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Background: Retinitis pigmentosa (RP) is a genetically heterogeneous group of degenerative disorders causing progressive vision loss due to photoreceptor death. RP affects other retinal cells, including the retinal pigment epithelium (RPE). MicroRNAs (miRs) are implicated in RP pathogenesis, and downregulating miR-181a/b has shown therapeutic benefit in RP mouse models by improving mitochondrial function. This study investigates the expression profile of miR-181a/b in RPE cells and the neural retina during RP disease progression. We also evaluate how miR-181a/b downregulation, by knocking out miR-181a/b-1 cluster in RPE cells, confers therapeutic efficacy in an RP mouse model and explore the mechanisms underlying this process. Results: Our findings reveal distinct expression profiles, with downregulated miR-181a/b in RPE cells suggesting a protective response and upregulated miR-181a/b in the neural retina indicating a role in disease progression. We found that miR-181a/b-2, encoded in a separate genomic cluster, compensates for miR-181a/b-1 ablation in RPE cells at late time points. The transient downregulation of miR-181a/b in RPE cells at post-natal week 6 (PW6) led to improved RPE morphology, retarded photoreceptor degeneration and decreased RPE aerobic glycolysis. Conclusions: Our study elucidates the underlying mechanisms associated with the therapeutic modulation of miR-181a/b, providing insights into the metabolic processes linked to its RPE-specific downregulation. Our data further highlights the impact of compensatory regulation between miR clusters with implications for the development of miR-based therapeutics. [ABSTRACT FROM AUTHOR]

Purpose To develop a universal gene therapy to overcome the genetic heterogeneity in retinitis pigmentosa (RP) resulting from mutations in rhodopsin ( RHO ). Design Experimental study for a combination gene therapy that uses both gene ablation and gene replacement. Participants This study included 2 kinds of human RHO mutation knock-in mouse models: Rho P23H and Rho D190N . In total, 23 Rho P23H/P23H , 43 Rho P23H/+ , and 31 Rho D190N/+ mice were used for analysis. Methods This study involved gene therapy using dual adeno-associated viruses (AAVs) that (1) destroy expression of the endogenous Rho gene in a mutation-independent manner via an improved clustered regularly interspaced short palindromic repeats-based gene deletion and (2) enable expression of wild-type protein via exogenous cDNA. Main Outcome Measures Electroretinographic and histologic analysis. Results The thickness of the outer nuclear layer (ONL) after the subretinal injection of combination ablate-and-replace gene therapy was approximately 17% to 36% more than the ONL thickness resulting from gene replacement-only therapy at 3 months after AAV injection. Furthermore, electroretinography results demonstrated that the a and b waves of both Rho P23H and Rho D190N disease models were preserved more significantly using ablate-and-replace gene therapy ( P < 0.001), but not by gene replacement monotherapy. Conclusions As a proof of concept, our results suggest that the ablate-and-replace strategy can ameliorate disease progression as measured by photoreceptor structure and function for both of the human mutation knock-in models. These results demonstrate the potency of the ablate-and-replace strategy to treat RP caused by different Rho mutations. Furthermore, because ablate-and-replace treatment is mutation independent, this strategy may be used to treat a wide array of dominant diseases in ophthalmology and other fields. Clinical trials using ablate-and-replace gene therapy would allow researchers to determine if this strategy provides any benefits for patients with diseases of interest. [ABSTRACT FROM AUTHOR]

Defects in Membrane Frizzled-related Protein (MFRP) cause autosomal recessive retinitis pigmentosa (RP). MFRP codes for a retinal pigment epithelium (RPE)-specific membrane receptor of unknown function. In patient-specific induced pluripotent stem (iPS)-derived RPE cells, precise levels of MFRP, and its dicistronic partner CTRP5, are critical to the regulation of actin organization. Overexpression of CTRP5 in naïve human RPE cells phenocopied behavior of MFRP-deficient patient RPE (iPS-RPE) cells. AAV8 (Y733F) vector expressing human MFRP rescued the actin disorganization phenotype and restored apical microvilli in patient-specific iPS-RPE cell lines. As a result, AAV-treated MFRP mutant iPS-RPE recovered pigmentation and transepithelial resistance. The efficacy of AAV-mediated gene therapy was also evaluated in Mfrprd6/Mfrprd6 mice-an established preclinical model of RP-and long-term improvement in visual function was observed in AAV-Mfrp-treated mice. This report is the first to indicate the successful use of human iPS-RPE cells as a recipient for gene therapy. The observed favorable response to gene therapy in both patient-specific cell lines, and the Mfrprd6/Mfrprd6 preclinical model suggests that this form of degeneration caused by MFRP mutations is a potential target for interventional trials. [ABSTRACT FROM AUTHOR]

