Design, Heterologous Expression, and Application of an Immobilized Protein Kinase.

Protein kinase A (PKA) is a biologically important enzyme for cell regulation, often referred to as the "central kinase". An immobilized PKA that retains substrate specificity and activity would be a useful tool for laboratory scientists, enabling targeted phosphorylation without interference from downstream kinase contamination or kinase autophosphorylation in sensitive assays. Moreover, it might also provide the benefits of robustness and reusability that are often associated with immobilized enzyme preparations. In this work, we describe the creation of a recombinant PKA fusion protein that incorporates the HaloTag covalent immobilization system. We demonstrate that protein fusion design, including affinity tag placement, is critical for optimal heterologous expression in Escherichia coli. Furthermore, we demonstrate various applications of our immobilized PKA, including the phosphorylation of recombinant PKA substrates, such as vasodilator-stimulated phosphoprotein, and endogenous PKA substrates in a cell lysate. This immobilized PKA also possesses robust activity and reusability over multiple trials. This work holds promise as a generalizable strategy for the production and application of immobilized protein kinases.

Computational Fluid Dynamics Turbulence Model and Experimental Study for a Fontan Cavopulmonary Assist Device.

Head-flow HQ curves for a Fontan cavopulmonary assist device (CPAD) were measured using a blood surrogate in a mock circulatory loop and simulated with various computational fluid dynamics (CFD) models. The tests benchmarked the CFD tools for further enhancement of the CPAD design. Recommended Reynolds-Averaged Navier-Stokes (RANS) CFD approaches for the development of conventional ventricular assist devices (VAD) were found to have shortcomings when applied to the Fontan CPAD, which is designed to neutralize off-condition obstruction risks that could contribute to a major adverse event. The no-obstruction condition is achieved with a von Karman pump, utilizing large clearances and small blade heights, which challenge conventional VAD RANS-based CFD hemodynamic simulations. High-fidelity large eddy simulation (LES) is always recommended; however, this may be cost-inhibitive for optimization studies in commercial settings, thus the reliance on RANS models. This study compares head and power predictions of various RANS turbulence models, employing experimental measurements and LES results as a basis for comparison. The models include standard k-ϵ, re-normalization group k-ϵ, realizable k-ϵ, shear stress transport (SST) k-ω, SST with transitional turbulence, and Generalized k-ω. For the pressure head predictions, it was observed that the standard k-ϵ model provided far better agreement with experiment. For the rotor torque, k-ϵ predictions were 30% lower than LES, while the SST and LES torque values were near identical. For the Fontan CPAD, the findings support using LES for the final design simulations, k-ϵ model for head and general flow simulation, and SST for power, shear stress, hemolysis, and thrombogenicity predictions.

Clinical Evaluation of Large Diameter Rigid-Gas Permeable Versus Soft Toric Contact Lenses for the Correction of Refractive Astigmatism. A MultiCenter Study.

PURPOSE: This study aims to address the clinical performance of a large diameter rigid gas permeable lens (LRGP) in a group of subjects with low-to-moderate (0.75-2.75 D) refractive astigmatism. An additional goal was to determine whether soft toric or LRGP contact lenses performed better objectively in the correction of astigmatism and to determine which modality is preferred by subjects. METHODS: This was a multisite prospective cross-over clinical study. Ten asymptomatic contact lens wearers per site (four university clinics) were recruited and randomly assigned to group A or group B. Group A was assigned to start wearing Comfilcon A soft toric lens first, for two weeks, and then crossed over to LRGP lenses (Boston XO, 14.3 mm diameter miniscleral lens). Group B initially wore LRGP lenses and then crossed over to soft toric lenses. For each type of lens worn, low-contrast and high-contrast visual acuity (VA) were evaluated at distance. At the conclusion of the study, after two months, all subjects completed a questionnaire in which they were asked to indicate their preference for one type of lens (soft toric or LRGP) and to rate the quality of vision in day-to-day activities. RESULTS: Thirty-six of 38 (94.7%) subjects completed the study with 75% preferring the vision of the LRGP lens as compared to the soft toric lenses worn in the study. 52.7% expressed a preference to continue with this modality despite only 38.8% reporting that these LRGP lenses are easy or very easy to handle. Wear time, subjective comfort, and subjective vision ratings exhibited no significant difference between the two groups. CONCLUSIONS: In a population of asymptomatic contact lens wearers, LRGP lenses can be considered as a good alternative to soft toric lenses for the correction of refractive astigmatism.

Preconditioning c-Kit-positive Human Cardiac Stem Cells with a Nitric Oxide Donor Enhances Cell Survival through Activation of Survival Signaling Pathways.

