Transduction Efficiency of AAV 2/6, 2/8 and 2/9 Vectors for Delivering Genes in Human Corneal Fibroblasts

In the present study, cellular tropism and relative transduction efficiency of AAV2/6, AAV2/8 and AAV2/9 vectors have been tested for the cornea using primary cultures of human corneal fibroblasts. The AAV6, AAV8 and AAV9 serotypes having AAV2 ITR plasmid encoding for alkaline phosphatase (AP) gene were generated by transfecting HEK293 cell line with pHelper, pARAP4 and pRep/Cap plasmids. Primary cultures of human corneal fibroblasts were exposed to AAV infectious particles at two different doses (1×105 and 2×105 MOI). Cytochemistry and enzyme assays were used to measure delivered transgene expression in samples collected at 4 and 30 hours after AAV infection by counting AP-stained cells or quantifying AP enzyme activity. Cellular toxicity of AAVs was evaluated with TUNEL and trypan blue assays. All three AAV serotypes transduced human corneal fibroblasts. The order of transduction efficiency was AAV2/6\u3e\u3e\u3eAAV2/9\u3eAAV2/8. The transduction efficiency of AAV2/6 was 30-50 fold higher (p \u3c0.001) for the human corneal fibroblasts compared to the AAV2/8 or AAV2/9 at two tested doses. The level of transgene expression at 4 hrs was considerably low compared to 30 hrs suggesting that the transgene delivery did not reach its peak at 4 hrs. Cultures exposed to any of the three AAV serotypes showed more than 97% cellular viability and less than 5 TUNEL positive cells suggesting that tested AAV serotypes do not induce significant cell death and are safe for corneal gene therapy

The Masses of Population II White Dwarfs

Globular star clusters are among the first stellar populations to have formed in the Milky Way, and thus only a small sliver of their initial spectrum of stellar types are still burning hydrogen on the main-sequence today. Almost all of the stars born with more mass than 0.8 M_sun have evolved to form the white dwarf cooling sequence of these systems, and the distribution and properties of these remnants uniquely holds clues related to the nature of the now evolved progenitor stars. With ultra-deep HST imaging observations, rich white dwarf populations of four nearby Milky Way globular clusters have recently been uncovered, and are found to extend an impressive 5 - 8 magnitudes in the faint-blue region of the H-R diagram. In this paper, we characterize the properties of these population II remnants by presenting the first direct mass measurements of individual white dwarfs near the tip of the cooling sequence in the nearest of the Milky Way globulars, M4. Based on Gemini/GMOS and Keck/LRIS multiobject spectroscopic observations, our results indicate that 0.8 M_sun population II main-sequence stars evolving today form 0.53 +/- 0.01 M_sun white dwarfs. We discuss the implications of this result as it relates to our understanding of stellar structure and evolution of population II stars and for the age of the Galactic halo, as measured with white dwarf cooling theory.Comment: Accepted for Publication in Astrophys. J. on Aug. 05th, 2009. 19 pages including 9 figures and 2 tables (journal format

Spatial and Temporal Ontogenies of Glutathione Peroxidase and Glutathione Disulfide Reductase During Development of the Prenatal Rat

Spatial and temporal expression and regulation of the antioxidant enzymes, glutathione peroxidase(GSH-Px), glutathione disulfide reductase (GSSG-Rd) may be important in determining cell-specificsusceptibility to embryotoxicants. Creation of tissue-specific ontogenies for antioxidant enzyme activities during development isan important first step in understanding regulatory relationships. Early organogenesis-stage embryos were groupedaccording to the somite number (GD 9–13), and fetuses were evaluated by gestational day (GD 14–21). GSH-Px activities in thevisceral yolk sac (VYS) increased on consecutive days from GD 9 to GD 13, representing a 5.7-fold increase during this period of development. GSH-Pxactivities in VYS decreased after GD 13, ultimately constituting a 37% decrease at GD 21. Head, heart, and trunk specific activities generallyincreased from GD 9 to GD 13 albeit not to the same magnitude as detected in the VYS. GSSG-Rd activities showed substantial increases in the VYS from GD 9 to GD13, 6.3-fold and decreased thereafter to 50% by GD 21. The greatest changes in enzyme activities were noted in the period between GD 10 and GD 11,where the embryo establishes an active cardiovascular system and begins to convert to aerobic metabolism. Generally, from GD 14–21, embryonic organGSH-Px and GSSG-Rd activities either remained constant or increased as gestation progressed. These studies suggest the importance of the VYS in dealing withROS and protecting the embryo. Furthermore, understanding the consequences of lower antioxidant activities during organogenesis may help to pinpoint periods ofteratogenic susceptibility to xenobiotics and increased oxygen. © 2001 John Wiley & Sons, Inc. J Biochem Mol Toxicol 15:197–206, 2001Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35226/1/17_ftp.pd

