of reference lists of primary articles were performed. RESULTS: Fifteen primary research articles were identified. Four of the included papers used microarrays and qRT-PCR to identify up- or down-regulated microRNA (miRNA, miR) profiles and direct further study, with the remainder focusing on miRNAs or long noncoding RNAs (lncRNAs) based on previous work in other organs or disease processes. The results of the reviewed papers identified miR-26a, -29b, -139, -155, and -200a as having anti-fibrotic effects. In contrast, miRs-200b and -216b may play pro-fibrotic roles in filtration surgery fibrosis. lncRNAs including H19, NR003923, and 00028 have demonstrated pro-fibrotic effects. CONCLUSIONS: Noncoding RNAs including miRNAs and lncRNAs are emerging and promising therapeutic targets in the prevention of post-glaucoma filtration surgery fibrosis.

Quantum dots trace lymphatic drainage from the mouse eye.

Glaucoma is a leading cause of blindness in the world, often associated with elevated eye pressure. Currently, all glaucoma treatments aim to lower eye pressure by improving fluid exit from the eye. We recently reported the presence of lymphatics in the human eye. The lymphatic circulation is known to drain fluid from organ tissues and, as such, lymphatics may also play a role in draining fluid from the eye. We investigated whether lymphatic drainage from the eye is present in mice by visualizing the trajectory of quantum dots once injected into the eye. Whole-body hyperspectral fluorescence imaging was performed in 17 live mice. In vivo imaging was conducted prior to injection, and 5, 20, 40 and 70 min, and 2, 6 and 24 h after injection. A quantum dot signal was observed in the left neck region at 6 h after tracer injection into the eye. Examination of immunofluorescence-labelled sections using confocal microscopy showed the presence of a quantum dot signal in the left submandibular lymph node. This is the first direct evidence of lymphatic drainage from the mouse eye. The use of quantum dots to image this lymphatic pathway in vivo is a novel tool to stimulate new treatments to reduce eye pressure and prevent blindness from glaucoma.

Estimation of Backscatter Coefficients Using an In Situ Calibration Source.

The objective of this article is to demonstrate the feasibility of estimating the backscatter coefficient (BSC) using an in situ calibration source. Traditional methods of estimating the BSC in vivo using a reference phantom technique do not account for transmission losses due to intervening layers between the ultrasonic source and the tissue region to be interrogated, leading to increases in bias and variance of BSC-based estimates. To account for transmission losses, an in situ calibration approach is proposed. The in situ calibration technique employs a titanium sphere that is well-characterized ultrasonically, biocompatible, and embedded inside the sample. A set of experiments was conducted to evaluate the embedded titanium spheres as in situ calibration targets for BSC estimation. The first experiment quantified the backscattered signal strength from titanium spheres of three sizes: 0.5, 1, and 2 mm in diameter. The second set of experiments assessed the repeatability of BSC estimates from the titanium spheres and compared these BSCs to theory. The third set of experiments quantified the ability of the titanium bead to provide an in situ reference spectrum in the presence of a lossy layer on top of the sample. The final set of experiments quantified the ability of the bead to provide a calibration spectrum over multiple depths in the sample. All experiments were conducted using an L9-4/38 linear array connected to a SonixOne system. The strongest signal was observed from the 2-mm titanium bead with the signal-to-noise ratio (SNR) of 11.6 dB with respect to the background speckle. Using an analysis bandwidth of 2.5-5.5 MHz, the mean differences between the experimentally derived BSCs and BSCs derived from the Faran theory were 0.54 and 0.76 dB using the array and a single-element transducer, respectively. The BSCs estimated using the in situ calibration approach without the layer and with the layer and using the reference phantom approach with the layer were compared to the reference phantom approach without the layer present. The mean differences in BSCs were 0.15, 0.73, and -9.69 dB, respectively. The mean differences of the BSCs calculated from data blocks located at depths that were either 30 pulse lengths above or below the actual bead depth compared to the BSC calculated at bead depth were -1.55 and -1.48 dB, respectively. The results indicate that an in situ calibration target can account for overlaying tissue losses, thereby improving the robustness of BSC-based estimates.

AFM study: Cell cycle and probe geometry influences nanomechanical characterization of Panc1 cells.

Atomic force microscope (AFM) is emerging as an immensely promising tool to study the cellular morphology with a nanometer scale resolution and to analyze nanomechanical properties (NPs) at various physiological conditions. Advancement of AFM technology enables studying living cells and differentiating cancer cell from normal cells based on topography and NPs. Though the trend overlaps from different literature; numerical values of nanomechanical readouts depict variations over a wide range. These anomalies are associated with the experimental setup under study. In this manuscript, we have identified heterogeneity in cell culture system in addition to the selection of AFM probe with specific tip geometry as the major contributors to the above mentioned anomalies. To test our hypothesis, we have used Panc1 cells, which is a pancreatic ductal adenocarcinoma cell type. Our results suggest that the cellular morphology, membrane roughness and NPs calculated from AFM study are distinctly influenced by cell cycle. Furthermore, we found that the NPs readout is also significantly associated with AFM tip geometries. The cells were found to be softer in their early resting phase when indented with pyramidal probe and became increasingly stiffer as they progressed through the cell cycles. On the contrary, when indented with the spherical probe, cells in G0/G1 phase were observed to be the stiffest. Such an exhaustive study of the role of cell cycle in influencing the NPs in Panc1 cell line along with the impact of tip geometry on NPs is the first of its kind, to the best of our knowledge.