Cardiac stem cell therapy has shown very promising potential to repair the infarcted heart but is severely limited by the poor survival of donor cells. Nitric oxide (NO) has demonstrated cytoprotective properties in various cells, but its benefits are unknown specifically for human cardiac stem cells (hCSCs). Therefore, we investigated whether pretreatment of hCSCs with a widely used NO donor, diethylenetriamine nitric oxide adduct (DETA-NO), promotes cell survival. Results from lactate dehydrogenase release assays showed a dose- and time-dependent attenuation of cell death induced by oxidative stress after DETA-NO preconditioning; this cytoprotective effect was abolished by the NO scavenger. Concomitant up-regulation of several cell signaling molecules after DETA-NO preconditioning was observed by Western blotting, including elevated phosphorylation of NRF2, NFκB, STAT3, ERK, and AKT, as well as increased protein expression of HO-1 and COX2. Furthermore, pharmaceutical inhibition of ERK, STAT3, and NFκB activities significantly diminished NO-induced cytoprotection against oxidative stress, whereas inhibition of AKT or knockdown of NRF2 only produced a minor effect. Blocking PI3K activity or knocking down COX2 expression did not alter the protective effect of DETA-NO on cell survival. The crucial roles of STAT3 and NFκB in NO-mediated signaling pathways were further confirmed by stable expression of gene-specific shRNAs in hCSCs. Thus, preconditioning hCSCs with DETA-NO promotes cell survival and resistance to oxidative stress by activating multiple cell survival signaling pathways. These results will potentially provide a simple and effective strategy to enhance survival of hCSCs after transplantation and increase their efficacy in repairing infarcted myocardium.

Off-Pump Coronary Artery Bypass Grafting and Transaortic Transcatheter Aortic Valve Replacement.

We sought to demonstrate the effectiveness of off-pump coronary artery bypass grafting and transcatheter aortic valve replacement in two patients with porcelain aortas and lesions that could not be optimally treated with percutaneous coronary intervention. Patients with aortic stenosis and coronary artery disease who are too high-risk for conventional surgical aortic valve replacement and coronary artery bypass grafting due to comorbidities and porcelain aorta, and who do not have the appropriate anatomy for percutaneous coronary intervention should be considered for concomitant transcatheter aortic valve replacement and off-pump coronary artery bypass grafting. doi: 10.1111/jocs.12762 (J Card Surg 2016;31:435-438).

Transcatheter Aortic and Mitral Valve Implantation (TAMVI) in Native Rheumatic Valves.

A 68-year-old female with moderate to severe aortic stenosis and severe mitral stenosis, deemed too high risk for surgery (STS mortality risk = 12.3%) with a porcelain aorta, was successfully treated with a transcatheter aortic and mitral valve implantation (TAMVI) via a transapical approach. A 23 mm Sapien valve (Edwards Lifesciences, Irvine, CA, USA) was placed in the aortic position and a 29 mm inverted Sapien valve (Edwards Lifesciences) in the mitral position.

A comparison of the short-term settling of three scleral lens designs.

PURPOSE: Scleral gas-permeable lenses are rapidly gaining international popularity. Unlike corneal gas-permeable lenses, scleral lenses are fitted to the bulbar conjunctiva and settle into the tissue after insertion. To date, we are unaware of any studies examining the settling behavior of three varying scleral lens designs. The purpose of this study was to quantify the mean total settling and mean rates of settling for three scleral lens designs: Onefit P&A (Blanchard Contact Lens, Inc), Mini Scleral Design (msd) (Blanchard Contact Lens, Inc), and Jupiter (Visionary Optics). METHODS: A randomized, repeated-measures study of Onefit P&A, msd, and Jupiter was performed. Subjects were fitted from a trial lens set, according to the manufacturer's guidelines. After insertion, lens settling was measured at 0 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, and 8 hours. Statistical analyses were performed using analysis of variance and two-stage, nonlinear regression analyses of settling over time. RESULTS: On average, the Onefit P&A, msd, and Jupiter lenses settled a total of 113.7, 133.7, and 88.1 µm, respectively. Although the rates of settling were not linear, analysis of variance revealed a significant difference in the average settling rates (p < 0.05) among the groups. Tukey analyses revealed a significant difference in the settling rates for msd and Jupiter comparison (p < 0.05). Nonlinear regression analyses predicted that the lenses settled to about 80% of the final estimated values for the Onefit P&A lens, 90% for msd, and 99% for the Jupiter, after 8 hours. CONCLUSION: We concluded that the amount of settling varied significantly among the three lens designs. Settling rates were greatest shortly after insertion. Larger lenses were estimated to be settled by 8 hours. Based on the results of this short-term study, careful consideration to the settling patterns of individual scleral lens designs should be given.