Deep ACS Imaging in the Globular Cluster NGC 6397: The Cluster Color Magnitude Diagram and Luminosity Function

We present the CMD from deep HST imaging in the globular cluster NGC 6397. The ACS was used for 126 orbits to image a single field in two colors (F814W, F606W) 5 arcmin SE of the cluster center. The field observed overlaps that of archival WFPC2 data from 1994 and 1997 which were used to proper motion (PM) clean the data. Applying the PM corrections produces a remarkably clean CMD which reveals a number of features never seen before in a globular cluster CMD. In our field, the main sequence stars appeared to terminate close to the location in the CMD of the hydrogen-burning limit predicted by two independent sets of stellar evolution models. The faintest observed main sequence stars are about a magnitude fainter than the least luminous metal-poor field halo stars known, suggesting that the lowest luminosity halo stars still await discovery. At the bright end the data extend beyond the main sequence turnoff to well up the giant branch. A populous white dwarf cooling sequence is also seen in the cluster CMD. The most dramatic features of the cooling sequence are its turn to the blue at faint magnitudes as well as an apparent truncation near F814W = 28. The cluster luminosity and mass functions were derived, stretching from the turn off down to the hydrogen-burning limit. It was well modeled with either a very flat power-law or a lognormal function. In order to interpret these fits more fully we compared them with similar functions in the cluster core and with a full N-body model of NGC 6397 finding satisfactory agreement between the model predictions and the data. This exercise demonstrates the important role and the effect that dynamics has played in altering the cluster IMF.Comment: 43 pages including 4 tables and 12 diagrams. Figures 2 and 3 have been bitmapped. Accepted for publication in the Astronomical Journa

The White Dwarf Cooling Sequence of NGC6397

We present the results of a deep Hubble Space Telescope (HST) exposure of the nearby globular cluster NGC6397, focussing attention on the cluster's white dwarf cooling sequence. This sequence is shown to extend over 5 magnitudes in depth, with an apparent cutoff at magnitude F814W=27.6. We demonstrate, using both artificial star tests and the detectability of background galaxies at fainter magnitudes, that the cutoff is real and represents the truncation of the white dwarf luminosity function in this cluster. We perform a detailed comparison between cooling models and the observed distribution of white dwarfs in colour and magnitude, taking into account uncertainties in distance, extinction, white dwarf mass, progenitor lifetimes, binarity and cooling model uncertainties. After marginalising over these variables, we obtain values for the cluster distance modulus and age of \mu_0 = 12.02 \pm 0.06 and T_c = 11.47 \pm 0.47Gyr (95% confidence limits). Our inferred distance and white dwarf initial-final mass relations are in good agreement with other independent determinations, and the cluster age is consistent with, but more precise than, prior determinations made using the main sequence turnoff method. In particular, within the context of the currently accepted \Lambda CDM cosmological model, this age places the formation of NGC6397 at a redshift z=3, at a time when the cosmological star formation rate was approaching its peak.Comment: 56 pages, 30 figure

TIGIT Marks Exhausted T Cells, Correlates with Disease Progression, and Serves as a Target for Immune Restoration in HIV and SIV Infection.

