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Find video protocols related to scientific articles indexed in Pubmed.
High submuscular versus space of Retzius placement of inflatable penile prosthesis reservoirs: results of a surgeon survey.
Can J Urol
PUBLISHED: 10-28-2014
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High submuscular (HSM) inflatable penile prosthesis (IPP) reservoir insertion is a new technique that involves placing the reservoir high beneath the muscles of the abdominal wall. We queried a variety of surgeons to assess their impressions of how HSM reservoir placement compares with traditional space of Retzius (SOR) placement.
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Dilemmas Families Face in Talking With Returning U.S. Military Service Members About Seeking Professional Help for Mental Health Issues.
Health Commun
PUBLISHED: 08-30-2014
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Drawing on Goldsmith's (2004) normative theory, this article maps dilemmas family members experience when talking with returning service members (SMs) about seeking mental health care. Eighty family members of United States SMs who served in Iraq or Afghanistan read a scenario where their SM was displaying posttraumatic stress disorder (PTSD) or depression symptoms. Participants described goals they would pursue, barriers they might encounter, and advice they would give others in the situation. Four dilemmas of talking about mental health emerged: (a) getting you to recognize the problem without implying you're not normal, (b) convincing you to seek help without implying you're weak, (c) being persistent but patient, and (d) wanting you to open up without implying I can understand. Family members reported using four groups of strategies to manage these dilemmas. Directions for expanding the concept of dilemmas as "paradoxes" and for supporting military families as well as rethinking policy assumptions are discussed.
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Screening of refractive surgery candidates for LASIK and PRK.
Cornea
PUBLISHED: 07-12-2014
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The aim of this study was to evaluate exclusion criteria in screening patients for refractive surgery.
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RNA interference-mediated knockdown of long-form phosphodiesterase-4D (PDE4D) enzyme reverses amyloid-?42-induced memory deficits in mice.
J. Alzheimers Dis.
PUBLISHED: 06-28-2014
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Phosphodiesterase-4 (PDE4) inhibitors enhance memory, increase hippocampal neurogenesis, and reverse amyloid-? (A?)-induced memory deficits. Here, we examined whether long-form PDE4D knockdown by lentiviral RNA construct containing a specific microRNA/miRNA-mir hairpin structure (4DmiRNA) reversed memory impairment caused by amyloid-?1-42 (A?42) in mice using the Morris water maze (MWM) and novelty object recognition tests. Western blotting analysis was used to assess protein levels of cAMP response element-binding protein (CREB, unphosphorylated and phosphorylated [pCREB]), brain-derived neurotrophic factor (BDNF), interleukin-1? (IL-1?), tumor necrosis factor-? (TNF-?), and nuclear factor-?B (NF-?B) to explore the neurochemical mechanisms. Aggregated A?42 (0.5 ?g/side) bilaterally infused in dentate gyrus decreased cAMP levels (p < 0.01) and produced memory deficits in the MWM (p < 0.01) and object recognition tests (p < 0.01). Microinfusions of lentiviruses resulted in downregulated expression of PDE4D4 and 4D5 proteins and reversed A?42-induced cAMP decline (p < 0.05) and memory deficits. Treatment also concomitantly increased pCREB (p < 0.05) and BDNF (p < 0.01) and reduced IL-1? (p < 0.05), TNF-? (p < 0.01), and NF-?B (p65) (p < 0.05) in the hippocampus of A?42-challenged mice. These results suggest that long-form PDE4D knockdown may offer a promising treatment for memory loss associated with Alzheimer's disease.
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Use of (18)F-fluorodeoxyglucose positron emission tomography-computed tomography to aid in diagnosing intestinal adenocarcinoma in 2 rhesus macaques (Macaca mulatta).
Comp. Med.
PUBLISHED: 06-24-2014
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Two aged female rhesus macaques (Macaca mulatta) presented with weight loss and intermittent inappetence. The signalment and constellation of clinical signs led clinicians to suspect the presence of intestinal adenocarcinoma. Because of each animal's advanced age and inconclusive radiographic findings, a noninvasive diagnostic tool was preferred over exploratory laparotomy to assist in determining a diagnosis. Consequently, 2-[(18)F]fluoro-2-deoxy-d-glucose (FDG) positron emission tomography-CT (FDG-PET-CT) was chosen to aid in confirming a suspicion of gastrointestinal adenocarcinoma in both animals. FDG is a glucose analogue labeled with fluorine-18 and is taken up by highly metabolically active cells, as observed in many cancers. Tomography revealed an annular constriction of the small intestine with focal FDG uptake in one animal, and an FDG avid transmural mass in the ascending colon of the second animal. Necropsy later confirmed both sites to be adenocarcinomas. This report supports the use of FDG-PET-CT as an adjunct to conventional radiography in the diagnosis of intestinal adenocarcinoma in nonhuman primates.
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Highly automated nano-LC/MS-based approach for thousand cell-scale quantification of side chain-hydroxylated oxysterols.
J. Lipid Res.
PUBLISHED: 05-02-2014
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Iso-octyl chain-hydroxylated oxysterols were determined in attomoles per 10,000 cells concentrations in 10,000-80,000 cultured pancreatic adenocarcinoma cells, using a sensitive, highly automated nano-LC-ESI-MS-based method. Identified oxysterols included 24S hydroxycholesterol (24S-OHC), 25 hydroxycholesterol (25-OHC), and 27 hydroxycholesterol (27-OHC), while 20S hydroxycholesterol and 22S hydroxycholesterol were not detected. Lower mass limit of quantification was 23 fg (65 amol) for 25-OHC and 27-OHC (100 times lower than our previous method) and 54 fg (135 amol) for 24S-OHC, after derivatization into Girard T hydrazones and online sample cleanup using simplified and robust automatic filtration and filter back flushing solid phase extraction LC/MS/MS. The instrument configuration was easily installed using a commercial nano-LC/MS system. Recoveries in spiked sample were 96, 97, and 77% for 24S-OHC, 25-OHC, and 27-OHC, with within- and between-day repeatabilities of 1-21% and 2-20% relative SD, respectively. The study demonstrates the potential of nano-LC in lipidomics/sterolomics.
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Cellular and extracellular matrix modulation of corneal stromal opacity.
Exp. Eye Res.
PUBLISHED: 04-29-2014
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Stromal transparency is a critical factor contributing to normal function of the visual system. Corneal injury, surgery, disease and infection elicit complex wound healing responses that serve to protect against insults and maintain the integrity of the cornea, and subsequently to restore corneal structure and transparency. However, in some cases these processes result in prolonged loss of corneal transparency and resulting diminished vision. Corneal opacity is mediated by the complex actions of many cytokines, growth factors, and chemokines produced by the epithelial cells, stromal cells, bone marrow-derived cells, lacrimal tissues, and nerves. Myofibroblasts, and the disorganized extracellular matrix produced by these cells, are critical determinants of the level and persistence of stromal opacity after corneal injury. Decreases in corneal crystallins in myofibroblasts and corneal fibroblasts contribute to cellular opacity in the stroma. Regeneration of a fully functional epithelial basement membrane (BM) appears to have a critical role in the maintenance of corneal stromal transparency after mild injuries and recovery of transparency when opacity is generated after severe injuries. The epithelial BM likely has a regulatory function whereby it modulates epithelium-derived growth factors such as transforming growth factor (TGF) ? and platelet-derived growth factor (PDGF) that drive the development and persistence of myofibroblasts from precursor cells. The purpose of this article is to review the factors involved in the maintenance of corneal transparency and to highlight the mechanisms involved in the appearance, persistency and regression of corneal opacity after stromal injury.
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Changes in custom biomechanical variables after femtosecond laser in situ keratomileusis and photorefractive keratectomy for myopia.
J Cataract Refract Surg
PUBLISHED: 04-13-2014
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To analyze changes in new biomechanical descriptors with myopic femtosecond laser-assisted laser in situ keratomileusis (LASIK), compare them with the biomechanical response after photorefractive keratectomy (PRK) with similar levels of myopic ablation, and evaluate correlations between changes in custom variables and biomechanically relevant variables.
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Topical cyclosporine a treatment in corneal refractive surgery and patients with dry eye.
J Refract Surg
PUBLISHED: 04-10-2014
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To evaluate preoperative and postoperative dry eye and the effect of cyclosporine A treatment in patients screened for corneal refractive surgery and treated with photorefractive keratectomy (PRK) or LASIK.
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BAC-EDTA transepithelial riboflavin-UVA crosslinking has greater biomechanical stiffening effect than standard epithelium-off in rabbit corneas.
Exp. Eye Res.