Mitochondrial Membrane Potential Identifies a Subpopulation of Mesenchymal Progenitor Cells to Promote Angiogenesis and Myocardial Repair.

Identifying effective donor cells is one of obstacles that limits cell therapy for heart disease. In this study, we sorted a subpopulation of human mesenchymal progenitor cells (hMPCs) from the right atrial appendage using the low mitochondrial membrane potential. Compared to the non-sorted cells, hMPCs hold the capacity for stemness and enrich mesenchymal stem cell markers. The hMPCs display better ability for survival, faster proliferation, less production of reactive oxygen species (ROS), and greater release of cytoprotective cytokines. The hMPCs exhibit decreased expression of senescence genes and increased expression of anti-apoptotic and antioxidant genes. Intramyocardial injection of hMPCs into the infarcted heart resulted in increased left ventricular ejection fraction and reduced cardiac remodeling and infarct size in the group of animals receiving hMPCs. Both in vitro and in vivo studies indicated hMPCs have the potential to differentiate into endothelial cells and smooth muscle cells. Immunohistochemistry staining showed that cell therapy with hMPCs enhances cardiac vascular regeneration and cardiac proliferation, and decreases cardiac cell apoptosis, which is associated with the increased secretion of cytoprotective and pro-angiogenic cytokines. Overall, we discovered a subpopulation of human mesenchymal progenitor cells via their low mitochondrial membrane potential, which might provide an alternative donor cell source for cellular therapy for ischemic heart disease.

Effects of Oral Antihistamines on Tear Volume, Tear Stability, and Intraocular Pressure.

The goal of this study was to investigate the effects of two commonly used oral antihistamines-diphenhydramine and loratadine-on tear volume, tear breakup time, and intraocular pressure. Placebo, diphenhydramine, and loratadine were administered for one week to 33 subjects experimentally blind to the treatment given. All the subjects received all three treatments over a period of six weeks. The outcome measures were the change in phenol red thread test (PRT), the tear breakup time (TBUT), and the intraocular pressure (IOP) of both eyes evaluated by experimentally masked observers. Neither of the mean changes in TBUT or IOP depended on the treatment given, but there was a significant monocular decrease in tear volume from diphenhydramine use. While we used an adequate treatment washout period of seven days, our investigation was limited by the short treatment times and inclusion of only young healthy patients. Overall, however, these results suggest that systemic diphenhydramine use should be limited to increase the effectiveness of conventional therapies. Clinicians should have fewer reservations about recommending the use of loratadine concurrent with dry eye treatments.

Long-term outcomes of coronary-artery bypass grafting versus stent implantation.

BACKGROUND: Several studies have compared outcomes for coronary-artery bypass grafting (CABG) and percutaneous coronary intervention (PCI), but most were done before the availability of stenting, which has revolutionized the latter approach. METHODS: We used New York's cardiac registries to identify 37,212 patients with multivessel disease who underwent CABG and 22,102 patients with multivessel disease who underwent PCI from January 1, 1997, to December 31, 2000. We determined the rates of death and subsequent revascularization within three years after the procedure in various groups of patients according to the number of diseased vessels and the presence or absence of involvement of the left anterior descending coronary artery. The rates of adverse outcomes were adjusted by means of proportional-hazards methods to account for differences in patients' severity of illness before revascularization. RESULTS: Risk-adjusted survival rates were significantly higher among patients who underwent CABG than among those who received a stent in all of the anatomical subgroups studied. For example, the adjusted hazard ratio for the long-term risk of death after CABG relative to stent implantation was 0.64 (95 percent confidence interval, 0.56 to 0.74) for patients with three-vessel disease with involvement of the proximal left anterior descending coronary artery and 0.76 (95 percent confidence interval, 0.60 to 0.96) for patients with two-vessel disease with involvement of the nonproximal left anterior descending coronary artery. Also, the three-year rates of revascularization were considerably higher in the stenting group than in the CABG group (7.8 percent vs. 0.3 percent for subsequent CABG and 27.3 percent vs. 4.6 percent for subsequent PCI). CONCLUSIONS: For patients with two or more diseased coronary arteries, CABG is associated with higher adjusted rates of long-term survival than stenting.

Contact lens correction of presbyopia.