Consumer demand for use of fewer traditional antimicrobial agents in foods has driven research interest in development of plant based antimicrobial agents for use in food and food processing. The purpose of the present study was to investigate Ib-AMP1, a plant antimicrobial peptide (pAMP), isolated from seeds of Impatiens balsamina. Activity against foodborne pathogens, cytotoxicity to select human cells, and residual activity were investigated. Results of these experiments aid in determining the feasibility of using Ib-AMP1 as an antimicrobial agent to control foodborne pathogens. Ib-AMP1 exhibited bactericidal activity against Staphylococcus aureus, Escherichia coli O157:H7, Salmonella enteric serovar Newport, Pseudomonas aeruginosa, and Bacillus cereus. When tested using low (103 CFU mL−1) and intermediate (106 CFU mL−1) E. coli O157:H7 cell numbers, an approximately 1.46–2.69log reduction in cell numbers occurred at the 1× and 2× minimum inhibitory concentration (MIC) of Ib-AMP1. The results suggest that a concentration of Ib-AMP1 several fold greater than the MIC would be required in foods with high levels of commensal bacteria. A separate experiment showed no residual activity of Ib-AMP1 was apparent following interaction of the peptide with bacteria. Results of the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] cell proliferation assay indicated that Ib-AMP1 at 200, 400 and 600 μg mL−1 inhibited by 50% cell proliferation activity of Hep G2, FHs 74 Int and HT29 cells, respectively. Taken together, these data suggest that Ib-AMP1 has potential application as an antimicrobial agent in food systems. [ABSTRACT FROM AUTHOR]

Purpose: Consuming even low numbers of the foodborne pathogen, Listeria monocytogenes, places the elderly at risk for severe illness. the impact of immunomodulation on the development of listerial infection within a young and aged population after low dose challenge with L. monocytogenes was investigated. Methods: Animals received daily supplementation of vitamin E for a period of 21 days to promote immunomodulation, and were then orally challenged with 100 CFU of L. monocytogenes. Levels of CD8+, CD4+ and CD3+ T cells were used as markers to determine the influence of daily supplementation with vitamin E on immune response; the spleen and liver were harvested for microbiological analysis. Results: Higher numbers of animals became infected in control groups than in vitamin Etreated group. During the post-challenge period, vitamin E-treated aged animals showed faster CD8+ T cell proliferation than control aged animals. Conclusion: Daily supplementation with vitamin E was more benefcial in young compared with aged animals in mitigating listerial infection. Results suggest exposure to even low numbers of L. monocytogenes can result in infection in both healthy young and aged populations. [ABSTRACT FROM AUTHOR]

Purpose: Consuming even low numbers of the foodborne pathogen, Listeria monocytogenes, places the elderly at risk for severe illness. The impact of immunomodulation on the development of listerial infection within a young and aged population after low dose challenge with L. monocytogenes was investigated.Methods: Animals received daily supplementation of vitamin E for a period of 21 days to promote immunomodulation, and were then orally challenged with 100 CFU of L. monocytogenes. Levels of CD8+, CD4+ and CD3+ T cells were used as markers to determine the influence of daily supplementation with vitamin E on immune response; the spleen and liver were harvested for microbiological analysis.Results: Higher numbers of animals became infected in control groups than in vitamin E-treated group. During the post-challenge period, vitamin E-treated aged animals showed faster CD8+ T cell proliferation than control aged animals.Conclusion: Daily supplementation with vitamin E was more beneficial in young compared with aged animals in mitigating listerial infection. Results suggest exposure to even low numbers of L. monocytogenes can result in infection in both healthy young and aged populations. [ABSTRACT FROM AUTHOR]