PUBLISHED: 03-26-2014
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Studies suggest that standard corneal collagen crosslinking (CXL) is a safe and effective treatment to stiffen the cornea for keratoconus and other ectatic corneal disorders. The purpose of the present study was to compare the biomechanical effects of transepithelial benzalkonium chloride-EDTA (BAC-EDTA) riboflavin-UVA crosslinking to standard epithelium-off riboflavin-UVA crosslinking in a rabbit model. Corneal stiffness was quantified using optical coherence elastography at two months after treatment. The mean lateral-to-axial displacement ratio for the BAC-EDTA transepithelial CXL group was lower (greater stiffness) [0.062 ± 0.042, mean ± SD] than epithelium-off CXL (mean ± SD: 0.065 ± 0.045) or untreated control eyes (0.069 ± 0.044). Using ANOVA with Tukey correction, a statistically significant difference was found between the BAC-EDTA transepithelial CXL group and standard epithelium-off CXL (p = 0.0019) or the untreated control (p < 0.0001) groups. A graph of the probability density functions for biomechanical stiffness also showed a greater shift in stiffening in the BAC-EDTA transepithelial CXL group than the standard epithelium-off CXL or untreated control group. These results demonstrated that the biomechanical stiffening effect produced by BAC-EDTA transepithelial CXL was greater than that produced by standard epithelium-off CXL in a rabbit model.
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Association between the percent tissue altered and post-laser in situ keratomileusis ectasia in eyes with normal preoperative topography.
Am. J. Ophthalmol.
PUBLISHED: 02-05-2014
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To investigate the association of a novel metric, percent tissue altered, with the occurrence of ectasia after laser in situ keratomileusis (LASIK) in eyes with normal corneal topography and to compare this metric with other recognized risk factors.
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Designer receptors show role for ventral pallidum input to ventral tegmental area in cocaine seeking.
Nat. Neurosci.
PUBLISHED: 01-28-2014
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The ventral pallidum is centrally positioned within mesocorticolimbic reward circuits, and its dense projection to the ventral tegmental area (VTA) regulates neuronal activity there. However, the ventral pallidum is a heterogeneous structure, and how this complexity affects its role within wider reward circuits is unclear. We found that projections to VTA from the rostral ventral pallidum (RVP), but not the caudal ventral pallidum (CVP), were robustly Fos activated during cue-induced reinstatement of cocaine seeking--a rat model of relapse in addiction. Moreover, designer receptor-mediated transient inactivation of RVP neurons, their terminals in VTA or functional connectivity between RVP and VTA dopamine neurons blocked the ability of drug-associated cues (but not a cocaine prime) to reinstate cocaine seeking. In contrast, CVP neuronal inhibition blocked cocaine-primed, but not cue-induced, reinstatement. This double dissociation in ventral pallidum subregional roles in drug seeking is likely to be important for understanding the mesocorticolimbic circuits underlying reward seeking and addiction.
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Dietary restriction reverses obesity-induced anhedonia.
Physiol. Behav.
PUBLISHED: 01-22-2014
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Obesity-induced changes in the metabolic and endocrine milieu elicit deficits in neuroplasticity, including increased risk for development of neuropsychiatric disorders such as depressive illness. We previously demonstrated that downregulation of hypothalamic insulin receptors (hypo-IRAS) elicits a phenotype that is consistent with features of the metabolic syndrome (MetS) and that rats with this phenotype exhibit deficits in neuronal plasticity, including depressive-like behaviors such as anhedonia. Since food restriction paradigms effectively inhibit obesity-induced neuroplasticity deficits, the aim of the current study was to determine whether food restriction could reverse obesity-induced anhedonia in hypo-IRAS rats. Compared to hypo-IRAS rats provided ad lib food access, food restriction paradigms that were initiated either prior to increases in body weight or following development of the MetS/obesity phenotype effectively restored sucrose intake in hypo-IRAS rats. Moreover, food restriction paradigms were able to prevent and reverse the changes in the endocrine/metabolic/inflammatory milieu observed in hypo-IRAS, such as increases in plasma leptin and triglyceride levels and increases in pro-inflammatory cytokines such as IL-1?, IL-6 and C-reactive protein (CRP). Collectively, these results demonstrate that obesity-induced anhedonia is a reversible process and identify some potential mechanistic mediators that may be responsible for co-morbid depression in obesity.
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Confounders of mortality and hospitalization rate calculations for profit and nonprofit dialysis facilities: analytic augmentation.
BMC Nephrol
PUBLISHED: 01-21-2014
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Patient outcomes have been compared on the basis of the profit status of the dialysis provider (for-profit [FP] and not-for-profit [NFP]). In its annual report, United States Renal Data System (USRDS) provides dialysis provider level death and hospitalization rates adjusted by age, race, sex, and dialysis vintage; however, recent analyses have suggested that other variables impact these outcomes. Our current analysis of hospitalization and mortality rates of hemodialysis patients included adjustments for those used by the USRDS plus other potential confounders: facility geography (end-stage renal disease network), length of facility ownership, vascular access at first dialysis session, and pre-dialysis nephrology care.
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TGF? and PDGF-B signaling blockade inhibits myofibroblast development from both bone marrow-derived and keratocyte-derived precursor cells in vivo.
Exp. Eye Res.
PUBLISHED: 01-20-2014
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Myofibroblasts, the primary cells associated with corneal stromal haze (opacity), can be derived from both cornea-derived and bone marrow-derived precursor cells. In the present study, the role of TGF? or PDGF blockage on bone marrow-derived myofibroblast development was investigated using a green fluorescent protein (GFP) chimeric bone marrow mouse model and plasmid vectors that blocked TGF? or PDGF signaling. At the peak of corneal haze one month after irregular phototherapeutic keratectomy the central stroma had significantly less alpha-smooth muscle actin (?-SMA)-positive cells derived from GFP+ bone marrow-derived cells or GFP- keratocyte/corneal fibroblast-derived cells when corneas were treated with the TGF? blocking vector pGFPC1.TGFRBKDEL or the PDGF blocking vector pCMV.PDGFRB.23KDEL compared with the corresponding empty vector treated or untreated control groups. In individual animals, 30-60% of myofibroblasts were derived from bone marrow-derived precursor cells and 40-70% of myofibroblasts were derived from keratocyte-derived precursor cells. TGF? and PDGF regulate corneal myofibroblast development from bone marrow-derived precursor cells and keratocyte/corneal fibroblast-derived precursor cells.
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Metabolites in vertebrate Hedgehog signaling.
Biochem. Biophys. Res. Commun.
PUBLISHED: 01-15-2014
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The Hedgehog (HH) signaling pathway is critical in embryonic development, stem cell biology, tissue homeostasis, chemoattraction and synapse formation. Irregular HH signaling is associated with a number of disease conditions including congenital disorders and cancer. In particular, deregulation of HH signaling has been linked to skin, brain, lung, colon and pancreatic cancers. Key mediators of the HH signaling pathway are the 12-pass membrane protein Patched (PTC), the 7-pass membrane protein Smoothened (SMO) and the GLI transcription factors. PTC shares homology with the RND family of small-molecule transporters and it has been proposed that it interferes with SMO through metabolites. Although a conclusive picture is lacking, substantial efforts are made to identify and understand natural metabolites/sterols, including cholesterol, vitamin D3, oxysterols and glucocorticoides, that may be affected by, or influence the HH signaling cascade at the level of PTC and SMO. In this review we will elaborate the role of metabolites in HH signaling with a focus on oxysterols, and discuss advancements in modern analytical approaches in the field.
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Transforming growth factor ? and platelet-derived growth factor modulation of myofibroblast development from corneal fibroblasts in vitro.
Exp. Eye Res.
PUBLISHED: 01-04-2014
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The purpose of this study was to test the hypotheses that development of mature vimentin+/?-smooth muscle actin+/desmin+ (V+A+D+) myofibroblasts from corneal fibroblasts is regulated by transforming growth factor (TGF) ? and platelet-derived growth factor (PDGF); and that myofibroblast development in vitro follows a similar developmental pathway as it does in vivo. Mouse corneal stromal fibroblasts (MSF) were isolated from the corneas of Swiss Webster mice and cultured in serum-free media augmented with DMEM/F12 and varying doses of TGF? (0.1-2.0 ng/ml), with and without mouse PDGF-AA and/or PDGF-BB (2.0 ng/ml), to study the transition of the MSF to V+A+D+ myofibroblasts. The mean percentage of vimentin+, ?-SMA+ and desmin+ cells was determined at each time point (2-15 days), with each growth factor concentration. MSF in vitro were noted to undergo the same developmental transition from V+A-D- to V+A+D- to V+A+D+ myofibroblasts as precursors undergo in vivo. TGF? at a dose of 0.5 ng/ml and 1.0 ng/ml with 2.0 ng/ml PDGF-AA and 2.0 ng/ml PDGF-BB in DMEM/F12 serum-free media was optimal for the development of V+A+D+ myofibroblasts. This study defines optimal in vitro conditions to monitor the development of MSF into myofibroblasts. The combined effects of TGF? and PDGF promote the full development of V+A+D+ myofibroblasts from MSF.