Canadians with visual impairment utilize home care services more frequently.

OBJECTIVE: To assess the impact of presenting visual impairment (PVI) on the utilization of formal home care services in Canada. Formal home care services were defined as services provided by professionals. Presenting visual impairment was self-reported impaired vision. DESIGN: Cross-sectional survey. PARTICIPANTS: Respondents to the Canadian Community Health Survey - Healthy Aging 2008/2009 (n = 30 865). METHODS: Self-reported utilization of formal home care services were compared by PVI status and sociodemographic groups. RESULTS: Overall, 23.9% of Canadians aged 45+ years with PVI utilized home care services versus 4.8% of those without (p < 0.0001). Canadians aged 65+ years (11.5%) used home care services more frequently than those aged 45-64 years (2.3%; p < 0.001). Among Canadians aged 65+ years, Caucasians (11.8%) were more frequent users than non-Caucasians (2.9%; p = 0.0002). Among Caucasians aged 65+ years, higher levels of utilization were documented in seniors with PVI (32.3%) versus those without (11.0%; p < 0.0001), females (14.5%) versus males (8.5%; p < 0.0001), seniors without a partner (18.6%) versus those with a partner (7.9%; p < 0.001), and seniors with incomes under the 20th percentile (14.7%) versus their peers with incomes ≥20th percentile (9.8%; p < 0.0001). Seniors with PVI were 30% more likely than those without to utilize home care services after adjusting for confounding effects (prevalence ratio = 1.3, 95% CI 1.1-2.5). Among nonusers of home care services, unmet needs for home care services were reported by 10.0% of Caucasian seniors with PVI versus 3.1% of those without PVI (p < 0.0001). CONCLUSIONS: Canadians with visual impairment are more likely to utilize formal home care services compared to those without.

Characterizing Fatty Liver in vivo in Rabbits, Using Quantitative Ultrasound.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease and can often lead to fibrosis, cirrhosis, cancer and complete liver failure. Liver biopsy is the current standard of care to quantify hepatic steatosis, but it comes with increased patient risk and only samples a small portion of the liver. Imaging approaches to assess NAFLD include proton density fat fraction estimated via magnetic resonance imaging (MRI) and shear wave elastography. However, MRI is expensive and shear wave elastography is not proven to be sensitive to fat content of the liver (Kramer et al. 2016). On the other hand, ultrasonic attenuation and the backscatter coefficient (BSC) have been observed to be sensitive to levels of fat in the liver (Lin et al. 2015; Paige et al. 2017). In this study, we assessed the use of attenuation and the BSC to quantify hepatic steatosis in vivo in a rabbit model of fatty liver. Rabbits were maintained on a high-fat diet for 0, 1, 2, 3 or 6 wk, with 3 rabbits per diet group (total N = 15). An array transducer (L9-4) with a center frequency of 4.5 MHz connected to a SonixOne scanner was used to gather radio frequency (RF) backscattered data in vivo from rabbits. The RF signals were used to estimate an average attenuation and BSC for each rabbit. Two approaches were used to parameterize the BSC (i.e., the effective scatterer diameter and effective acoustic concentration using a spherical Gaussian model and a model-free approach using a principal component analysis [PCA]). The 2 major components of the PCA from the BSCs, which captured 96% of the variance of the transformed data, were used to generate input features to a support vector machine for classification. Rabbits were separated into two liver fat-level classes, such that approximately half of the rabbits were in the low-lipid class (≤9% lipid liver level) and half of the rabbits in the high-lipid class (>9% lipid liver level). The slope and the midband fit of the attenuation coefficient provided statistically significant differences (p value = 0.00014 and p value = 0.007, using a two-sample t test) between low and high-lipid fat classes. The proposed model-free and model-based parameterization of the BSC and attenuation coefficient parameters yielded classification accuracies of 84.11 %, 82.93 % and 78.91 % for differentiating low-lipid versus high-lipid classes, respectively. The results suggest that attenuation and BSC analysis can differentiate low-fat versus high-fat livers in a rabbit model of fatty liver disease.

Self-perceived Impact of Glaucomatous Visual Field Loss and Visual Disabilities on Driving Difficulty and Cessation.