Macular degeneration (MD) is characterized by the progressive deterioration of the macula and represents one of the most prevalent causes of blindness worldwide. Abnormal intracellular accumulation of lipid droplets and pericellular deposits of lipid-rich material in the retinal pigment epithelium (RPE) called drusen are clinical hallmarks of different forms of MD including Doyne honeycomb retinal dystrophy (DHRD) and age-related MD (AMD). However, the appropriate molecular therapeutic target underlying these disorder phenotypes remains elusive. Here, we address this knowledge gap by comparing the proteomic profiles of induced pluripotent stem cell (iPSC)-derived RPEs (iRPE) from individuals with DHRD and their isogenic controls. Our analysis and follow-up studies elucidated the mechanism of lipid accumulation in DHRD iRPE cells. Specifically, we detected significant downregulation of carboxylesterase 1 (CES1), an enzyme that converts cholesteryl ester to free cholesterol, an indispensable process in cholesterol export. CES1 knockdown or overexpression of EFEMP1 R345W , a variant of EGF-containing fibulin extracellular matrix protein 1 that is associated with DHRD and attenuated cholesterol efflux and led to lipid droplet accumulation. In iRPE cells, we also found that EFEMP1R345W has a hyper-inhibitory effect on epidermal growth factor receptor (EGFR) signaling when compared to EFEMP1WT and may suppress CES1 expression via the downregulation of transcription factor SP1. Taken together, these results highlight the homeostatic role of cholesterol efflux in iRPE cells and identify CES1 as a mediator of cholesterol efflux in MD. [ABSTRACT FROM AUTHOR]

D190N, a missense mutation in rhodopsin, causes photoreceptor degeneration in patients with autosomal dominant retinitis pigmentosa (adRP). Two competing hypotheses have been developed to explain why D190N rod photoreceptors degenerate: (a) defective rhodopsin trafficking prevents proteins from correctly exiting the endoplasmic reticulum, leading to their accumulation, with deleterious effects or (b) elevated mutant rhodopsin expression and unabated signaling causes excitotoxicity. A knock-in D190N mouse model was engineered to delineate the mechanism of pathogenesis. Wild type (wt) and mutant rhodopsin appeared correctly localized in rod outer segments of D190N heterozygotes. Moreover, the rhodopsin glycosylation state in the mutants appeared similar to that in wt mice. Thus, it seems plausible that the injurious effect of the heterozygous mutation is not related to mistrafficking of the protein, but rather from constitutive rhodopsin activity and a greater propensity for chromophore isomerization even in the absence of light. [ABSTRACT FROM AUTHOR]