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Open tubular lab-on-column/mass spectrometry for targeted proteomics of nanogram sample amounts.
PLoS ONE
PUBLISHED: 01-01-2014
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A novel open tubular nanoproteomic platform featuring accelerated on-line protein digestion and high-resolution nano liquid chromatography mass spectrometry (LC-MS) has been developed. The platform features very narrow open tubular columns, and is hence particularly suited for limited sample amounts. For enzymatic digestion of proteins, samples are passed through a 20 µm inner diameter (ID) trypsin + endoproteinase Lys-C immobilized open tubular enzyme reactor (OTER). Resulting peptides are subsequently trapped on a monolithic pre-column and transferred on-line to a 10 µm ID porous layer open tubular (PLOT) liquid chromatography LC separation column. Wnt/ß-catenein signaling pathway (Wnt-pathway) proteins of potentially diagnostic value were digested+detected in targeted-MS/MS mode in small cell samples and tumor tissues within 120 minutes. For example, a potential biomarker Axin1 was identifiable in just 10 ng of sample (protein extract of ?1,000 HCT15 colon cancer cells). In comprehensive mode, the current OTER-PLOT set-up could be used to identify approximately 1500 proteins in HCT15 cells using a relatively short digestion+detection cycle (240 minutes), outperforming previously reported on-line digestion/separation systems. The platform is fully automated utilizing common commercial instrumentation and parts, while the reactor and columns are simple to produce and have low carry-over. These initial results point to automated solutions for fast and very sensitive MS based proteomics, especially for samples of limited size.
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Introduction to the special section on communication and wartime deployment.
Health Commun
PUBLISHED: 10-17-2013
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Over the past decade, the wars in Iraq and Afghanistan have taken a heavy toll on the physical, psychological, and relational health of military service members and their families. The articles included in this special section of Health Communication add to the robust, interdisciplinary body of research on the health consequences of wartime deployment by examining how communication enables the recovery process of service members and their families. Because communication processes can signal health problems, construct and promote family resiliency, and shape the content and delivery of health interventions, our disciplines theory and research can help inform ongoing efforts to support military families as the wars in Iraq and Afghanistan wind down.
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The corneal epithelial basement membrane: structure, function, and disease.
Invest. Ophthalmol. Vis. Sci.
PUBLISHED: 10-01-2013
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The corneal epithelial basement membrane (BM) is positioned between basal epithelial cells and the stroma. This highly specialized extracellular matrix functions not only to anchor epithelial cells to the stroma and provide scaffolding during embryonic development but also during migration, differentiation, and maintenance of the differentiated epithelial phenotype. Basement membranes are composed of a diverse assemblage of extracellular molecules, some of which are likely specific to the tissue where they function; but in general they are composed of four primary components--collagens, laminins, heparan sulfate proteoglycans, and nidogens--in addition to other components such as thrombospondin-1, matrilin-2, and matrilin-4 and even fibronectin in some BM. Many studies have focused on characterizing BM due to their potential roles in normal tissue function and disease, and these structures have been well characterized in many tissues. Comparatively few studies, however, have focused on the function of the epithelial BM in corneal physiology. Since the normal corneal stroma is avascular and has relatively low keratocyte density, it is expected that the corneal BM would be different from the BM in other tissues. One function that appears critical in homeostasis and wound healing is the barrier function to penetration of cytokines from the epithelium to stroma (such as transforming growth factor ?-1), and possibly from stroma to epithelium (such as keratinocyte growth factor). The corneal epithelial BM is also involved in many inherited and acquired corneal diseases. This review examines this structure in detail and discusses the importance of corneal epithelial BM in homeostasis, wound healing, and disease.
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Integrated enzyme reactor and high resolving chromatography in "sub-chip" dimensions for sensitive protein mass spectrometry.
Sci Rep
PUBLISHED: 09-25-2013
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Reliable, sensitive and automatable analytical methodology is of great value in e.g. cancer diagnostics. In this context, an on-line system for enzymatic cleavage of proteins, subsequent peptide separation by liquid chromatography (LC) with mass spectrometric detection has been developed using "sub-chip" columns (10-20??m inner diameter, ID). The system could detect attomole amounts of isolated cancer biomarker progastrin-releasing peptide (ProGRP), in a more automatable fashion compared to previous methods. The workflow combines protein digestion using an 20??m ID immobilized trypsin reactor with a polymeric layer of 2-hydroxyethyl methacrylate-vinyl azlactone (HEMA-VDM), desalting on a polystyrene-divinylbenzene (PS-DVB) monolithic trap column, and subsequent separation of resulting peptides on a 10??m ID (PS-DVB) porous layer open tubular (PLOT) column. The high resolution of the PLOT columns was maintained in the on-line system, resulting in narrow chromatographic peaks of 3-5 seconds. The trypsin reactors provided repeatable performance and were compatible with long-term storage.
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A method to generate enhanced GFP+ chimeric mice to study the role of bone marrow-derived cells in the eye.
Exp. Eye Res.
PUBLISHED: 08-22-2013
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GFP-chimeric mice are important tools to study the role of bone marrow-derived cells in eye physiology. A method is described to generate GFP-chimeric mice using whole-body, sub-lethal radiation (600 rad) of wild-type C57BL/6 recipients followed by tail vein injection of bone marrow cells derived from GFP+ (GFP-transgenic C57/BL/6-Tg(UBC-GFP)30 Scha/J) mice. This method yields stable GFP+ chimeras with greater than 95% chimerism (range 95-99%), achieved within one month of bone marrow transfer confirmed by microscopy and fluorescence-assisted cell sorting (FACS) analysis, with lower mortality after irradiation than prior methods. To demonstrate the efficacy of GFP+ bone marrow chimeric mice, the role of circulating GFP+ bone marrow-derived cells in myofibroblast generation after irregular photo-therapeutic keratectomy (PTK) was analyzed. Many SMA+ myofibroblasts that were generated at one month after PTK were derived from GFP+ bone marrow-derived cells. The GFP+ bone marrow chimeric mouse provides an excellent model for studying the role of bone marrow-derived cells in corneal wound healing, glaucoma surgery, optic nerve head pathology and retinal pathophysiology and wound healing.
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RNA interference-mediated phosphodiesterase 4D splice variants knock-down in the prefrontal cortex produces antidepressant-like and cognition-enhancing effects.
Br. J. Pharmacol.
PUBLISHED: 07-17-2013
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Phosphodiesterase 4 (PDE4) inhibitors produce potent antidepressant-like and cognition-enhancing effects. However, their clinical utility is limited by the major side effect of emesis, which appears to be PDE4 isoform-specific. Although PDE4D subtype plays the pivotal role in these therapeutic profiles, it is also the primary subtype responsible for emesis. Therefore, the aim of present research was to investigate whether long-form PDE4D variants mediate antidepressant-like and cognition-enhancing effects, but are irrespective with emesis.
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Biogenesis of a bacterial organelle: the carboxysome assembly pathway.
Cell
PUBLISHED: 06-08-2013
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The carboxysome is a protein-based organelle for carbon fixation in cyanobacteria, keystone organisms in the global carbon cycle. It is composed of thousands of subunits including hexameric and pentameric proteins that form a shell to encapsulate the enzymes ribulose 1,5-bisphosphate carboxylase/oxygenase and carbonic anhydrase. Here, we describe the stages of carboxysome assembly and the requisite gene products necessary for progression through each. Our results demonstrate that, unlike membrane-bound organelles of eukaryotes, in carboxysomes the interior of the compartment forms first, at a distinct site within the cell. Subsequently, shell proteins encapsulate this procarboxysome, inducing budding and distribution of functional organelles within the cell. We propose that the principles of carboxysome assembly that we have uncovered extend to diverse bacterial microcompartments.
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Transmission electron microscopy analysis of epithelial basement membrane repair in rabbit corneas with haze.
Invest. Ophthalmol. Vis. Sci.
PUBLISHED: 05-23-2013
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To assess the ultrastructure of the epithelial basement membrane using transmission electron microscopy (TEM) in rabbit corneas with and without subepithelial stroma opacity (haze).
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Poly(lactide-co-glycolide)-rifampicin nanoparticles efficiently clear Mycobacterium bovis BCG infection in macrophages and remain membrane-bound in phago-lysosomes.