PURPOSE: To investigate if glaucoma severity and the presence of self-reported glare and difficulty with dark adaptation are associated with driving difficulty or cessation. PATIENTS AND METHODS: Individuals with glaucoma, age 50+ and visual acuity in the better eye ≥20/50 were included. Glaucoma severity was defined by the visual field mean deviation (MD) in the better eye and was classified into 2 groups: mild (MD>-6 dB) and moderate/severe (MD≤-6 dB). Patient responses to the glare and dark adaptation subscales in Glaucoma Quality of Life-15 questionnaire were used to measure relevant visual disability. Associations were assessed utilizing prevalence ratios (PR). RESULTS: A total of 99 participants (57% female) were included with 19% (19/99) reporting driving cessation. Patients with moderate/severe glaucoma when compared with mild glaucoma reported a significantly higher percentage of driving cessation (33% vs. 8%; P=0.002), presence of glare (27% vs. 6%; P=0.012), and difficulty with dark adaptation (31% vs. 10%; P=0.011).Individuals with self-perceived difficulty with dark adaptation were about 4 times more likely than those without to have difficulty driving at night (adjusted PR=3.94; P<0.0001) or in poor driving conditions (adjusted PR=4.09; P=0.0002). Self-reported glare was associated with an increased risk of driving difficulty in poor driving conditions (PR=4.17; P=0.05). CONCLUSIONS: Patients with moderate/severe glaucomatous visual field loss reported significantly higher percentage of driving cessation, presence of glare and difficulty with dark adaptation. Difficulty with dark adaptation was significantly associated with difficulty driving at night or in poor driving conditions. Further studies are needed to confirm these findings.

Bacterial Keratitis in Toronto: A 16-Year Review of the Microorganisms Isolated and the Resistance Patterns Observed.

PURPOSE: To review the incidence, distribution, current trends, and resistance patterns of bacterial keratitis isolates in Toronto over the past 16 years. METHODS: Microbiology records of suspected bacterial keratitis that underwent a diagnostic corneal scraping and cultures from January 1, 2000, through December 31, 2015, were retrospectively reviewed. The distribution of the main isolated pathogens and in vitro laboratory minimum inhibitory concentration testing results were used to identify resistance patterns. RESULTS: A total of 2330 corneal scrapings were taken over 16 years. A pathogen was recovered in 1335 samples (57.3%), with bacterial keratitis accounting for 1189 of the positive cultures (86.0% of all isolates). The total number of gram-positive and gram-negative isolates was 963 and 324, respectively. Coagulase-negative Staphylococcus and Pseudomonas aeruginosa were the most common gram-positive and gram-negative bacteria isolates, respectively. A decreasing trend in the number of isolates in gram-positive bacteria (P = 0.01), specifically among Staphylococcus aureus (P < 0.0001) and Streptococcus species (P = 0.005), was identified. When analyzing the susceptibilities of gram-positive and gram-negative isolates, an increasing trend in antibiotic resistance was observed in erythromycin (P = 0.018), ceftazidime (P = 0.046), and piperacillin/tazobactam (P = 0.005). The susceptibility of tested gram-positive microorganisms to vancomycin was 99.6%. CONCLUSIONS: There has been a decreasing trend in the number of isolates in gram-positive microorganisms over the past 16 years. An increasing trend in resistance for various antibiotics against gram-negative and gram-positive isolates was identified. High susceptibility to vancomycin reinforced the empirical use of fortified tobramycin and vancomycin in the initial management of severe bacterial keratitis.

Latanoprost Stimulates Ocular Lymphatic Drainage: An In Vivo Nanotracer Study.

PURPOSE: Ocular lymphatics have been recently shown to contribute to aqueous humor outflow. It is not yet known whether lymphatic outflow can be stimulated by pharmacological agents. Here we determine whether latanoprost, a prostaglandin F2 alpha analog commonly used to lower IOP to treat glaucoma, increases lymphatic drainage from the eye. METHODS: Lymphatic drainage in mice was assessed in vivo, in 11 latanoprost-treated and 11 control animals using hyperspectral imaging at multiple times following quantum dot (QD) injection into the eye. QD signal intensity was also measured in tissue sections using hyperspectral imaging. RESULTS: In the latanoprost-treated group, lymphatic drainage rate into the submandibular lymph node was increased compared with controls (1.23 ± 1.06 hours-1 vs. 0.30 ± 0.17 hours-1, mean ± SD, P < 0.02). Total QD signal intensity in the submandibular lymph node was greater in the latanoprost-treated group compared with controls (10.55 ± 1.12 vs. 9.48 ± 1.24, log scale, P < 0.05). CONCLUSIONS: This is the first evidence that latanoprost increases lymphatic drainage from the eye. The pharmacological manipulation of this newly identified lymphatic outflow pathway may be relevant to treatments aimed at lowering intraocular pressure in glaucoma. TRANSLATIONAL RELEVANCE: This is the first evidence that a prostaglandin drug widely prescribed for glaucoma, enhances lymphatic drainage from the eye. The pharmacological stimulation of this newly identified outflow pathway may be highly relevant to treatments aimed at lowering IOP to prevent blindness from glaucoma.