Retinitis pigmentosa (RP) encompasses a diverse group of Mendelian disorders leading to progressive degeneration of rods and then cones. For reasons that remain unclear, diseased RP photoreceptors begin to deteriorate, eventually leading to cell death and, consequently, loss of vision. Here, we have hypothesized that RP associated with mutations in phosphodiesterase-6 (PDE6) provokes a metabolic aberration in rod cells that promotes the pathological consequences of elevated cGMP and Ca2+, which are induced by the Pde6 mutation. Inhibition of sirtuin 6 (SIRT6), a histone deacetylase repressor of glycolytic flux, reprogrammed rods into perpetual glycolysis, thereby driving the accumulation of biosynthetic intermediates, improving outer segment (OS) length, enhancing photoreceptor survival, and preserving vision. In mouse retinae lacking Sirt6, effectors of glycolytic flux were dramatically increased, leading to upregulation of key intermediates in glycolysis, TCA cycle, and glutaminolysis. Both transgenic and AAV2/8 gene therapy-mediated ablation of Sirt6 in rods provided electrophysiological and anatomic rescue of both rod and cone photoreceptors in a preclinical model of RP. Due to the extensive network of downstream effectors of Sirt6, this study motivates further research into the role that these pathways play in retinal degeneration. Because reprogramming metabolism by enhancing glycolysis is not gene specific, this strategy may be applicable to a wide range of neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Background and Aims Acute exacerbation ( AE) of chronic hepatitis B virus ( HBV) infection is common and negatively impacts the clinical outcome. Although upsurge of viral load always precedes or coincides with AE, the underlying immunological mechanisms remain unclear and were investigated. Methods We prospectively followed the serum cytokine/chemokine profiles, viral load, and alanine aminotransferase ( ALT) levels in 250 patients and identified 44 consecutive patients (male: 72.7%; age: 40.4 ± 9.7 years; hepatitis B e antigen [ HBeAg] positivity: 63.6%; genotype B/ C: 75%/25%) who developed AE during the follow-up in a medical center. The impact of clinical characteristics (age, gender, HBeAg, ALT, HBV genotype), cytokines (tumor necrosis factor-alpha, interferon gamma, interleukin [ IL]-2, IL-4, IL-6, and IL-10), and chemokines ( CXCL10/interferon gamma-induced protein [ IP]-10, CCL2/ MCP-1, CXCL9/ MIG, CCL5/ RANTES, and CXCL8/ IL-8) on the serum HBV DNA dynamics at different time points (baseline, peak of serum HBV DNA level, peak of serum ALT level, and after AE) were analyzed. Results Of 44 patients, serum HBV DNA level surged before the peak of serum ALT level in 23 (52.3%), and coincided with the peak of ALT in 21 (47.7%). The upsurge of serum viral load significantly correlated with the increase of IL-10 ( P = 0.0037) and CXCL10/ IP-10 ( P = 0.0044). Upsurge of serum viral load was preceded by an increase in serum IL-4 ( P < 0.05), IL-6 ( P < 0.05), and IL-10 ( P < 0.05). Combination of HBV genotype, IL-6 level at baseline, and ALT level at the peak of serum HBV DNA reliably predicted subsequent AE pattern ( P = 0.0116). Conclusions Enhanced Th2 activity is likely involved in the surge of HBV DNA level before hepatitis exacerbation. [ABSTRACT FROM AUTHOR]

Purpose: The objective of this study was to determine whether cells from the conjunctiva could be reprogrammed into induced pluripotent stem (iPS) cells, providing an alternative source of stem cells. Methods: We employed a doxycycline-induced reprogrammable mouse strain to generate iPS cells from conjunctiva. The identity of the stem cells was confirmed by reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence assays. Immunocytochemistry and teratoma assays are established means for scoring stem cell pluripotency. The reprogramming efficiencies of conjunctival cells and ear fibroblasts were compared. Results: We confirmed the identity of the stem cells and demonstrated expression of pluripotency markers (OCT4, SOX2, NANOG, and SSEA1), as tested by RT-PCR and immunofluorescence assays. In addition, derived iPS cells differentiated successfully into embryoid bodies, and showed teratoma formation when injected into immunodeficient mice. Reprogramming conjunctival tissue is as efficient as reprogramming ear fibroblasts. Conjunctiva-iPS exhibited classic features of embryonic stem (ES) cells with respect to morphology, expression of surface antigens, and pluripotency-associated transcription factors, capacity to differentiate in vitro, and the ability to form all three germ layers in vivo. Conclusion: The present study demonstrated that conjunctival cells, which are readily obtained during the course of many routine conjunctival biopsies and ophthalmic procedures, can be another reliable source of iPS cells. [ABSTRACT FROM AUTHOR]

The limited symptom relief and side effects of current Alzheimer’s disease (AD) medications warrant urgent discovery and study of new anti-AD agents. The “cholinergic hypothesis” of AD prompts us to search for plant-derived acetylcholineesterase (AChE) inhibitors such as galanthamine that has been licensed in Europe for AD treatment. We used the unique amyloid β-expressing transgenic C. elegans CL4176, which exhibits paralysis when human Aβ1–42 is induced, to study two natural benzylphenethylamine alkaloids isolated from Lycoris radiata (L’ Her.) Herb, galanthamine and haemanthidine, and their synthetic derivatives 1,2-Di-O-acetyllycorine and 1-O-acetyllycorine for their anti-paralysis effects. Our data indicate that these Lycoris compounds effectively delay the paralysis of CL4176 worms upon temperature up-shift, and prolong the lives of these transgenic worms. Lycoris compounds were shown to significantly inhibit the gene expression of ace-1 and ace-2. Additionally, the Lycoris compounds may modulate inflammatory and stress-related gene expressions to combat the Aβ-toxicity in C. elegans. [ABSTRACT FROM AUTHOR]