The contact lens wearing presbyopic population is underserved worldwide. There are several available options, including single vision contact lenses and spectacle over-correction for near, monovision, rigid gas permeable (GP) multifocal and bifocal lens designs and soft bifocal lens designs. This paper presents an overview of the latest research and clinical experience as it pertains to these forms of correction. Recent comparative studies concluded that contact lens multifocals perform better in many different forms of visual measurements, in some cases performing similar to best spectacle correction, than monovision wearers. The future appears to be very promising for multifocal GP and soft lens modalities.

Inhibition of p16INK4A to Rejuvenate Aging Human Cardiac Progenitor Cells via the Upregulation of Anti-oxidant and NFκB Signal Pathways.

Autologous human cardiac stem/progenitor cell (hCPC) therapy is a promising treatment that has come into use in recent years for patients with cardiomyopathy. Though innovative in theory, a major hindrance to the practical application of this treatment is that the hCPCs of elderly patients, who are most susceptible to myocardial disease, are senescent and prone to cell death. Rejuvenating hCPCs from elderly patients may help overcome this obstacle, and can be accomplished by reversing entry into the cellular stage of senescence. p16INK4A, a cyclin dependent kinase inhibitor, is an important player in the regulation of cell senescence. In this study, we investigated whether knockdown of p16INK4A will rejuvenate aging hCPCs to a youthful phenotype. Our data indicated that upregulation of p16INK4A is associated with hCPC senescence. Both cell proliferation and survival capacity were significantly increased in hCPCs infected with lentivirus expressing p16INK4A shRNA when compared to control hCPCs. The knockdown of p16INK4A also induced antioxidant properties as indicated by a 50% decrease in ROS generation at basal cell metabolism, and a 25% decrease in ROS generation after exposure to oxidative stress. Genes associated with cell senescence (p21CIP1), anti-apoptosis (BCL2 and MCL1), anti-oxidant (CYGB, PRDX1 and SRXN1), and NFκB signal pathway (p65, IKBKB, HMOX1, etc.), were significantly upregulated after the p16INK4A knockdown. Knocking down the NFĸB-p65 expression also significantly diminished the cytoprotective effect caused by the p16INK4A knockdown. Our results suggest that genetic knockdown of p16INK4A may play a significant role in inducing antioxidant effects and extending lifespan of aging hCPCs. This genetic modification may enhance the effectiveness of autologous hCPC therapy for repair of infarcted myocardium.

Sulfiredoxin-1 enhances cardiac progenitor cell survival against oxidative stress via the upregulation of the ERK/NRF2 signal pathway.

Cardiac stem/progenitor cells (CPCs) have recently emerged as a potentially transformative regenerative medicine to repair the infarcted heart. However, the limited survival of donor cells is one of the major challenges for CPC therapy. Our recent research effort on preconditioning human CPCs (hCPCs) with cobalt protoporphyrin (CoPP) indicated that sulfiredoxin-1 (SRXN1) is upregulated upon preconditioning aldehyde dehydrogenase bright hCPCs (ALDHbr-hCPCs) with CoPP. Further studies demonstrated that overexpressing SRXN1 enhanced the survival capacity for ALDHbr-hCPCs. This was associated with the up-regulation of anti-apoptotic factors, including BCL2 and BCL-xL. Meanwhile, overexpressing SRXN1 decreased the ROS generation and mitochondrial membrane potential, concomitant with the up-regulated primary antioxidant systems, such as PRDX1, PRDX3, TXNRD1, Catalase and SOD2. It was also observed that overexpressing SRXN1 increased the migration, proliferation, and cardiac differentiation of ALDHbr-hCPCs. Interestingly, SRXN1 activated the ERK/NRF2 cell survival signaling pathway, which may be the underlying mechanism through which overexpressing SRXN1 lead to protection of hCPCs against oxidative stress-induced apoptosis. Taken together, these results provide a rationale for the exploration of SRXN1 as a novel molecular target that can be used to enhance the effectiveness of cardiac stem/progenitor cell therapy for ischemic heart disease.

The safety and efficacy of contact lens wear in the industrial and chemical workplace.

BACKGROUND: The use and safety of contact lenses in the industrial and chemical workplace has often been questioned since the 1960s because of many unconfirmed reports of ocular injury resulting from contact lens wear. Because of these urban legends, contact lens wear has been banned or wearers have been required to wear additional personal protective equipment (PPE) not required of non-contact lens wearers. METHODS: Literature review via Medline and Google search. RESULTS: Research has shown that contact lenses typically provide protective benefits that decrease the severity of ocular injury and improve worker performance. While contact lens wear contraindications do exist, in most cases, and with proper precautions, contact lens wear is still possible. CONCLUSIONS: Industrial and chemical companies need to establish written contact lens use policies based on current studies that have shown the safety of workplace contact lens wear when combined with the same PPE required of non-contact lens wearers. Practitioners need to discuss, with their contact lens patients, the additional responsibilities required to maintain proper lens hygiene and proper PPE in the workplace.

