Site-specific integration in mammalian cells mediated by a new hybrid baculovirus-adeno-associated virus vector

Baculovirus can transiently transduce primary human and rat hepatocytes, as well as a subset of stable celllines. To prolong transgene expression, we have developed new hybrid vectors which associate key elementsfrom adeno-associated virus (AAV) with the elevated transducing capacity of baculovirus. The hybrid vectorscontain a transgene cassette composed of the !-galactosidase (!-Gal) reporter gene and the hygromycin resistance(Hygr) gene flanked by the AAV inverted terminal repeats (ITRs), which are necessary for AAV replicationand integration in the host genome. Constructs were derived both with and without the AAV rep geneunder the p5 and p19 promoters cloned in different positions with respect to the baculovirus polyheidrinpromoter. A high-titer preparation of baculovirus-AAV (Bac-AAV) chimeric virus containing the ITR–Hygr–!-Gal sequence was obtained with insect cells only when the rep gene was placed in an antisense orientationto the polyheidrin promoter. Infection of 293 cells with Bac-AAV virus expressing the rep gene results in a 10-to 50-fold increase in the number of Hygr stable cell clones. Additionally, rep expression determined the localizationof the transgene cassette in the aavs1 site in approximately 41% of cases as detected by bothSouthern blotting and fluorescent in situ hybridization analysis. Moreover, site-specific integration of the ITRflankedDNA was also detected by PCR amplification of the ITR-aavs1 junction in transduced human fibroblasts.These data indicate that Bac-AAV hybrid vectors can allow permanent, nontoxic gene delivery of DNAconstructs for ex vivo treatment of primary human cells

Study of the neutron-rich region in the vicinity of 208Pb via multinucleon transfer reactions

The multinucleon transfer reaction mechanism was employed to populate isotopes around the doubly- magic 208 Pb nucleus. We used an unstable 94 Rb beam on 208 Pb targets of different thickness. Transfer channels were studied via the fragment-γ and γ-γ coincidences, by using MINIBALL γ spectrometer coupled to a particle detector. Gamma transitions associated to the different Pb isotopes, populated by the neutron transfers, are discussed in terms of excitation energy and spin. Fragment angular distributions were extracted, andcompared with the reaction model

Don’t forget the porpoise: acoustic monitoring reveals fine scale temporal variation between bottlenose dolphin and harbour porpoise in Cardigan Bay SAC

Populations of bottlenose dolphin and harbour porpoise inhabit Cardigan Bay, which was designated a Special Area of Conservation (SAC), with bottlenose dolphin listed as a primary feature for its conservation status. Understanding the abundance, distribution and habitat use of species is fundamental for conservation and the implementation of management. Bottlenose dolphin and harbour porpoise usage of feeding sites within Cardigan Bay SAC was examined using passive acoustic monitoring. Acoustic detections recorded with calibrated T-PODs (acoustic data loggers) indicated harbour porpoise to be present year round and in greater relative abundance than bottlenose dolphin. Fine-scale temporal partitioning between the species occurred at three levels: (1) seasonal differences, consistent between years, with porpoise detections peaking in winter months and dolphin detections in summer months; (2) diel variation, consistent across sites, seasons and years, with porpoise detections highest at night and dolphin detections highest shortly after sunrise; and (3) tidal variation was observed with peak dolphin detections occurring during ebb at the middle of the tidal cycle and before low tide, whereas harbour porpoise detections were highest at slack water, during and after high water with a secondary peak recorded during and after low water. General Additive Models (GAMs) were applied to better understand the effects of each covariate. The reported abundance and distribution of the two species, along with the temporal variation observed, have implications for the design and management of protected areas. Currently, in the UK, no SACs have been formally designated for harbour porpoise while three exist for bottlenose dolphins. Here, we demonstrate a need for increased protection and species-specific mitigation measures for harbour porpoise

Morphological and Functional Changes in the Retina after Chronic Oxygen-Induced Retinopathy

The mouse model of oxygen-induced retinopathy (OIR) has been widely used for studies of retinopathy of prematurity (ROP). This disorder, characterized by abnormal vascularization of the retina, tends to occur in low birth weight neonates after exposure to high supplemental oxygen. Currently, the incidence of ROP is increasing because of increased survival of these infants due to medical progress. However, little is known about changes in the chronic phase after ROP. Therefore, in this study, we examined morphological and functional changes in the retina using a chronic OIR model. Both the a- and b-waves in the OIR model recovered in a time-dependent manner at 4 weeks (w), 6 w, and 8 w, but the oscillatory potential (OP) amplitudes remained depressed following a return to normoxic conditions. Furthermore, decrease in the thicknesses of the inner plexiform layer (IPL) and inner nuclear layer (INL) at postnatal day (P) 17, 4 w, and 8 w and hyperpermeability of blood vessels were observed in conjunction with the decrease in the expression of claudin-5 and occludin at 8 w. The chronic OIR model revealed the following: (1) a decrease in OP amplitudes, (2) morphological abnormalities in the retinal cells (limited to the IPL and INL) and blood vessels, and (3) an increase in retinal vascular permeability via the impairment of the tight junction proteins. These findings suggest that the experimental animal model used in this study is suitable for elucidating the pathogenesis of ROP and may lead to the development of potential therapeutic agents for ROP treatment