Ferulic acid (FA) is an antioxidant and photoprotectiveagent usedin biomedical and cosmetic formulations to prevent skin cancer andsenescence. Although FA exhibits numerous health benefits, physicochemicalinstability leading to decomposition hinders its efficacy. To minimizeinherent decomposition, a FA-containing biodegradable polymer wasprepared via solution polymerization to chemically incorporate FAinto a poly(anhydride-ester). The polymer was characterized usingnuclear magnetic resonance and infrared spectroscopies. The molecularweight and thermal properties were also determined. In vitro studiesdemonstrated that the polymer was hydrolytically degradable, thusproviding controlled release of the chemically incorporated bioactivewith no detectable decomposition. The polymer degradation productswere found to exhibit antioxidant and antibacterial activity comparableto that of free FA, and in vitro cell viability studies demonstratedthat the polymer is noncytotoxic toward fibroblasts. This rendersthe polymer a potential candidate for use as a controlled releasesystem for skin care formulations. [ABSTRACT FROM AUTHOR]

The U.S. Food and Drug Administration recently approved phase I/II clinical trials for embryonic stem (ES) cell-based retinal pigmented epithelium (RPE) transplantation, but this allograft transplantation requires lifelong immunosuppressive therapy. Autografts from patient-specific induced pluripotent stem (iPS) cells offer an alternative solution to this problem. However, more data are required to establish the safety and efficacy of iPS transplantation in animal models before moving iPS therapy into clinical trials. This study examines the efficacy of iPS transplantation in restoring functional vision in Rpe65rd12/Rpe65rd12 mice, a clinically relevant model of retinitis pigmentosa (RP). Human iPS cells were differentiated into morphologically and functionally RPE-like tissue. Quantitative real-time polymerase chain reaction (RT-PCR) and immunoblots confirmed RPE fate. The iPS-derived RPE cells were injected into the subretinal space of Rpe65rd12/Rpe65rd12 mice at 2 d postnatally. After transplantation, the long-term surviving iPS-derived RPE graft colocalized with the host native RPE cells and assimilated into the host retina without disruption. None of the mice receiving transplants developed tumors over their lifetimes. Furthermore, electroretinogram, a standard method for measuring efficacy in human trials, demonstrated improved visual function in recipients over the lifetime of this RP mouse model. Our study provides the first direct evidence of functional recovery in a clinically relevant model of retinal degeneration using iPS transplantation and supports the feasibility of autologous iPS cell transplantation for retinal and macular degenerations featuring significant RPE loss. [ABSTRACT FROM AUTHOR]

Cold rollings of A1008 steel specimens are carried out in this study to analyze the influences of roll’s surface pattern and specimen’s reduction ratio on surface morphologies, optical properties, and mechanical properties of rolled samples. A 3D fractal formula, along with dedicated algorithms developed, is applied to solve the periodic (characteristics) lengths and fractal dimensions for roll and rolled surfaces along and perpendicular to the rolling directions. The correlation for every fractal parameter between roll and the rolled specimen is developed to be a function of roll’s surface pattern and reduction ratio first. The light tracking simulations were then carried out for rolled samples only. Rockwell hardness and tensile tests were conducted to evaluate the specimen’s hardness and Young’s modulus. A roll having a higher fractal dimension, or a longer periodic length, can result in a higher fractal dimension and a longer periodic length of the rolled specimen, respectively. A larger reduction ratio also results in the rolled surface with higher fractal dimensions and longer periodic lengths. Increasing the fractal dimensions of a rolled specimen can elevate the maximum illuminance and, conversely, lower the minimum illuminance, while an increase in periodic lengths can increase the minimum illuminance and decrease the maximum illuminance. A decrease in the fractal dimensions and/or increase in the periodic lengths of rolled surfaces in the two directions can raise the illuminance uniformity. For the specimens prepared with the same roll pattern, an increase in reduction ratio can bring an increment in illuminance uniformity and hardness of rolled samples. This study provides an efficient way to determine the roll pattern and reduction ratio that satisfies the specific demands in optical properties. [ABSTRACT FROM AUTHOR]