Cytoglobin Promotes Cardiac Progenitor Cell Survival against Oxidative Stress via the Upregulation of the NFκB/iNOS Signal Pathway and Nitric Oxide Production.

Human cardiac stem/progenitor cells (hCPCs) may serve in regenerative medicine to repair the infarcted heart. However, this approach is severely limited by the poor survival of donor cells. Recent studies suggest that the mammalian globin cytoglobin (CYGB) regulates nitric oxide (NO) metabolism and cell death. In the present study, we found that CYGB is expressed in hCPCs. Through molecular approaches aimed at increasing or decreasing CYGB expression in hCPCs, we found that CYGB functions as a pro-survival factor in response to oxidative stress. This was associated with the upregulation of primary antioxidant systems such as peroxiredoxins-1, heme oxygenase-1, and anti-apoptotic factors, including BCL2, BCL-XL, and MCL1. Most significantly, we established that CYGB increased the expression of NFкB-dependent genes including iNOS, and that iNOS-dependent NO production was required for a feedforward loop that maintains CYGB expression. Our study delineates for the first time a role for a globin in regulating hCPC survival and establishes mechanistic insights in the function of CYGB. It provides a rationale for the exploration of the CYGB pathway as a molecular target that can be used to enhance the effectiveness of cardiac stem/progenitor cell therapy for ischemic heart disease.

Gas permeable toric use and applications: survey of Section on Cornea and Contact Lens Diplomates of the American Academy of Optometry.

BACKGROUND: The purpose of this study was to survey Diplomates in the Section on Cornea and Contact Lenses of the American Academy of Optometry (AAO) to determine their fitting preferences for astigmatic patients, lens materials used, and their perception of simplicity or complexity of bitoric fitting. METHODS: A total of 180 practitioners, representing all active Diplomates in the Cornea and Contact Lens Section, were sent a survey of 11 questions pertaining to their astigmatic contact lens prescribing habits. Sixty-eight (38%) of those surveyed responded to the questionnaire. RESULTS: The results of this survey included the following: (1) gas permeable (GP) lenses represented 28.7% of all contact lenses fit; (2) a spherical power effect (SPE) bitoric lens was preferred (versus four other options) by 58.8% of respondents for a 3.5 diopter (D) refractive astigmat with no residual astigmatism, whereas a cylinder power effect (CPE) bitoric was preferred by 55.9% for a 3.5 D refractive astigmatic with more than 1 D of residual astigmatism; (3) only 10.4% of responding practitioners consider GP torics to be difficult to fit. CONCLUSIONS: (1) Back surface toric and bitoric GP lenses are considered easy to design and fit by most Diplomates. (2) The Mandell-Moore guide is the preferred empirical method, and the Polycon SPE is the preferred diagnostic fitting method for bitoric lenses for those responding to the survey.

Do hospitals and surgeons with higher coronary artery bypass graft surgery volumes still have lower risk-adjusted mortality rates?

BACKGROUND: Studies that are the basis of recommended volume thresholds for CABG surgery are outdated and not reflective of recent advances in the field. This study examines both hospital and surgeon volume-mortality relations for CABG surgery through the use of a population-based clinical data set. METHODS AND RESULTS: Data from New York's clinical CABG surgery registry from 1997 to 1999 (total number of procedures, 57 150) were used to examine the individual and combined impact of annual hospital volume and annual surgeon volume on in-hospital mortality rates after adjusting for differences in severity of illness. Significantly lower risk-adjusted mortality rates occurred above all annual hospital volume thresholds between 200 and 800 and above all surgeon volume thresholds between 50 and 200. The number needed to treat (NNT) at higher-volume providers to avoid a death was minimized for a hospital threshold volume of 100 (NNT=50) and a surgeon threshold volume of 50 (NNT=118). The risk-adjusted mortality rate (RAMR) for patients undergoing surgery performed by surgeons with volumes of > or =125 in hospitals with volumes of > or =600 was 1.89%. The RAMR was significantly higher (2.67%) for patients undergoing surgery performed by surgeons with volumes of <125 in hospitals with volumes of <600. CONCLUSIONS: Higher-volume surgeons and hospitals continue to have lower risk-adjusted mortality rates, and patients undergoing surgery performed by higher-volume surgeons in higher-volume hospitals have the lowest mortality rates.