J. Cell. Sci.
PUBLISHED: 05-17-2013
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Nanoparticles (NPs) are increasingly used as biodegradable vehicles to selectively deliver therapeutic agents such as drugs or antigens to cells. The most widely used vehicle for this purpose is based on copolymers of lactic acid and glycolic acid (PLGA) and has been extensively used in experiments aimed at delivering antibiotics against Mycobacterium tuberculosis in animal models of tuberculosis. Here, we describe fabrication of PLGA NPs containing either a high concentration of rifampicin or detectable levels of the green fluorescent dye, coumarin-6. Our goal here was twofold: first to resolve the controversial issue of whether, after phagocytic uptake, PLGA NPs remain membrane-bound or whether they escape into the cytoplasm, as has been widely claimed. Second, we sought to make NPs that enclosed sufficient rifampicin to efficiently clear macrophages of infection with Mycobacterium bovis BCG. Using fluorescence microscopy and immuno-electron microscopy, in combination with markers for lysosomes, we show that BCG bacteria, as expected, localized to early phagosomes, but that at least 90% of PLGA particles were targeted to, and remained in, low pH, hydrolase-rich phago-lysosomes. Our data collectively argue that PLGA NPs remain membrane-enclosed in macrophages for at least 13 days and degrade slowly. Importantly, provided that the NPs are fabricated with sufficient antibiotic, one dose given after infection is sufficient to efficiently clear the BCG infection after 9-12 days of treatment, as shown by estimates of the number of bacterial colonies in vitro.
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Separation optimization of long porous-layer open-tubular columns for nano-LC-MS of limited proteomic samples.
J Sep Sci
PUBLISHED: 05-11-2013
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The single-run resolving power of current 10 ?m id porous-layer open-tubular (PLOT) columns has been optimized. The columns studied had a poly(styrene-co-divinylbenzene) porous layer (~0.75 ?m thickness). In contrast to many previous studies that have employed complex plumbing or compromising set-ups, SPE-PLOT-LC-MS was assembled without the use of additional hardware/noncommercial parts, additional valves or sample splitting. A comprehensive study of various flow rates, gradient times, and column length combinations was undertaken. Maximum resolution for <400 bar was achieved using a 40 nL/min flow rate, a 400 min gradient and an 8 m long column. We obtained a 2.3-fold increase in peak capacity compared to previous PLOT studies (950 versus previously obtained 400, when using peak width = 2? definition). Our system also meets or surpasses peak capacities obtained in recent reports using nano-ultra-performance LC conditions or long silica monolith nanocolumns. Nearly 500 proteins (1958 peptides) could be identified in just one single injection of an extract corresponding to 1000 BxPC3 beta catenin (-/-) cells, and ~1200 and 2500 proteins in extracts of 10,000 and 100,000 cells, respectively, allowing detection of central members and regulators of the Wnt signaling pathway.
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Mouse strain variation in SMA(+) myofibroblast development after corneal injury.
Exp. Eye Res.
PUBLISHED: 04-26-2013
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The purpose of present study was to investigate differences in myofibroblast development after haze-generating injury in different commonly used strains of mice. The inbred mouse strains used in this study were Balb/c, C57BL/6, C3H/HeJ and DBA/1J. All mice had uniform irregular phototherapeutic keratectomy with an excimer laser according to a previously published method to generate stromal haze. DBA/1J mice generated significantly greater density of alpha smooth muscle actin (SMA)-positive myofibroblasts in the anterior stroma compared to Balb/c (p < 0.05), C57BL/6 (p < 0.05) and C3H/HeJ (p < 0.01) mice. The C3H/HeJ strain had significantly lower density of SMA-positive myofibroblasts compared to other three mouse strains. These results indicate that mouse strain must be considered in designing experiments and interpreting the results of experiments in which corneal haze and myofibroblast generation is studied in mice. Further investigation of genetics underlying mouse strain variation could provide insight into the corneal wound healing and haze generation processes.
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Two new high-resolution crystal structures of carboxysome pentamer proteins reveal high structural conservation of CcmL orthologs among distantly related cyanobacterial species.
Photosyn. Res.
PUBLISHED: 04-01-2013
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Cyanobacteria have evolved a unique carbon fixation organelle known as the carboxysome that compartmentalizes the enzymes RuBisCO and carbonic anhydrase. This effectively increases the local CO2 concentration at the active site of RuBisCO and decreases its relatively unproductive side reaction with oxygen. Carboxysomes consist of a protein shell composed of hexameric and pentameric proteins arranged in icosahedral symmetry. Facets composed of hexameric proteins are connected at the vertices by pentameric proteins. Structurally homologous pentamers and hexamers are also found in heterotrophic bacteria where they form architecturally related microcompartments such as the Eut and Pdu organelles for the metabolism of ethanolamine and propanediol, respectively. Here we describe two new high-resolution structures of the pentameric shell protein CcmL from the cyanobacteria Thermosynechococcus elongatus and Gloeobacter violaceus and provide detailed analysis of their characteristics and comparison with related shell proteins.
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Structural basis and SAR for G007-LK, a lead stage 1,2,4-triazole based specific tankyrase 1/2 inhibitor.
J. Med. Chem.
PUBLISHED: 03-29-2013
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Tankyrases 1 and 2 (TNKS1/2) are promising pharmacological biotargets with possible applications for the development of novel anticancer therapeutics. A focused structure-activity relationship study was conducted based on the tankyrase inhibitor JW74 (1). Chemical analoging of 1 improved the 1,2,4-triazole based core and led to 4-{5-[(E)-2-{4-(2-chlorophenyl)-5-[5-(methylsulfonyl)pyridin-2-yl]-4H-1,2,4-triazol-3-yl}ethenyl]-1,3,4-oxadiazol-2-yl}benzonitrile (G007-LK), a potent, "rule of 5" compliant and a metabolically stable TNKS1/2 inhibitor. G007-LK (66) displayed high selectivity toward tankyrases 1 and 2 with biochemical IC50 values of 46 nM and 25 nM, respectively, and a cellular IC50 value of 50 nM combined with an excellent pharmacokinetic profile in mice. The PARP domain of TNKS2 was cocrystallized with 66, and the X-ray structure was determined at 2.8 Å resolution in the space group P3221. The structure revealed that 66 binds to unique structural features in the extended adenosine binding pocket which forms the structural basis for the compounds high target selectivity and specificity. Our study provides a significantly optimized compound for targeting TNKS1/2 in vitro and in vivo.
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On-line solid phase extraction-liquid chromatography, with emphasis on modern bioanalysis and miniaturized systems.
J Pharm Biomed Anal
PUBLISHED: 03-24-2013
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On-line solid phase extraction (SPE)-liquid chromatography (LC) allows for automated, sensitive, precise and selective bioanalysis. It is a common feature in miniaturized- or nano LC systems, which are well suited for applications requiring high sensitivity and/or treatment of limited samples (laser micro-dissection samples, rare cancer stem cells, etc.). Traditionally, particles with reversed phase (RP) functional groups are used for the columns in SPE-LC systems. There is however an expanding diversity in SPE-LC combinations applied to meet todays bioanalytical challenges. Current online SPE-LC combinations employ, e.g. porous graphitic carbon (PGC) and hydrophilic interaction liquid chromatography (HILIC) materials for metabolomics and glycomics, restricted access media (RAM) columns coupled with nano LC for peptidomics, immunoaffinity trap columns for targeted proteomics and metal oxide affinity phases for phosphopeptide analysis. However, issues can arise when combining different phases in on-line SPE-LC, e.g. due to solvent incompatibilities between enrichment/separation principles and sample solvent requirements. Consequences can be low recovery and poor resolution, or need for additional instrumentation. On-line SPE-LC with very narrow columns (10-20?m inner diameters) can be appropriate to obtain maximum sensitivity and information. In such highly miniaturized systems, non-particulate columns are arguably more suited (e.g. monolithic or porous layer open tubular (PLOT) columns) as e.g. hardware contributions resulting in extra column volumes are reduced. Basic SPE-LC systems can be configured/modified to perform quite complex analytical operations, and certain columns, configurations and hardware can improve robustness.
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Biological and biomechanical responses to traditional epithelium-off and transepithelial riboflavin-UVA CXL techniques in rabbits.
J Refract Surg
PUBLISHED: 02-11-2013
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To compare the biological effects of riboflavin-ultraviolet A (UVA) corneal cross-linking (CXL) performed with a traditional epithelium-off method to several transepithelial methods in a rabbit model. Preliminary experiments on biomechanical rigidity were also performed.