HIV infection induces phenotypic and functional changes to CD8+ T cells defined by the coordinated upregulation of a series of negative checkpoint receptors that eventually result in T cell exhaustion and failure to control viral replication. We report that effector CD8+ T cells during HIV infection in blood and SIV infection in lymphoid tissue exhibit higher levels of the negative checkpoint receptor TIGIT. Increased frequencies of TIGIT+ and TIGIT+ PD-1+ CD8+ T cells correlated with parameters of HIV and SIV disease progression. TIGIT remained elevated despite viral suppression in those with either pharmacological antiretroviral control or immunologically in elite controllers. HIV and SIV-specific CD8+ T cells were dysfunctional and expressed high levels of TIGIT and PD-1. Ex-vivo single or combinational antibody blockade of TIGIT and/or PD-L1 restored viral-specific CD8+ T cell effector responses. The frequency of TIGIT+ CD4+ T cells correlated with the CD4+ T cell total HIV DNA. These findings identify TIGIT as a novel marker of dysfunctional HIV-specific T cells and suggest TIGIT along with other checkpoint receptors may be novel curative HIV targets to reverse T cell exhaustion

The role of Nrf1 and Nrf2 in the regulation of glutathione and redox dynamics in the developing zebrafish embryo

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Redox Biology 13 (2017): 207–218, doi:10.1016/j.redox.2017.05.023.Redox signaling is important for embryogenesis, guiding pathways that govern processes crucial for embryo patterning, including cell polarization, proliferation, and apoptosis. Exposure to pro-oxidants during this period can be deleterious, resulting in altered physiology, teratogenesis, later-life diseases, or lethality. We previously reported that the glutathione antioxidant defense system becomes increasingly robust, including a doubling of total glutathione and dynamic shifts in the glutathione redox potential at specific stages during embryonic development in the zebrafish, Danio rerio. However, the mechanisms underlying these changes are unclear, as is the effectiveness of the glutathione system in ameliorating oxidative insults to the embryo at different stages. Here, we examine how the glutathione system responds to the model pro-oxidants tert-butylhydroperoxide and tert-butylhydroquinone at different developmental stages, and the role of Nuclear factor erythroid 2-related factor (Nrf) proteins in regulating developmental glutathione redox status. Embryos became increasingly sensitive to pro-oxidants after 72 h post-fertilization (hpf), after which the duration of the recovery period for the glutathione redox potential was increased. To determine whether the doubling of glutathione or the dynamic changes in glutathione redox potential are mediated by zebrafish paralogs of Nrf transcription factors, morpholino oligonucleotides were used to knock down translation of Nrf1 and Nrf2 (nrf1a, nrf1b, nrf2a, nrf2b). Knockdown of Nrf1a or Nrf1b perturbed glutathione redox state until 72 hpf. Knockdown of Nrf2 paralogs also perturbed glutathione redox state but did not significantly affect the response of glutathione to pro-oxidants. Nrf1b morphants had decreased gene expression of glutathione synthesis enzymes, while hsp70 increased in Nrf2b morphants. This work demonstrates that despite having a more robust glutathione system, embryos become more sensitive to oxidative stress later in development, and that neither Nrf1 nor Nrf2 alone appear to be essential for the response and recovery of glutathione to oxidative insults.This research was supported by several NIH grants, including F32ES028085 (to KES), F32ES017585 (to ART-L), F32ES019832 (to LMW), P20GM103423 (to LMW), R01ES025748 (to ART-L), R01ES015912 (JJS), and R01ES016366 (MEH). Additional research support was provided by the J. Seward Johnson Fund at WHOI and the WHOI Postdoctoral Scholar Award with funding from Walter A. and Hope Noyes Smith (to ART-L)