Pars plana vitrectomy for diabetic macular edema. Internal limiting membrane delamination vs posterior hyaloid removal. A prospective randomized trial

To access publisher full text version of this article. Please click on the hyperlink in Additional Links field.BACKGROUND: Diabetes mellitus, as well as subsequent ocular complications such as cystoid macular edema (CME), are of fundametal socio-economic relevance. Therefore, we evaluated the influence of internal limiting membrane (ILM) removal on longterm morphological and functional outcome in patients with diabetes mellitus (DM) type 2 and chronic CME without evident vitreomacular traction. METHOD: Forty eyes with attached posterior hyaloid were included in this prospective trial and randomized intraoperatively. Prior focal (n = 31) or panretinal (n = 25) laser coagulation was permitted. Group I (n = 19 patients) underwent surgical induction of posterior vitreous detachment (PVD), group II (n = 20 patients) PVD and removal of the ILM. Eleven patients with detached posterior hyaloid (group III) were not randomized, and ILM removal was performed. One eye had to be excluded from further analysis. Examinations included ETDRS best-corrected visual acuity (BCVA), fluorescein angiography (FLA) and OCT at baseline, 3 and 6 months postoperatively. Main outcome measure was BCVA at 6 months, secondary was foveal thickness. RESULTS: Mean BCVA over 6 months remained unchanged in 85% of patients of group II, and decreased in 53% of patients of group I. Results were not statistically significant different [group I: mean decrease log MAR 95% CI (0.06; 0.32), group II: (-0.02; 0.11)]. OCT revealed a significantly greater reduction of foveal thickness following PVD with ILM removal [group I: mean change: 95% CI (-208.95 μm; -78.05 μm), group II: (-80.90 μm: +59.17 μm)]. CONCLUSION: Vitrectomy, PVD with or without ILM removal does not improve vision in patients with DM type 2 and cystoid diabetic macular edema without evident vitreoretinal traction. ILM delamination shows improved morphological results, and appears to be beneficial in eyes with preexisting PVD

Evidence for a spin-aligned neutron-proton paired phase from the level structure of 92^{92}Pd

The general phenomenon of shell structure in atomic nuclei has been understood since the pioneering work of Goeppert-Mayer, Haxel, Jensen and Suess.They realized that the experimental evidence for nuclear magic numbers could be explained by introducing a strong spin-orbit interaction in the nuclear shell model potential. However, our detailed knowledge of nuclear forces and the mechanisms governing the structure of nuclei, in particular far from stability, is still incomplete. In nuclei with equal neutron and proton numbers ($N = Z$), the unique nature of the atomic nucleus as an object composed of two distinct types of fermions can be expressed as enhanced correlations arising between neutrons and protons occupying orbitals with the same quantum numbers. Such correlations have been predicted to favor a new type of nuclear superfluidity; isoscalar neutron-proton pairing, in addition to normal isovector pairing (see Fig. 1). Despite many experimental efforts these predictions have not been confirmed. Here, we report on the first observation of excited states in $N = Z = 46$ nucleus $^{92}$Pd. Gamma rays emitted following the $^{58}$Ni($^{36}$Ar,2$n$)$^{92}$Pd fusion-evaporation reaction were identified using a combination of state-of-the-art high-resolution {\gamma}-ray, charged-particle and neutron detector systems. Our results reveal evidence for a spin-aligned, isoscalar neutron-proton coupling scheme, different from the previous prediction. We suggest that this coupling scheme replaces normal superfluidity (characterized by seniority coupling) in the ground and low-lying excited states of the heaviest N = Z nuclei. The strong isoscalar neutron- proton correlations in these $N = Z$ nuclei are predicted to have a considerable impact on their level structures, and to influence the dynamics of the stellar rapid proton capture nucleosynthesis process.Comment: 13 pages, 3 figure

The Cholesterol Metabolite 25-Hydroxycholesterol Activates Estrogen Receptor α-Mediated Signaling in Cancer Cells and in Cardiomyocytes

The hydroxylated derivatives of cholesterol, such as the oxysterols, play important roles in lipid metabolism. In particular, 25-hydroxycholesterol (25 HC) has been implicated in a variety of metabolic events including cholesterol homeostasis and atherosclerosis. 25 HC is detectable in human plasma after ingestion of a meal rich in oxysterols and following a dietary cholesterol challenge. In addition, the levels of oxysterols, including 25 HC, have been found to be elevated in hypercholesterolemic serum.Here, we demonstrate that the estrogen receptor (ER) α mediates gene expression changes and growth responses induced by 25 HC in breast and ovarian cancer cells. Moreover, 25 HC exhibits the ERα-dependent ability like 17 β-estradiol (E2) to inhibit the up-regulation of HIF-1α and connective tissue growth factor by hypoxic conditions in cardiomyocytes and rat heart preparations and to prevent the hypoxia-induced apoptosis.The estrogen action exerted by 25 HC may be considered as an additional factor involved in the progression of breast and ovarian tumors. Moreover, the estrogen-like activity of 25 HC elicited in the cardiovascular system may play a role against hypoxic environments