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Phase I trial of recombinant modified vaccinia ankara encoding Epstein-Barr viral tumor antigens in nasopharyngeal carcinoma patients.
Cancer Res.
PUBLISHED: 01-24-2013
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Epstein-Barr virus (EBV) is associated with several malignancies including nasopharyngeal carcinoma, a high incidence tumor in Chinese populations, in which tumor cells express the two EBV antigens EB nuclear antigen 1 (EBNA1) and latent membrane protein 2 (LMP2). Here, we report the phase I trial of a recombinant vaccinia virus, MVA-EL, which encodes an EBNA1/LMP2 fusion protein designed to boost T-cell immunity to these antigens. The vaccine was delivered to Hong Kong patients with nasopharyngeal carcinoma to determine a safe and immunogenic dose. The patients, all in remission more than 12 weeks after primary therapy, received three intradermal MVA-EL vaccinations at three weekly intervals, using five escalating dose levels between 5 × 10(7) and 5 × 10(8) plaque-forming unit (pfu). Blood samples were taken during prescreening, immediately before vaccination, one week afterward and at intervals up to one year later. Immunogenicity was tested by IFN-? ELIspot assays using complete EBNA1 and LMP2 15-mer peptide mixes and known epitope peptides relevant to patient MHC type. Eighteen patients were treated, three per dose level one to four and six at the highest dose, without dose-limiting toxicity. T-cell responses to one or both vaccine antigens were increased in 15 of 18 patients and, in many cases, were mapped to known CD4 and CD8 epitopes in EBNA1 and/or LMP2. The range of these responses suggested a direct relationship with vaccine dose, with all six patients at the highest dose level giving strong EBNA1/LMP2 responses. We concluded that MVA-EL is both safe and immunogenic, allowing the highest dose to be forwarded to phase II studies examining clinical benefit.
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Examining HPV threat-to-efficacy ratios in the Extended Parallel Process Model.
Health Commun
PUBLISHED: 01-22-2013
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The Extended Parallel Process Model (EPPM) posits that an effective fear appeal includes both threat and efficacy components; however, research has not addressed whether there is an optimal threat-to-efficacy ratio. It is possible that varying levels of threat and efficacy in a persuasive message could yield different effects on attitudes, beliefs, and behaviors. In a laboratory experiment, women (n?=?442) were exposed to human papilloma virus (HPV) prevention messages containing one of six threat-to-efficacy ratios and one of two message frames (messages emphasizing the connection between HPV and cervical cancer or HPV and genital warts). Multiple mediation analysis revealed that a 1-to-1 ratio of threat to efficacy was most effective at increasing prevention intentions, primarily because it caused more fear and risk susceptibility than other message ratios. Response efficacy significantly mediated the relationship between message framing and intentions, such that participants exposed to a genital warts message reported significantly higher intentions, and this association can be explained in part through response efficacy. Implications for future theoretical research as well as campaigns and intervention research are discussed.
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An outcomes analysis of over 200 revision surgeries for penile prosthesis implantation: a multicenter study.
J Sex Med
PUBLISHED: 11-14-2011
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Inflatable penile prosthesis (IPP) implantation is a well-established treatment for medically refractory erectile dysfunction, with long-term reliability. Overall survival is 96% at 5 years and 60% at 15 years for primary (virgin) implantation.
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Blood-borne angiotensin II acts in the brain to influence behavioral and endocrine responses to psychogenic stress.
J. Neurosci.
PUBLISHED: 10-22-2011
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This study elucidates the neural circuits by which circulating angiotensin II (ANGII) acts in the brain to influence humoral and behavioral responses to psychological stressors. To test the hypothesis that systemic ANGII mediates stress responding via the subfornical organ (SFO), we first found that the timing of increased systemic ANGII in response to 60 min restraint coincides with increased c-fos mRNA expression in the SFO. Next, we administered an anterograde neuronal tract tracer into the SFO and found that fibers originating there make appositions onto neurons in the paraventricular nucleus of the hypothalamus that are also c-fos positive following restraint stress. To determine whether circulating ANGII stimulates the release of stress hormones via activation of angiotensin type 1 receptors (AT1R) within the SFO, we delivered lentivirus to knockdown AT1R expression locally in the SFO. Inhibition of AT1R specifically within the SFO blunted the release of adrenocorticotrophin-releasing hormone and corticosterone in response to restraint stress and caused rats to spend more time in the open arms of an elevated-plus maze than controls, indicating that inhibition of AT1R within the SFO is anxiolytic. Collectively, these results suggest that circulating ANGII acts on AT1R in the SFO to influence responding to psychological stressors.
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Biofilm and penile prosthesis infections in the era of coated implants: a review.
J Sex Med
PUBLISHED: 09-22-2011
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The numbers of inflatable penile prosthesis (IPP) implanted has increased yearly due to the large numbers of patients treated for prostate cancer, patients becoming refractory to the five phosphodiesterase inhibitors and Peyronies disease.
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Flaporhexis: rapid and effective technique to limit epithelial ingrowth after LASIK enhancement.
J Cataract Refract Surg
PUBLISHED: 08-30-2011
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We describe a method of lifting and replacing the laser in situ keratomileusis (LASIK) flap to reduce the incidence of epithelial ingrowth beneath the flap after LASIK enhancement. In the rapid flaporhexis technique, the flap edge is opened by 1 clock hour with a Sinskey hook and the flap is peeled back after the exposed edge is grasped with a forceps. When necessary, further blunt retraction of the flap is performed with a triangular polyvinyl acetate sponge. After ablation and before the flap is replaced, a triangular sponge is used to clear epithelial remnants from the interface. This method consistently produces a smooth epithelial dissection and decreases the possibility that epithelium is retained beneath the flap.
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Chronic reduction of insulin receptors in the ventromedial hypothalamus produces glucose intolerance and islet dysfunction in the absence of weight gain.
Am. J. Physiol. Endocrinol. Metab.
PUBLISHED: 08-09-2011
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Insulin is believed to regulate glucose homeostasis mainly via direct effects on the liver, muscle, and adipose tissues. The contribution of insulins central nervous system effects to disorders of glucose metabolism has received less attention. To evaluate whether postnatal reduction of insulin receptors (IRs) within the ventromedial hypothalamus (VMH), a brain region critical for glucose sensing, contributes to disorders of peripheral glucose metabolism, we microinjected a lentiviral vector expressing an antisense sequence to knockdown IRs or a control lentiviral vector into the VMH of nonobese nondiabetic rats. After 3-4 mo, we assessed 1) glucose tolerance, 2) hepatic insulin sensitivity, and 3) insulin and glucagon secretion, using the glucose clamp technique. Knockdown of IRs locally in the VMH caused glucose intolerance without altering body weight. Increments of plasma insulin during a euglycemic clamp study failed to suppress endogenous glucose production and produced a paradoxical rise in plasma glucagon in the VMH-IR knockdown rats. Unexpectedly, these animals also displayed a 40% reduction (P < 0.05) in insulin secretion in response to an identical hyperglycemic stimulus (?220 mg/dl). Our data demonstrate that chronic suppression of VMH-IR gene expression is sufficient to impair glucose metabolism as well as ?-cell and ?-cell function in nondiabetic, nonobese rats. These data suggest that insulin resistance within the VMH may be a significant contributor to the development of type 2 diabetes.
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Expert training with standardized operative technique helps establish a successful penile prosthetics program for urologic resident education.
J Sex Med
PUBLISHED: 08-03-2011
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The challenge of resident education in urologic surgery programs is to overcome disparity imparted by diverse patient populations, limited training times, and inequalities in the availability of expert surgical educators. Specifically, in the area of prosthetic urology, only a small proportion of programs have full-time faculty available to train residents in this discipline.
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High efficiency, high temperature separations on silica based monolithic columns.
J Chromatogr A
PUBLISHED: 06-30-2011
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The effect of temperature on separation using reversed-phase monolithic columns has been investigated using a nano-LC pumping system for gradient separation of tryptic peptides with MS detection. A goal of this study was to find optimal conditions for high-speed separations. The chromatographic performance of the columns was evaluated by peak capacity and peak capacity per time unit. Column lengths ranging from 20 to 100 cm and intermediate gradient times from 10 to 30 min were investigated to assess the potential of these columns in a final step separation, e.g. after fractionation or specific sample preparation. Flow rates from 250 to 2000 nL/min and temperatures from 20 to 120°C were investigated. Temperature had a significant effect on fast separations, and a flow rate of 2000 nL/min and a temperature of 80°C gave the highest peak capacity per time unit. These settings produced 70% more protein identifications in a biological sample compared to a conventional packed column. Alternatively, an equal amount of protein identifications was obtained with a 40% reduction in run time compared to the conventional packed column.