Deep ACS Imaging in the Globular Cluster NGC6397: Dynamical Models

We present N-body models to complement deep imaging of the metal-poor core-collapsed cluster NGC6397 obtained with the Hubble Space Telescope. All simulations include stellar and binary evolution in-step with the stellar dynamics and account for the tidal field of the Galaxy. We focus on the results of a simulation that began with 100000 objects (stars and binaries), 5% primordial binaries and Population II metallicity. After 16 Gyr of evolution the model cluster has about 20% of the stars remaining and has reached core-collapse. We compare the color-magnitude diagrams of the model at this age for the central region and an outer region corresponding to the observed field of NGC6397 (about 2-3 half-light radii from the cluster centre). This demonstrates that the white dwarf population in the outer region has suffered little modification from dynamical processes - contamination of the luminosity function by binaries and white dwarfs with non-standard evolution histories is minimal and should not significantly affect measurement of the cluster age. We also show that the binary fraction of main-sequence stars observed in the NGC6397 field can be taken as representative of the primordial binary fraction of the cluster. For the mass function of the main-sequence stars we find that although this has been altered significantly by dynamics over the cluster lifetime, especially in the central and outer regions, that the position of the observed field is close to optimal for recovering the initial mass function of the cluster stars (below the current turn-off mass). More generally we look at how the mass function changes with radius in a dynamically evolved stellar cluster and suggest where the best radial position to observe the initial mass function is for clusters of any age.Comment: 34 pages, 11 figures, submitted to AJ, companion paper to 0708.403

Effects of diet form and feeder adjustment on growth performance of nursery and finishing pigs

Citation: Nemechek, J. E., Tokach, M. D., Dritz, S. S., Fruge, E. D., Hansen, E. L., Goodband, R. D., . . . Woodworth, J. C. (2015). Effects of diet form and feeder adjustment on growth performance of nursery and finishing pigs. Journal of Animal Science, 93(8), 4172-4180. doi:10.2527/jas2015-9028Three experiments were conducted to determine the effects of feeder adjustment and diet form on growth performance of nursery (Exp. 1 and 2) and finishing (Exp. 3) pigs. Treatments were arranged as a 2 x 3 factorial with the main effects of feeder adjustment and diet form. The 2 feeder adjustments were a narrow and wide feeder adjustment (minimum gap opening of 1.27 and 2.54 cm, respectively). The 3 diet forms were meal, poor-quality pellets (70% pellets and 30% fines for Exp. 1 and 2 and 50% pellets and 50% fines for Exp. 3), and screened pellets with minimal fines (3 to 10%). In Exp. 1, 210 pigs (initially 11.9 kg BW) were used in a 21-d trial with 7 pigs per pen and 5 pens per treatment. No feeder adjustment x diet form interactions were observed. There were no differences in ADG, ADFI, or G:F due to feeder adjustment. Pigs fed the meal diet had increased (P < 0.05) ADG and ADFI compared with pigs fed the poor-quality or screened pellets. Pigs fed meal or poor-quality pellets had decreased (P < 0.05) G: F compared with pigs fed screened pellets. In Exp. 2, 1,005 nursery pigs (initially 14.1 kg BW) were used in a 28-d trial with 26 to 28 pigs per pen and 6 pens per treatment. Pigs fed from the narrow feeder adjustment had decreased (P < 0.05) ADG and ADFI compared with pigs fed from the wide adjustment with no differences in G: F. Pigs fed the meal diet had decreased (P < 0.05) ADG compared with pigs fed poor-quality or screened pellets. Pigs fed meal or poor-quality pellets had decreased (P < 0.05) G: F compared with pigs fed screened pellets. In Exp. 3, 246 pigs (initially 56.8 kg BW) were used in a 69-d trial with 5 pens per treatment and 6 or 7 pigs per pen. Overall, ADFI decreased (P < 0.05) and G: F increased (P < 0.05) for pigs fed from the narrow adjusted feeders compared with the wide adjustment with no differences in ADG. Overall, pigs fed meal diets tended to have decreased (P < 0.10) ADG and had decreased (P < 0.05) G: F compared with pigs fed screened pellets; ADG and G: F in those fed poor-quality pellets were intermediate. Feeding meal or poor-quality pellets increased (P < 0.05) ADFI compared with pigs fed screened pellets. In conclusion, feeding nursery pigs from a wide feeder gap may increase ADG and ADFI with no negative effects on G: F. For finishing pigs, reducing feeder gap reduced feed disappearance and improved G: F. In all experiments, the greatest G: F improvements from pelleting were observed when the percentage of fines was minimized

Diminishing Effectiveness of Long-Term Maintenance Topical Steroid Therapy in PPI Non-Responsive Eosinophilic Esophagitis

While topical corticosteroids are first-line therapy for eosinophilic esophagitis (EoE), the data regarding long-term effectiveness are lacking. We aimed to determine long-term histologic and endoscopic outcomes of maintenance therapy in EoE steroid responders