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Automatic filtration and filter flush for robust online solid-phase extraction liquid chromatography.
J Sep Sci
PUBLISHED: 06-27-2011
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Online solid-phase extraction-liquid chromatography (SPE-LC) with microbore or capillary columns was significantly improved regarding robustness, as an easily installed automatic filtration and filter flushing (AFFL) procedure was added to avoid system clogging. Specifically, an injected sample is passed through a union containing a stainless steel filter prior to SPE trapping. The filter stops any particulate matter from reaching the SPE. When the SPE is subsequently connected to the LC column by column switching, a separate pump backflushes the filter-union, removing the particulate matter off the filter after each injection. This feature greatly reduced backpressure buildup over the entire system.
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Anti-infection dip suggestions for the Coloplast Titan Inflatable Penile Prosthesis in the era of the infection retardant coated implant.
J Sex Med
PUBLISHED: 06-23-2011
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Infection is the worst complication seen with inflatable penile prosthesis (IPP). Both the American Medical Systems (AMS) and Coloplast IPP have infection retardant coatings. AMS is coated at the factory with rifampicin and minocycline (InhibiZone). The Coloplast IPP has a hydrophilic coating covalently bonded to its components that will absorb any aqueous solution before implantation and provides increased surface lubricity to decrease bacterial adherence.
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Short-term cell death and inflammation after intracorneal inlay implantation in rabbits.
J Refract Surg
PUBLISHED: 06-21-2011
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To investigate the cell death and inflammatory response to insertion of the KAMRA inlay (AcuFocus Inc) for presbyopia.
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Visual performance of an apodized diffractive multifocal intraocular lens with +3.00-d addition: 1-year follow-up.
J Refract Surg
PUBLISHED: 06-01-2011
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To determine whether implantation of a multifocal intraocular lens (IOL) with a lower addition (+3.00 diopters [D]) at the lens plane results in better intermediate visual acuity 1 year after surgery compared with a multifocal IOL with higher addition (+4.00 D).
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Dialyzer reuse with peracetic acid does not impact patient mortality.
Clin J Am Soc Nephrol
PUBLISHED: 05-12-2011
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Numerous studies have shown the overall benefits of dialysis filter reuse, including superior biocompatibility and decreased nonbiodegradable medical waste generation, without increased risk of mortality. A recent study reported that dialyzer reprocessing was associated with decreased patient survival; however, it did not control for sources of potential confounding. We sought to determine the effect of dialyzer reprocessing with peracetic acid on patient mortality using contemporary outcomes data and rigorous analytical techniques.
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A novel synthetic smoothened antagonist transiently inhibits pancreatic adenocarcinoma xenografts in a mouse model.
PLoS ONE
PUBLISHED: 04-20-2011
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Hedgehog (Hh) signaling is over-activated in several solid tumors where it plays a central role in cell growth, stroma recruitment and tumor progression. In the Hh signaling pathway, the Smoothened (SMO) receptor comprises a primary drug target with experimental small molecule SMO antagonists currently being evaluated in clinical trials.
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Modulation transfer function and optical quality after bilateral implantation of a +3.00 D versus a +4.00 D multifocal intraocular lens.
J Cataract Refract Surg
PUBLISHED: 04-19-2011
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To determine whether the improvement in intermediate vision after bilateral implantation of an aspheric multifocal intraocular lens (IOL) with a +3.00 diopter (D) addition (add) occurs at the expense of optical quality compared with the previous model with a +4.00 D add.
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Maropitant citrate for treatment of ulcerative dermatitis in mice with a C57BL/6 background.
J. Am. Assoc. Lab. Anim. Sci.
PUBLISHED: 03-29-2011
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Murine ulcerative dermatitis (UD) is a common progressive condition of mice with a C57BL/6 background. Typically, mice present with scabs and crusts on the skin of the dorsal neck and ears, and are often severely pruritic. Animals tend to scratch the lesions, causing additional trauma to the already ulcerated and inflamed skin. Therapeutic intervention largely has been unsuccessful, in part due to the lack of a known cause for the disease. Though the exact etiology of UD has not been elucidated, substance P (SP) has recently been demonstrated as an important neuropeptide linked to the itch-scratch cycle. SP functions at the tachykinin neurokinin 1 (NK1) receptor. We hypothesized that inhibition of SP binding to the NK1 receptor would decrease the itch sensation, thus decreasing scratching behavior and subsequent skin trauma. The purpose of this study was to evaluate the effectiveness of an NK1 receptor antagonist, maropitant citrate, as a treatment for murine UD. Treatment with 1 mg/kg maropitant citrate significantly reduced the size of UD lesions in mice.
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CRIP1a switches cannabinoid receptor agonist/antagonist-mediated protection from glutamate excitotoxicity.
Neurosci. Lett.
PUBLISHED: 03-10-2011
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A shared pathology among many neurological and neurodegenerative disorders is neuronal loss. Cannabinoids have been shown to be neuroprotective in multiple systems. However, both agonists and antagonists of the CB(1) cannabinoid receptor are neuroprotective, but the mechanisms responsible for these actions remain unclear. Recently a CB(1) receptor interacting protein, CRIP1a, was identified and found to alter CB(1) activity. Here we show that in an assay of glutamate neurotoxicity in primary neuronal cortical cultures CRIP1a disrupts agonist-induced neuroprotection and confers antagonist-induced neuroprotection.
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Downregulation of hypothalamic insulin receptor expression elicits depressive-like behaviors in rats.
Behav. Brain Res.
PUBLISHED: 03-07-2011
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Ongoing epidemiological studies estimate that greater than 60% of the adult US population may be categorized as either overweight or obese. There is a growing appreciation that the complications of obesity extend to the central nervous system (CNS) and may result in increased risk for neurological co-morbidities like depressive illness. One potential mechanistic mediator linking obesity and depressive illness is the adipocyte derived hormone leptin. We previously demonstrated that lentivirus-mediated downregulation of hypothalamic insulin receptors increases body weight, adiposity and plasma leptin levels, which is consistent with features of the metabolic syndrome. Using this novel model of obesity, we examined performance in the forced swim test (FST), the sucrose preference test and the elevated plus maze (EPM), approaches that are often used as measures of depressive-like and anxiety-like behaviors, in rats that received third ventricular injections of either an insulin receptor antisense lentivirus (hypo-IRAS) or a control lentivirus (hypo-Con). Hypo-IRAS rats exhibited significant increases in immobility time and corresponding decreases in active behaviors in the FST and exhibited anhedonia as measured by decreased sucrose intake compared to hypo-Con rats. Hypo-IRAS rats also exhibited increases in anxiety-like behaviors in the EPM. Plasma, hippocampal and amygdalar brain-derived neurotrophic factor (BDNF) levels were reduced in hypo-IRAS rats, suggesting that the obesity/hyperleptinemic phenotype may elicit this behavioral phenotype through modulation of neurotrophic factor expression. Collectively, these data support the hypothesis for an increased risk for mood disorders in obesity, which may be related to decreased expression of hippocampal and amygdalar BDNF.
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Obesity/hyperleptinemic phenotype impairs structural and functional plasticity in the rat hippocampus.
Physiol. Behav.
PUBLISHED: 02-15-2011
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Epidemiological studies estimate that greater than 60% of the adult US population may be categorized as either overweight or obese, and there is a growing appreciation that the complications of obesity extend to the central nervous system (CNS). While the vast majority of these studies have focused on the hypothalamus, more recent studies suggest that the complications of obesity may also affect the structural and functional integrity of the hippocampus. A potential contributor to obesity-related CNS abnormalities is the adipocyte-derived hormone leptin. In this regard, decreases in CNS leptin activity may contribute to deficits in hippocampal synaptic plasticity and suggest that leptin resistance, a well-described phenomenon in the hypothalamus, may also be observed in the hippocampus. Unfortunately, the myriad of metabolic and endocrine abnormalities in diabetes/obesity phenotypes makes it challenging to assess the role of leptin in hippocampal neuroplasticity deficits associated with obesity models. To address this question, we examined hippocampal morphological and behavioral plasticity following lentivirus-mediated downregulation of hypothalamic insulin receptors (hypo-IRAS). Hypo-IRAS rats exhibit increases in body weight, adiposity, plasma leptin and triglyceride levels. As such, hypo-IRAS rats develop a phenotype that is consistent with features of the metabolic syndrome. In addition, hippocampal morphological plasticity and performance of hippocampal-dependent tasks are adversely affected in hypo-IRAS rats. Leptin-mediated signaling is also decreased in hypo-IRAS rats. We will discuss these findings in the context of how hyperleptinemia and hypertriglyceridemia may represent mechanistic mediators of the neurological consequences of impaired hippocampal synaptic plasticity in obesity.
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Selective targeting of ASIC3 using artificial miRNAs inhibits primary and secondary hyperalgesia after muscle inflammation.
Pain
PUBLISHED: 02-04-2011
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Acid-sensing ion channels (ASICs) are activated by acidic pH and may play a significant role in the development of hyperalgesia. Earlier studies show ASIC3 is important for induction of hyperalgesia after muscle insult using ASIC3-/- mice. ASIC3-/- mice lack ASIC3 throughout the body, and the distribution and composition of ASICs could be different from wild-type mice. We therefore tested whether knockdown of ASIC3 in primary afferents innervating muscle of adult wild-type mice prevented development of hyperalgesia to muscle inflammation. We cloned and characterized artificial miRNAs (miR-ASIC3) directed against mouse ASIC3 (mASIC3) to downregulate ASIC3 expression in vitro and in vivo. In CHO-K1 cells transfected with mASIC3 cDNA in culture, the miR-ASIC3 constructs inhibited protein expression of mASIC3 and acidic pH-evoked currents and had no effect on protein expression or acidic pH-evoked currents of ASIC1a. When miR-ASIC3 was used in vivo, delivered into the muscle of mice using a herpes simplex viral vector, both muscle and paw mechanical hyperalgesia were reduced after carrageenan-induced muscle inflammation. ASIC3 mRNA in DRG and protein levels in muscle were decreased in vivo by miR-ASIC3. In CHO-K1 cells co-transfected with ASIC1a and ASIC3, miR-ASIC3 reduced the amplitude of acidic pH-evoked currents, suggesting an overall inhibition in the surface expression of heteromeric ASIC3-containing channels. Our results show, for the first time, that reducing ASIC3 in vivo in primary afferent fibers innervating muscle prevents the development of inflammatory hyperalgesia in wild-type mice, and thus, may have applications in the treatment of musculoskeletal pain in humans.
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Hydrophilic interaction chromatography of nucleoside triphosphates with temperature as a separation parameter.
J Chromatogr A
PUBLISHED: 01-18-2011
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Eight deoxynucleoside triphosphates (dNTPs) and nucleoside triphosphates (NTPs): ATP, CTP, GTP, UTP, dATP, dCTP, dGTP and dTTP, were separated with two 15 cm ZIC-pHILIC columns coupled in series, using LC-UV instrumentation. The polymer-based ZIC-pHILIC column gave significantly better separations and peak shape than a silica-based ZIC-HILIC column. Better separations were obtained with isocratic elution as compared to gradient elution. The temperature markedly affected the selectivity and could be used to fine tune separation. The analysis time was also affected by temperature, as lower temperatures surprisingly reduced the retention of the nucleotides. dNTP/NTP standards could be separated in 35 min with a flow rate of 200 ?L/min. In Escherichia coli cell culture samples dNTP/NTPs could be selectively separated in 7 0min using a flow rate of 100 ?L/min.
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Phosphodiesterase-4D knock-out and RNA interference-mediated knock-down enhance memory and increase hippocampal neurogenesis via increased cAMP signaling.
J. Neurosci.
PUBLISHED: 01-07-2011
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Phosphodiesterase-4 (PDE4) plays an important role in mediating memory via the control of intracellular cAMP signaling; inhibition of PDE4 enhances memory. However, development of PDE4 inhibitors as memory enhancers has been hampered by their major side effect of emesis. PDE4 has four subtypes (PDE4A-D) consisting of 25 splice variants. Mice deficient in PDE4D displayed memory enhancement in radial arm maze, water maze, and object recognition tests. These effects were mimicked by repeated treatment with rolipram in wild-type mice. In addition, similarly as rolipram-treated wild-type mice, PDE4D-deficient mice also displayed increased hippocampal neurogenesis and phosphorylated cAMP response element-binding protein (pCREB). Furthermore, microinfusion of lentiviral vectors that contained microRNAs (miRNAs) targeting long-form PDE4D isoforms into bilateral dentate gyri of the mouse hippocampus downregulated PDE4D4 and PDE4D5, enhanced memory, and increased hippocampal neurogenesis and pCREB. Finally, while rolipram and PDE4D deficiency shortened ?2 adrenergic receptor-mediated anesthesia, a surrogate measure of emesis, miRNA-mediated PDE4D knock-down in the hippocampus did not. The present results suggest that PDE4D, in particular long-form PDE4D, plays a critical role in the mediation of memory and hippocampal neurogenesis, which are mediated by cAMP/CREB signaling; reduced expression of PDE4D, or at least PDE4D4 and PDE4D5, in the hippocampus enhances memory but appears not to cause emesis. These novel findings will aid in the development of PDE4 subtype- or variant-selective inhibitors for treatment of disorders involving impaired cognition, including Alzheimers disease.
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Novel synthetic antagonists of canonical Wnt signaling inhibit colorectal cancer cell growth.
Cancer Res.
PUBLISHED: 01-05-2011
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Canonical Wnt signaling is deregulated in several types of human cancer where it plays a central role in tumor cell growth and progression. Here we report the identification of 2 new small molecules that specifically inhibit canonical Wnt pathway at the level of the destruction complex. Specificity was verified in various cellular reporter systems, a Xenopus double-axis formation assay and a gene expression profile analysis. In human colorectal cancer (CRC) cells, the new compounds JW67 and JW74 rapidly reduced active ?-catenin with a subsequent downregulation of Wnt target genes, including AXIN2, SP5, and NKD1. Notably, AXIN2 protein levels were strongly increased after compound exposure. Long-term treatment with JW74 inhibited the growth of tumor cells in both a mouse xenograft model of CRC and in Apc(Min) mice (multiple intestinal neoplasia, Min). Our findings rationalize further preclinical and clinical evaluation of these new compounds as novel modalities for cancer treatment.
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Obesity/hyperleptinemic phenotype adversely affects hippocampal plasticity: effects of dietary restriction.
Physiol. Behav.
PUBLISHED: 09-01-2010
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Epidemiological studies estimate that greater than 60% of the adult US population may be categorized as either overweight or obese and there is a growing appreciation that obesity affects the functional integrity of the central nervous system (CNS). We recently developed a lentivirus (LV) vector that produces an insulin receptor (IR) antisense RNA sequence (IRAS) that when injected into the hypothalamus selectively decreases IR signaling in hypothalamus, resulting in increased body weight, peripheral adiposity and plasma leptin levels. To test the hypothesis that this obesity/hyperleptinemic phenotype would impair hippocampal synaptic transmission, we examined short term potentiation (STP) and long term potentiation (LTP) in the hippocampus of rats that received the LV-IRAS construct or the LV-Control construct in the hypothalamus (hypo-IRAS and hypo-Con, respectively). Stimulation of the Schaffer collaterals elicits STP that develops into LTP in the CA1 region of hypo-Con rats; conversely, hypo-IRAS rats exhibit STP that fails to develop into LTP. To more closely examine the potential role of hyperleptinemia in these electrophysiological deficits, hypo-IRAS were subjected to mild food restriction paradigms that would either: 1) prevent the development of the obesity phenotype; or 2) reverse an established obesity phenotype in hypo-IRAS rats. Both of these paradigms restored LTP in the CA1 region and reversed the decreases in the phosphorylated/total ratio of GluA1 Ser845 AMPA receptor subunit expression observed in the hippocampus of hypo-IRAS rats. Collectively, these data support the hypothesis that obesity impairs hippocampal synaptic transmission and support the hypothesis that these deficits are mediated through the impairment of hippocampal leptin activity.
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New enhancements of the scrotal one-incision technique for placement of artificial urinary sphincter allow proximal cuff placement.
J Sex Med
PUBLISHED: 06-30-2010
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Urinary incontinence impairs sexual functioning and sexual satisfaction. Traditional artificial urinary sphincter (AUS) implantation requires perineal incision for cuff placement and a second inguinal incision for reservoir and pump placement. We believed AUS could be placed easier and quicker through one scrotal incision. Aim.? In an effort to effect more proximal placement of the cuff while keeping the advantages of the one scrotal incision technique, we report enhancements to the original surgical technique.
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Differences in the early biomechanical effects of hyperopic and myopic laser in situ keratomileusis.
J Cataract Refract Surg
PUBLISHED: 05-25-2010
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To compare changes in corneal hysteresis (CH) and the corneal resistance factor (CRF) in myopic and hyperopic laser in situ keratomileusis (LASIK) and evaluate their relationship to the number of photoablative pulses delivered, a surrogate for ablation volume.
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Use of mitomycin-C for phototherapeutic keratectomy and photorefractive keratectomy surgery.
Curr Opin Ophthalmol
PUBLISHED: 05-22-2010
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Experience with mitomycin C (MMC) application during corneal surface ablation procedures such as phototherapeutic keratectomy and photorefractive keratectomy has grown over the last decade. This review will highlight our understanding of the utility, mechanism of action, and safety profile of MMC.
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Corneal wound healing after ultraviolet-A/riboflavin collagen cross-linking: a rabbit study.
J Refract Surg
PUBLISHED: 04-19-2010
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To investigate corneal wound healing following ultraviolet-A (UVA)/riboflavin corneal collagen cross-linking (CXL) in rabbit corneas.
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Hedgehog antagonists cyclopamine and dihydroveratramine can be mistaken for each other in Veratrum album.
J Pharm Biomed Anal
PUBLISHED: 03-16-2010
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A toxic plant, Veratrum album (ssp. viriscens), was found to have an inhibitory effect on Hedgehog (Hh), a developmental signaling pathway that has been shown to be active during development, in adult stem cells and in numerous human tumors. Based on earlier studies it was believed that the known Hh inhibitor cyclopamine was present in V. album (ssp. viriscens). Here we show that instead of cyclopamine, dihydroveratramine (DHV) was found in V. album (ssp. viriscens). These compounds are easily mistaken for each other, as both substances share the same molecular weight, and the same main MS/MS fragments. DHV was found to be a less potent Hh inhibitor compared to cyclopamine. This is the first reported occurrence of DVH in nature.
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Changes in expression of NMDA-NR1 receptor subunits in the rostral ventromedial medulla modulate pain behaviors.
Pain
PUBLISHED: 03-11-2010
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NMDA receptors have an important role in pain facilitation in rostral ventromedial medulla (RVM) and the NR1 subunit is essential for its function. Studies suggest that the NMDA receptors in RVM are critical to modulate both cutaneous and muscle hypersensitivity induced by repeated intramuscular acid injections. We propose that increased expression of the NR1 subunit in the RVM is critical for the full development of hypersensitivity. To test this we used recombinant lentiviruses to over-express the NR1 subunit in the RVM and measured nociceptive sensitivity to cutaneous and muscle stimuli. We also downregulated the expression of NR1 in the RVM and measured the hyperalgesia produced by repeated-acid injections. Increasing the expression of NR1 in the RVM reduces cutaneous and muscle withdrawal threshold, and decreasing the expression of NR1 in the RVM increases the muscle withdrawal threshold and prevents the development of hyperalgesia in an animal model of muscle pain. These results suggest that the NR1 subunits in the RVM are critical for modulating NMDA receptor function, which in turn sets the tone of the nervous systems response to noxious stimuli and tissue injury.
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Femtosecond laser in laser in situ keratomileusis.
J Cataract Refract Surg
PUBLISHED: 03-01-2010
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Flap creation is a critical step in laser in situ keratomileusis (LASIK). Efforts to improve the safety and predictability of the lamellar incision have fostered the development of femtosecond lasers. Several advantages of the femtosecond laser over mechanical microkeratomes have been reported in LASIK surgery. In this article, we review common considerations in management and complications of this step in femtosecond laser-LASIK and concentrate primarily on the IntraLase laser because most published studies relate to this instrument.
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Corneal myofibroblast generation from bone marrow-derived cells.
Exp. Eye Res.
PUBLISHED: 02-27-2010
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The purpose of this study was to determine whether bone marrow-derived cells can differentiate into myofibroblasts, as defined by alpha-smooth muscle actin (SMA) expression, that arise in the corneal stroma after irregular phototherapeutic keratectomy and whose presence within the cornea is associated with corneal stromal haze. C57BL/6J-GFP chimeric mice were generated through bone marrow transplantation from donor mice that expressed enhanced green fluorescent protein (GFP) in a high proportion of their bone marrow-derived cells. Twenty-four GFP chimeric mice underwent haze-generating corneal epithelial scrape followed by irregular phototherapeutic keratectomy (PTK) with an excimer laser in one eye. Mice were euthanized at 2 weeks or 4 weeks after PTK and the treated and control contralateral eyes were removed and cryo-preserved for sectioning for immunocytochemistry. Double immunocytochemistry for GFP and myofibroblast marker alpha-smooth muscle actin (SMA) were performed and the number of SMA+GFP+, SMA+GFP-, SMA-GFP+ and SMA-GFP- cells, as well as the number of DAPI+ cell nuclei, per 400x field of stroma was determined in the central, mid-peripheral and peri-limbal cornea. In this mouse model, there were no SMA+ cells and only a few GFP+ cells detected in unwounded control corneas. No SMA+ cells were detected in the stroma at two weeks after irregular PTK, even though there were numerous GFP+ cells present. At 4 weeks after irregular PTK, all corneas developed mild to moderately severe corneal haze. In each of the three regions of the corneas examined, there were on average more than 9x more SMA+GFP+ than SMA+GFP- myofibroblasts. This difference was significant (p < 0.01). There were significantly more (p < 0.01) SMA-GFP+ cells, which likely include inflammatory cells, than SMA+GFP+ or SMA+GFP- cells, although SMA-GFP- cells represent the largest population of cells in the corneas. In this mouse model, the majority of myofibroblasts developed from bone marrow-derived cells. It is possible that all myofibroblasts in these animals developed from bone marrow-derived cells since mouse chimeras produced using this method had only 60-95% of bone marrow-derived cells that were GFP+ and it is not possible to achieve 100% chimerization. This model, therefore, cannot exclude the possibility of myofibroblasts also developed from keratocytes and/or corneal fibroblasts.
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Separation of intact proteins on porous layer open tubular (PLOT) columns.
J Chromatogr A
PUBLISHED: 02-09-2010
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Porous layer open tubular (PLOT) polystyrene divinylbenzene columns have been used for separating intact proteins with gradient elution. The 10 microm I.D. x 3 m columns were easily coupled to standard liquid chromatography-mass spectrometry (LC-MS) instrumentation with commercially available fittings. Standard proteins separated on PLOT columns appeared as narrow and symmetrical peaks with good resolution. Average peak width increased linearly with gradient time (tG) from 0.14 to 0.33 min (tG 20 and 120 min, respectively) using a 3 m column. With shorter columns, peak widths were larger and increased more steeply with gradient time. Theoretical peak capacity (nc) increased with column length (tested up to 3 m). The nc increased with tG until a plateau was reached. The highest peak capacity achieved (nc=185) was obtained with a 3 m column, where a plateau was reached with tG 90 min. The within- and between column retention time repeatabilities were below 0.6% and below 2.5% (relative standard deviation, RSD), respectively. The carry-over following injection of 0.5 ng per protein was less than 1.1%. The retention time dependence on column temperature was investigated in the range 20-50 degrees C. Proteins in a skimmed milk sample were separated using the method.
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Hedgehog antagonist cyclopamine isomerizes to less potent forms when acidified.
J Pharm Biomed Anal
PUBLISHED: 02-08-2010
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The effect of acid treatment of cyclopamine, a natural antagonist of the hedgehog (Hh) signaling pathway and a potential anti-cancer drug, has been studied. Previous reports have shown that under acidic conditions, as in the stomach, cyclopamine is less effective. Also, it has been stated that cyclopamine converts to veratramine, which has side effects such as hemolysis. In this study, we examined in detail the influence of acidification on structure and activity of cyclopamine. We found that of acidified cyclopamine converts to two previously unreported isomers, which we have called cyclopamine (S) and cyclopamine (X). These have likely gone undetected because cyclopamine is often analyzed with fast and hence lower resolving chromatographic methods. Compared to natural cyclopamine, these cyclopamine isomers have a significantly reduced effect on the ciliary transport of the Hh receptor smoothened, and reduced inhibition on the Hedgehog signaling pathway. The side effects of these isomers are unknown. Our findings can partly explain a reduced efficiency of cyclopamine in a gastric environment, and may help with the rational design of more pH independent cyclopamine analogues.
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Minimal clinically important difference for the ocular surface disease index.
Arch. Ophthalmol.
PUBLISHED: 01-13-2010
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To assess the minimal clinically important difference (MCID) for the Ocular Surface Disease Index (OSDI; Allergan Inc, Irvine, California, holds the copyright), a 12-item patient-reported outcome questionnaire designed to quantify ocular disability due to dry eye disease.
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Effect of femtosecond laser energy level on corneal stromal cell death and inflammation.
J Refract Surg
PUBLISHED: 10-12-2009
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To analyze the effects of variations in femtosecond laser energy level on corneal stromal cell death and inflammatory cell influx following flap creation in a rabbit model.
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